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A new and scalable method for the isolation of extracellular vesicles (EV) from Citrus lemon juice samples was developed. The methodology included preliminary preconcentration of the sample using ultrafiltration (UF) followed by size-exclusion chromatography (SEC) purification and final preconcentration of the eluates. Transmission electron microscopy and proteomic analysis showed that isolates contained exosome-like vesicles, exocyst-positive organelle (EXPO), and microvesicles. The efficiency of certain isolation steps was evaluated with total protein content assay (bicinchoninic acid assay, BCA), nanoparticles tracking analysis (NTA), and capillary electrophoresis (CE). A good correlation between CE, BCA, and NTA results was shown. The application of CE enabled the detection of soluble contaminants, macromolecular aggregates, and vesicles' heterogeneity. The fluorescent staining of encapsulated nucleic acids was proposed for the identity confirmation of EV detected in CE. The study demonstrates the CE as a comprehensive tool for monitoring of the EV isolation process., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Aleksandra Stec, Leszek Kalinowski reports financial support was provided by Polish Ministry of Education and Science., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Multiple molecular pathways including the receptor for advanced glycation end-products-diaphanous related formin 1 (RAGE-Diaph1) signaling are known to play a role in diabetic peripheral neuropathy (DPN). Evidence suggests that neuropathological alterations in type 1 diabetic spinal cord may occur at the same time as or following peripheral nerve abnormalities. We demonstrated that DPN was associated with perturbations of RAGE-Diaph1 signaling pathway in peripheral nerve accompanied by widespread spinal cord molecular changes. More than 500 differentially expressed genes (DEGs) belonging to multiple functional pathways were identified in diabetic spinal cord and of those the most enriched was RAGE-Diaph1 related PI3K-Akt pathway. Only seven of spinal cord DEGs overlapped with DEGs from type 1 diabetic sciatic nerve and only a single gene cathepsin E (CTSE) was common for both type 1 and type 2 diabetic mice. In silico analysis suggests that molecular changes in spinal cord may act synergistically with RAGE-Diaph1 signaling axis in the peripheral nerve. KEY MESSAGES: Molecular perturbations in spinal cord may be involved in the progression of diabetic peripheral neuropathy. Diabetic peripheral neuropathy was associated with perturbations of RAGE-Diaph1 signaling pathway in peripheral nerve accompanied by widespread spinal cord molecular changes. In silico analysis revealed that PI3K-Akt signaling axis related to RAGE-Diaph1 was the most enriched biological pathway in diabetic spinal cord. Cathepsin E may be the target molecular hub for intervention against diabetic peripheral neuropathy., (© 2023. The Author(s).)

This study investigated the effect of low-intensity blue light on the albino Wistar rat retina, including intrinsically photosensitive retinal ganglion cells (ipRGCs). Three groups of nine albino Wistar rats were used. One group was continuously exposed to blue light (150 lx) for 2 d (STE); one was exposed to 12 h of blue light and 12 h of darkness for 10 d (LTE); one was maintained in 12 h of white light (150 lx) and 12 h of darkness for 10 d (control). Melanopsin (Opn4) was immunolabelled on retinal whole-mounts. To count and measure Opn4-positive ipRGC somas and dendrites (including Sholl profiles), Neuron J was used. Retinal cryosections were immunolabeled for glial fibrillary acid protein (GFAP) and with terminal deoxynucleotidyl transferase dUTP nick-end labelling for apoptosis detection. LTE reduced the length of Opn4-positive ipRGC dendrites ( p = 0.03) and decreased Opn4-immunoreactivity in ipRGC outer stratifying dendrites. LTE and STE decreased the complexity of dendritic arborization (Sholl profile; p < 0.001, p = 0.03, respectively), increased retinal GFAP immunoreactivity ( p < 0.001, p = 0.002, respectively), and caused outer segment vesiculation and outer nuclear layer apoptosis. Ultrastructural analysis showed that LTE damaged mitochondria in retinal ganglion cells and in the inner plexiform layer. Thus, LTE to low-intensity blue light harms the retinas of albino Wistar rats.

The European beaver is a herbivorous rodent whose diet changes seasonally, and in winter consists of large quantities of woody plants. It is distinguished among other mammals by a unique organization of the stomach that comprises the cardiogastric gland and by the unusual process of mucus formation in the gastric mucosa. The aim of study was to (i) characterize the structure of the beaver esophagus with particular attention to the mucosal epithelium; (ii) compare the histological structure of the esophagi collected in spring, summer, and winter; (iii) provide preliminary data on the structure of the esophagus in beaver fetuses. The study was conducted on esophagi of 18 adult beavers captured in Poland in April, August, and December, and on 3 fetal organs. The results obtained in adults show that the mucosa is lined with thick stratified squamous keratinized epithelium with a structure similar to that of the skin epidermis. Ultrastructural studies reveal the presence of multiple lamellar and non-lamellar bodies in granular cells, whose morphology and location gradually change while reaching the upper epithelial layers. The muscularis mucosa comprises a layer of longitudinally oriented bundles of smooth muscle cells. Both mucosa and submucosa do not comprise any glands. The thick muscularis externa consists mainly of internal circular and external longitudinal layers of striated muscle fibers. The keratinized layer of mucosa epithelium was 2-3-fold thicker in esophagi collected in winter than in those collected in spring and summer, while the epithelial cell layer thickness remained unchanged regardless of the season. Immunolabeling for proliferating cell nuclear antigen shows a higher index of epithelium proliferation in esophagi collected in winter than in spring and summer. No seasonal differences were noted in other layers of the esophagus. Fetal organs have epithelium covered with a keratinized layer, thinner than in adults, and the muscularis externa comprises both striated and smooth muscle cells.

The possibility of using chitin from the molts of an insect-ealworm ( Tenebrio molitor ) to remove anionic (RB5, RY84) and cationic dyes (BV10, BR46) from aqueous solutions was investigated. The scope of the research included, among others: Characteristics of chitin from mealworms (FTIR, SEM, pH PZC ), the effect of pH on sorption efficiency, sorption kinetics (pseudo-first, pseudo-second order, intramolecular diffusion models) and the determination of the maximum sorption capacity (Langmuir and Freundlich models). The sorption efficiency of anionic dyes on chitin from mealworm was the highest at pH 2-3, and for cationic dyes at pH 6. The equilibrium time of sorption of anionic dyes was 240-300 min and for cationic dyes it was 180-240 min. The experimental data on dye sorption kinetics was best described by the pseudo-second order model. The maximum sorption capacity of chitin from the mealworm for the anionic dyes RB5 and RY84 was 121.15 mg/g and 138.55 mg/g, respectively, and was higher than with some carbon-based materials (literature data). In the case of cationic dyes, the sorption capacity of the tested chitin was lower and reached 3.22 mg/g and 59.56 mg/g for BV10 and BR46, respectively.

Research on age-dependent changes in pineal activity has been limited almost exclusively to melatonin (MLT). This study determined, for the first time, the alterations occurring in the metabolic profile of MLT synthesis-related indoles during the post-embryonic development period in birds. Turkeys reared under a 12 h light/dark cycle were euthanized at 2 h intervals for 24 h at the ages of 2, 7, 14, and 28 days and 10, 20, 30, and 45 weeks. The results showed prominent changes in the metabolic profile of indoles during development and could be distinguished in four stages. The first stage, from hatching to the age of 2 weeks, was characterized by a decrease in the 5-hydroxytryptophan concentration and an increase in the concentrations of serotonin (5-HT), MLT, 5-methoxyindoleacetic acid, and 5-methoxytryptamine (5-MTAM). During the second stage, around the age of 1 month, the concentrations of N-acetylserotonin (NAS) and MLT reached a maximum. The synthesis and degradation of 5-HT were also the highest. The third stage, around the age of 10 weeks, was characterized by decreased levels of 5-HT (approximately 50%) and 5-hydroxyindoleacetic acid and a high level of 5-MTAM. The last stage, covering the age of 20 to 45 weeks, was characterized by a large decrease in the synthesis, content, and degradation of 5-HT. Despite these changes, there were no prominent differences in the nocturnal levels of NAS and MLT between the third and fourth stages. The concentrations of all tryptophan derivatives showed daily fluctuations until the age of 45 weeks.

Despite concern over potential retinal damage linked to exposure to light-emitting-diode (LED) light (particularly blue light), it remains unknown how exposure to low-intensity monochromatic LED light affects the expression of rhodopsin ( Rho , a photopigment that mediates light-induced retinal degeneration), melanopsin ( Opn4 , a blue-light sensitive photopigment), c-Fos (associated with retinal damage/degeneration), and Birc5 (anti-apoptotic). This study investigated the mRNA expression profiles of these genes under exposure to white and monochromatic light (blue, red, green) in the retinas of albino rats under a cycle of 12 h of light and 12 h of darkness. In each group, 32 Wistar rats were exposed to one type of monochromatic-LED or white-fluorescent light for 7 day (150 lx). Retinal samples were taken for qPCR analysis and light and electron microscopy. Blue and green light exposure markedly decreased expression of Rho and Opn4 mRNA and increased expression of Birc5 and c-Fos mRNA ( P < 0.05). In retinas from the blue-light group, loss and vesiculation of photoreceptor outer segments were visible, but not in retinas from the red-light and control group. Measurements of the photoreceptor inner and outer segments length revealed, that this length was significantly decreased in the blue- and green-light exposure groups ( P < 0.02), but not in the red-light exposure group. Increased expression of Birc5 and decreased expression of Rho and Opn4 after exposure to blue and green light may be early responses that help to reduce light-induced retinal damage., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ziółkowska and Lewczuk.)

The development of field-emission scanning electron microscopes for high-resolution imaging at very low acceleration voltages and equipped with highly sensitive detectors of backscattered electrons (BSE) has enabled transmission electron microscopy (TEM)-like imaging of the cut surfaces of tissue blocks, which are impermeable to the electron beam, or tissue sections mounted on the solid substrates. This has resulted in the development of methods that simplify and accelerate ultrastructural studies of large areas and volumes of biological samples. This article provides an overview of these methods, including their advantages and disadvantages. The imaging of large sample areas can be performed using two methods based on the detection of transmitted electrons or BSE. Effective imaging using BSE requires special fixation and en bloc contrasting of samples. BSE imaging has resulted in the development of volume imaging techniques, including array tomography (AT) and serial block-face imaging (SBF-SEM). In AT, serial ultrathin sections are collected manually on a solid substrate such as a glass and silicon wafer or automatically on a tape using a special ultramicrotome. The imaging of serial sections is used to obtain three-dimensional (3D) information. SBF-SEM is based on removing the top layer of a resin-embedded sample using an ultramicrotome inside the SEM specimen chamber and then imaging the exposed surface with a BSE detector. The steps of cutting and imaging the resin block are repeated hundreds or thousands of times to obtain a z-stack for 3D analyses.

The embryonic ontogeny of pineal secretory activity in birds has been investigated almost exclusively in chickens. This study aimed to characterize this process in domestic geese. The pineal organs of embryos aged 18-28 days were incubated in superfusion culture under different light conditions for 4-5 days and treated with norepinephrine (NE). Melatonin (MLT) was measured by radioimmunoassay and other indoles by HPLC with fluorescence detection. Additionally, pineal organs were collected from embryos at 14-28 days of age and used to measure catecholamines by HPLC with electrochemical detection. MLT secretion increased with embryo age, most intensively between the 22nd and 24th days of life. The daily changes in MLT secretion under the 12 L:12D cycle occurred on the first day of culture, starting from an embryonic age of 24 days. MLT secretion was controlled by the light-dark cycle in all age groups studied. However, exposure to light during the scotophase did not alter the secretion of MLT. The endogenous oscillator expressed its activity in regulating MLT secretion in the pineal organs of embryos aged 24 days and older but could not generate a rhythm after one cycle. The rhythm of 5-hydroxytryptophan release during the first day of culture was found in the pineal organs of all embryos, while the rhythmic release of N -acetylserotonin and 5-methoxyindole acetic acid started at the age of 24 days. The proportion of released indoles changed with embryo age. NE caused a decrease in MLT secretion and provoked an increase in serotonin release. Incubation of the pineal organs induced the development of MLT secretory machinery and its diurnal rhythmicity. The pineal content of catecholamines increased prominently at the end of embryonic development.

This study determined the effect of norepinephrine and light exposure on melatonin secretion in goose pineal explants. Additionally, it investigated changes in the content of norepinephrine, dopamine, and their metabolites [3,4-dihydroxyphenylacetic acid; vanillylmandelic acid (VMA); homovanillic acid] in goose pineal glands in vivo under 12 h of light and 12 h of darkness (LD), a reversed cycle (DL), constant light (LL), and constant darkness (DD). In vitro content of melatonin was measured by radioimmunoassay; contents of catecholamines and their metabolites were measured by high-performance liquid chromatography. Exposure of pineal explants to LD or DL established rhythmic melatonin secretion; this rhythm was much better entrained with norepinephrine exposure during photophase than without it. When the explants were kept in LL or DD, the rhythm was abolished, unless NE was administered during natural scotophase of a daily cycle. In vivo , norepinephrine and dopamine levels did not display rhythmic changes, but their respective metabolites, HMV and VMA, displayed well-entrained diurnal rhythms. These results indicate that norepinephrine and sympathetic innervation play key roles in regulation of pineal secretory activity in geese, and that pineal levels of VMA and HMV provide precise information about the activity of sympathetic nerve fibers in goose pineal glands., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Ziółkowska and Lewczuk.)

The metabolism of pineal indoles is closely related to alterations in the light and dark phases of a daily cycle. Recent research showed important interspecies differences in the pineal biochemistry, and a strong impact of monochromatic light on many physiological processes in birds. Therefore, the aims of study were to characterize the metabolism of melatonin-synthesis indoles in the pineal organ of the domestic turkey, and to determine the changes occurring in this metabolism under the influence of different wavelengths and intensities of light. For this purpose, 3-week-old turkeys were kept under 16 lx white light, or under blue, green, and red light with intensities of 16, 32, and 64 lx during the photophase, and after 7 d were sacrificed at 4 h intervals. The activities of melatonin-synthesizing enzymes and the contents of indoles were measured in the same pineal organ. The results revealed that the activities of tryptophan hydroxylase and arylalkylamine N-acetyltransferase, and the levels of all tryptophan derivatives had significant daily changes in birds kept under each light condition used. The profile of pineal indole metabolism in 4-week-old turkeys was characterized by high-amplitude rhythms in the activity of arylalkylamine N-acetyltransferase and the contents of N-acetylserotonin and melatonin, equal relative amounts of serotonin and 5-hydroxyindoleacetic acid, and higher content of melatonin than N-acetylserotonin. The monochromatic light significantly modified the pineal indole metabolism, and its effects were dependent on the color and intensity of light. Pronounced changes occurred in the level of serotonin synthesis and the daily rhythm course of melatonin synthesis.

The purpose of this study was to determine the effects of single and combined administrations of deoxynivalenol (DON) and zearalenone (ZEN) on the histology and ultrastructure of pig liver. The study was performed on immature gilts, which were divided into four equal groups. Animals in the experimental groups received DON at a dose of 12 μg/kg body weight (BW) per day, ZEN at 40 μg/kg BW per day, or a mixture of DON (12 μg/kg BW per day) and ZEN (40 μg/kg BW). The control group received vehicle. The animals were killed after 1, 3, and 6 weeks of experiment. Treatment with mycotoxins resulted in several changes in liver histology and ultrastructure, including: (1) an increase in the thickness of the perilobular connective tissue and its penetration to the lobules in gilts receiving DON and DON + ZEN; (2) an increase in the total microscopic liver score (histology activity index (HAI)) in pigs receiving DON and DON + ZEN; (3) dilatation of hepatic sinusoids in pigs receiving ZEN, DON and DON + ZEN; (4) temporary changes in glycogen content in all experimental groups; (5) an increase in iron accumulation in the hepatocytes of gilts treated with ZEN and DON + ZEN; (6) changes in endoplasmic reticulum organization in the hepatocytes of pigs receiving toxins; (7) changes in morphology of Browicz-Kupffer cells after treatment with ZEN, DON, and DON + ZEN. The results show that low doses of mycotoxins used in the present study, even when applied for a short period, affected liver morphology.

The extracellular vesicles (EVs) released by plant pathogens of the Pectobacterium genus were investigated. The isolates were obtained using differential centrifugation followed by filtration and were characterized in terms of total protein content and particle size distribution. The transmission electron microscopy (TEM) analysis revealed the presence of two morphologically differentiated subpopulations of vesicles in the obtained isolates. The proteomic analysis using matrix-assisted laser desorption ionization mass spectrometry with time of flight detector (MALDI-TOF/TOF-MS) enabled to identify 62 proteomic markers commonly found in EVs of Gram-negative rods from the Enterobacteriaceae family. Capillary electrophoresis (CE) was proposed as a novel tool for the characterization of EVs. The method allowed for automated and fast (<15 min per sample) separation of vesicles from macromolecular aggregates with low sample consumption (about 10 nL per analysis). The approach required simple background electrolyte (BGE) composed of 50 mM BTP and 75 mM glycine (pH 9.5) and standard UV detection. The report presents a new opportunity for quality control of samples containing EVs., Competing Interests: Declaration of Competing Interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper, (Copyright © 2020. Published by Elsevier B.V.)

The aim of this study was to characterize the diurnal rhythm of plasma melatonin (MLT) concentration and its regulation by light and endogenous oscillators in 10-week-old domestic turkeys. Three experiments were performed to examine (i) the course of daily changes in plasma MLT concentration in turkeys kept under a 12 h light: 12 h dark (12L:12D) cycle; (ii) the influence of night-time light exposure lasting 0.5, 1, 2, or 3 h on the plasma MLT level; and (iii) the occurrence of circadian fluctuations in plasma MLT levels in birds kept under continuous dim red light and the ability of turkeys to adapt their pineal secretory activity to a reversed light-dark cycle (12D:12L). The plasma MLT concentration was measured with a direct radioimmunoassay. The plasma MLT concentration in turkeys kept under a 12L:12D cycle changed significantly in a daily rhythm. It was low during the photophase and increased stepwise after the onset of darkness to achieve the maximal level in the middle of the scotophase. Next, it decreased during the second half of the night. The difference between the lowest level of MLT and the highest level was approximately 18-fold. The exposure of turkeys to light during the scotophase caused a rapid, large decrease in plasma MLT concentration. The plasma MLT concentration decreased approximately 3- and 10-fold after 0.5 and 1 h of light exposure, respectively, and reached the day-time level after 2 h of exposure. In turkeys kept under continuous darkness, the plasma MLT level was approximately 2.5-fold higher at 02:00 h than at 14:00 h. In birds kept under 12D:12L, the plasma MLT level was significantly higher at 14:00 h than at 02:00 h. The results showed that plasma MLT concentrations in 10-week-old turkeys have a prominent diurnal rhythm, which is endogenously generated and strongly influenced by environmental light.

The effect of constant light and constant darkness on intraocular pressure (IOP) in goats has not been investigated. We hypothesized that IOP variations would differ between goats kept under a cycle of 12 hours of light and 12 hours of darkness (LD), constant darkness (DD), and constant light (LL). To test this hypothesis, goats were exposed to these conditions for five days (LD, 30 goats; DD, 10 goats; LL, 10 goats). IOP was measured by applanation tonometry at 9 a.m. (beginning of photophase in LD) and 9 p.m. (beginning of scotophase in LD) on the fourth and fifth days of exposure. We found that changes in mean IOP from 9 a.m. to 9 p.m. differed significantly between groups (χ 2 (2) = 23.04, p < .0001). Most goats in LD showed a regular pattern of higher IOP in the morning and lower IOP in the evening, whereas those in DD and LL did not follow this pattern. In LD conditions, mean IOP was 2.4 mm Hg lower at 9 p.m. than at 9 a.m. (95% confidence interval for the difference (CI): -2.8 to -1.9 mm Hg, p < .0001). In DD conditions, mean IOP did not differ between 9 p.m. and 9 a.m. (CI: -0.9 to 0.8 mm Hg, p = .90). In LL conditions, it was 0.6 mm Hg lower at 9 p.m. (CI: -1.5 to 0.2 mm Hg, p = .12). Our results indicate that IOP in goats kept in LD is higher in the morning than in the evening, and that IOP variations are reduced in goats kept in DD and LL. These results suggest that exposure to alternating periods of light and darkness is important for maintaining rhythmic variations in IOP in this species.

The avian pineal organ is photosensitive because of the presence of photopigments, of which pinopsin seems to be one of the most important. This organ is subject to far-reaching changes during post-hatching development, but evidence regarding pinopsin presence and direct photoreception during this time is lacking. This study was carried out to demonstrate the following: 1) the structures showing immunoreactivity to pinopsin in the turkey pineal organ, 2) the changes of these structures during development, 3) the pinopsin localization in pinealocytes in monolayer cultures, and 4) the role of direct photoreception in the regulation of melatonin secretion in pineal organs in adult turkeys. Pinopsin immunoreactivity was localized in the apical extensions of columnar cells limiting the follicular lumen, in fiber-like structures located between columnar cells in the inner part of follicle wall, in string-shapes or small spherical structures distributed in the outer part of follicle wall and in amorphous material inside the follicle lumen. In young birds, immunoreactivity was also sporadically noted in cell bodies of rudimentary receptor pinealocytes. The distribution of pinopsin showed prominent age-dependent changes, including a subsequent increase in pinopsin-positive structures in the outer part of the follicle wall and a prominent reduction in the number and size of positive apical extensions in 40- and 56-week-old turkeys. These data demonstrate that the role of secretory pinealocytes in pineal photoreception increases with age. In monolayer cultures, all pinealocytes showed strong reactions in club- or bulbous-shaped prolongations. The pineal organs of adult birds were less sensitive to light exposition at night than those of young turkeys, which points to differences in light sensitivity between rudimentary receptor and secretory pinealocytes. However, direct photoreception could play an important role in the regulation of melatonin secretion in adult turkeys., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The regulation of melatonin secretion in the avian pineal organ is highly complex and shows prominent interspecies differences. The aim of this study was to determine the roles of direct photoreception and the internal oscillator in the regulation of melatonin secretion in the pineal organ of the domestic turkey. The pineal organs were collected from 12-, 13- and 14-week-old female turkeys reared under a 12 L:12 D cycle with the photophase from 07.00 to 19.00, and were incubated in superfusion culture for 3-6 days. The cultures were subjected to different light conditions including 12 L:12 D cycles with photophases between 07.00 and 19.00, 13.00 and 01.00 or 01.00 and 13.00, a reversed cycle 12 D:12 L, cycles with long (16 L:8 D) and short (8 L:16 D) photophases, and continuous darkness or illumination. The pineal organs were also exposed to light pulses of variable duration during incubation in darkness or to periods of darkness during the photophase. The secretion of melatonin was determined by direct radioimmunoassay. The turkey pineal organs secreted melatonin in a well-entrained diurnal rhythm with a very high amplitude. Direct photoreception as an independently acting mechanism was able to ensure quick and precise adaptation of the melatonin secretion rhythm to changes in light-dark conditions. The pineal organs secreted melatonin in circadian rhythms during incubation in continuous darkness or illumination. The endogenous oscillator of turkey pinealocytes was able to acquire and store information about the light-dark cycle and then to generate the circadian rhythm of melatonin secretion in continuous darkness according to the stored data. The obtained data suggest that the turkey pineal gland is highly autonomous in the generation and regulation of the melatonin secretion rhythm. They also demonstrate that the turkey pineal organ in superfusion culture is a valuable model for chronobiological studies, providing a highly precise clock and calendar. This system has several features which make it an attractive alternative to other avian pineal glands for circadian studies.

The aim of this study was to characterize the embryonic ontogeny of 5-hydroxyindoles and 5-methoxyindoles synthesis pathways in the goose pineal organ. The study was performed on embryos aged 14-28 days, which have been incubated under a 12L:12D cycle. The pineal organs were collected for measurements of indole content by HPLC every 6 h on embryonic day (ED) 14, ED 16, ED 18 and ED 22 or every 2 h on ED 24, ED 26 and ED 28. The level of tryptophan showed no significant changes during development and no day-night variations. The content of 5-hydroxytryptophan increased between ED 14 and ED 26. It was significantly higher during scotophase than during photophase starting from ED 14. The serotonin content was low during the early stages of development (ED 14-ED 18) and prominently increased from ED 20. The serotonin levels also showed day-night differences; however, they were less conspicuous than those of 5-hydroxytryptophan. The changes in the level of 5-hydroxyindole acetic acid were similar to those of serotonin. 5-Hydroxytryptophol was measurable from ED 18. Levels of N -acetylserotonin, which were detectable for the first time on ED 16, prominently increased between ED 22 and ED 28 and showed significant day-night differences from ED 20. Melatonin was detectable from ED 18. Like N -acetylserotonin, its content increased rapidly between ED 22 and ED 28, and from ED 20 showed diurnal variations. 5-Methoxyindole acetic acid and 5-methoxytryptophol occurred at measurable levels from ED 18 and ED 26, respectively. The obtained results showed that embryonic development of indole metabolism in the goose pineal organ starts with the beginning of serotonin synthesis. The processes of serotonin acetylation and 5-hydroxyindoles methylation were turned on later. Diurnal rhythmicity develops very early in the embryonic pineal organ of the goose when the eggs are incubated under a 12 h light: 12 h dark schedule. Two processes are responsible for generation of the diurnal rhythms of 5-hydroxyindoles and 5-methoxyindoles: (i) hydroxylation of tryptophan and (ii) acetylation of serotonin.

Our previous study showed that the turkey pineal organ, in contrast to that of the chicken, is characterized by a follicular structure throughout the entire period of post-hatching life. Despite the preservation of the follicular organization, the histological structure of the pineal follicles in turkeys changes prominently with age. The present research was performed to investigate the cellular composition and organization of the follicle wall as well as the ultrastructure of parenchymal cells in the turkey pineal organ during the period of post-hatching development. Pineal organs were collected from female turkeys at 2 days, 2 weeks, 4 weeks, 10 weeks, 20 weeks, 30 weeks, 40 weeks, and 56 weeks post-hatching. The organs were prepared for immunocytochemical studies using antibodies against N-acetylserotonin O-methyltransferase (ASMT), glial fibrillary acidic protein (GFAP) and proliferating cell nuclear antigen (PCNA) and for ultrastructural examination. The results showed that regardless of age, the pineal follicle was formed by ASMT-immunopositive cells, among which rudimentary photoreceptor and secretory pinealocytes were identified. The second component of the follicle wall consisted of GFAP-immunopositive cells, as represented by ependymal-like and astrocyte-like cells. Rudimentary photoreceptor pinealocytes and ependymal-like cells formed the inner part of the follicle wall, while secretory pinealocytes and astrocyte-like cells created the outer part. Three forms of the pineal follicle structure characteristic of young (two days to ten weeks), young adult (20-30 weeks) and adult (40-56 weeks) turkeys were distinguished. These forms primarily differed in the relative dimensions of the inner and outer parts of the follicle wall. Ultrastructural studies showed prominent changes in the organization of rudimentary receptor pinealocytes during the investigated period of life. These cells developed until the age of 20 weeks, at which time they appeared as strongly elongated cells with a stratified, highly regular distribution of organelles. In adult turkeys, rudimentary receptor pinealocytes showed pronounced regressive changes; however, we never observed their transformation into cells of the secretory type. Secretory pinealocytes increased in number and size during the post-hatching period, which was especially pronounced after 20 weeks of age. The most prominent changes in the supporting cells included the intensification of GFAP-immunoreactivity due to the accumulation of filaments in the cytoplasm and the development of astrocyte-like cells. The increase in the number of secretory pinealocytes and astrocyte-like supporting cells resulted in the formation of two distinct parts of the follicle wall in the pineal organs of young adult and adult turkeys., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The avian pineal gland is one of three central biological clocks that contain all the components of a circadian system: a photoreceptive input, oscillator, and rhythmically secreted melatonin (MEL) as an effector. The biosynthesis of MEL is regulated by the neurotransmitters noradrenaline (NA), vasoactive intestinal peptide (VIP), and pituitary adenylate cyclase-activating polypeptide (PACAP). The aim of the present study was to characterize the daily profile of neurotransmitters and their receptors in the pineal gland of male Hy-Line chickens housed under controlled light (12:12 light:dark) conditions. The pineal glands were isolated from 16-day-old birds every 2 h over a 24-h period, immediately after decapitation. The catecholamine content was measured using HPLC with electrochemical detection, whereas expression of VIP and PACAP was measured using quantitative real-time PCR (RT-qPCR) assays and Western blotting. Expression of the neurotransmitter receptors was also measured using RT-qPCR. We found daily changes in NA content, with elevated nocturnal levels, whereas the NA receptor was expressed in antiphase. Although we did not observe daily changes in VIP and PACAP protein levels, we found prominent diurnal changes in the expression of the Vip and Pacap genes. We also detected precursors of NA, 3,4-dihydroxy-L-phenylalanine (DOPA), and dopamine (DA) in the pineal glands, in addition to the DA metabolites. Our results provide the first evidence that the pineal gland itself may synthetize the neurotransmitters needed to regulate MEL biosynthesis.

Impact Statement: The delivery of short snippets of RNA, such as synthetic miRNA agents, is an essential step for achieving RNA-mediated knockdown, which has not been studied in sufficient detail in fish. Our results indicate that a MiR92b-3p mimic may be effectively delivered via intraperitoneal injection to the spleen and the liver of whitefish, and that it likely achieves functionality without causing any apparent toxic effects in the challenged animals. We report the novel finding that the MiR92b-3p mimic reduced the in vivo liver mRNA expression levels of its putative pro-apoptotic targets (p53, cdkn1a, and pcna), and important metabolic genes, e.g. cdo1. This shows that this methodology of MiR92b-3p mimic transfection in vivo may be a useful tool for studies that investigate the molecular pathways that confer pro-proliferative and anti-apoptotic phenotypes or those that regulate intracellular metabolism in fish and other vertebrates.

Several observations from experiments in rodents and human patients suggest that diabetes affects pineal gland function, including melatonin secretion; however, the accumulated data are not consistent. The aim of the present study was to determine the effects of streptozotocin-induced diabetes on the pineal gland in the domestic pig, a species widely used as a model in various biomedical studies. The study was performed on 10 juvenile pigs, which were divided into two groups: control and diabetic. Diabetes was evoked by administration of streptozotocin (150 mg/kg of body weight). After six weeks, the animals were euthanized between 12.00 and 14.00, and the pineal glands were removed and divided into two equal parts, which were used for biochemical analyses and for preparation of explants for the superfusion culture. The pineal contents (per 100 μg protein) of serotonin, 5-hydroxyindole acetic acid, 5-hydroxytryptophol, 5-methoxyindole acetic acid, 5-methoxytryptophol, and 5-methoxytryptamine were significantly lower in diabetic pigs than in control pigs. In contrast, the level of N-acetylserotonin was significantly higher in diabetic animals. No significant differences were found in the level of melatonin between control and experimental pigs. The amounts of 3,4-dihydroxyphenylalanine, dopamine, norepinephrine, and 3,4-dihydroxyphenylacetic acid were significantly lower in the pineal glands of diabetic animals. The level of vanillylmandelic acid was higher in diabetic pigs. No differences were observed in the level of basal and NE-stimulated release of N-acetylserotonin or melatonin between the pineal explants prepared from control and experimental animals. In vitro treatment with insulin was ineffective. In conclusion, streptozotocin-induced diabetes affects both indole metabolism and adrenergic neurotransmission in the pig pineal gland.

The contamination of feed with mycotoxins results in reduced growth, feed refusal, immunosuppression, and health problems. Deoxynivalenol (DON) and zearalenone (ZEN) are among the most important mycotoxins. The aim of the study was to examine the effects of low doses of these mycotoxins on the histological structure and ultrastructure of the large intestine in the pig. The study was performed on 36 immature gilts of mixed breed (White Polish Big &times; Polish White Earhanging), which were divided into four groups administrated per os with ZEN at 40 &micro;g/kg BW, DON at 12 &micro;g/kg BW, a mixture of ZEN (40 &micro;g/kg BW) and DON (12 &micro;g/kg BW) or a placebo. The pigs were killed by intravenous overdose of pentobarbital after one, three, and six weeks of treatment. The cecum, ascending and descending colon samples were prepared for light and electron microscopy. Administration of toxins did not influence the architecture of the mucosa and submucosa in the large intestine. ZEN and ZEN + DON significantly decreased the number of goblet cells in the cecum and descending colon. The mycotoxins changed the number of lymphocytes and plasma cells in the large intestine, which usually increased in number. However, this effect differed between the intestine segments and toxins. Mycotoxins induced some changes in the ultrastructure of the mucosal epithelium. They did not affect the expression of proliferative cell nuclear antigen and the intestinal barrier permeability. The obtained results indicate that mycotoxins especially ZEN may influence the defense mechanisms of the large intestine.

Feline infectious peritonitis (FIP) is a serious, widely distributed systemic disease caused by feline coronavirus (FCoV), in which ocular disease is common. However, questions remain about the patterns of ocular inflammation and the distribution of viral antigen in the eyes of cats with FIP. This study characterized the ocular lesions of FIP including the expression of glial fibrillary acidic protein and proliferating cell nuclear antigen by Müller cells in the retina in cases of FIP and to what extent macrophages are involved in ocular inflammation in FIP. Immunohistochemistry for FCoV, CD3, CD79a, glial fibrillary acidic protein, calprotectin, and proliferating cell nuclear antigen was performed on paraffin sections from 15 naturally occurring cases of FIP and from controls. Glial fibrillary acidic protein expression was increased in the retina in cases of FIP. Müller cell proliferation was present within lesions of retinal detachment. Macrophages were present in FIP-associated ocular lesions, but they were the most numerous inflammatory cells only within granulomas (2/15 cats, 13%). In cases of severe inflammation of the ciliary body with damage to blood vessel walls and ciliary epithelium (3/15, 20%), some macrophages expressed FCoV antigens, and immunolabeling for calprotectin on consecutive sections suggested that these FCoV-positive macrophages were likely to be recently derived from blood. In cases of severe and massive inflammation of most ocular structures (4/15, 26%), B cells and plasma cells predominated over T cells and macrophages. These results indicate that gliosis can be present in FIP-affected retinas and suggest that breakdown of the blood-ocular barrier can allow FCoV-bearing macrophages to access the eye.

Two structures, considered as secretory in nature, are present in the pinealocytes in of the domestic pig show the presence of two structures, which are considered as secretory in nature - the dense core vesicles (DCV) and the membrane bounded (dense) bodies (MBB). The latter are extremely numerous in pig pinealocytes (they occupy 6-20% of the cytoplasm), and the number of MBB changes under different physiological and experimental conditions. Norepinephrine is the main neurotransmitter that regulates the secretion of pineal melatonin. The present study was carried out to 1) clarify whether the DCV and their source - the Golgi apparatus (GA) - as well as the MBB are controlled by norepinephrine, 2) determine the effect of adrenergic stimulation on these structures, and 3) identify the receptors involved in the regulation of these structures. The studies were performed using a static organ culture of pig pineal explants. The explants were incubated in a control medium between 08:00 and 20:00 and in a medium with 10μM norepinephrine or alpha- or beta-adrenoceptor agonists between 20:00 and 08:00 on five consecutive days. The tissues were subsequently prepared for ultrastructural analysis. The results distinctly showed that the DCV, GA and MBB in pig pinealocytes are under adrenergic control. The stimulation of the beta-adrenoceptors resulted in an increase in the numerical density of the DCV and a decrease in the relative volume of the GA in the perikarya, while the incubation with agonists of the alpha1-adrenoceptors was ineffective. The relative volume of the MBB in the perikarya significantly decreased after treatment with both beta-agonists and alpha1-agonists, which suggested the involvement of two types of adrenoceptors in the regulation of these structures., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

To date, there has been no systematic approach comprehensively describing the sequence of pathological changes in fish during prolonged exposure to microcystin-LR (MC-LR). Towards this aim, juvenile whitefish individuals received an intraperitoneal injection with pure MC-LR, and the injection was repeated every week to maintain continuous exposure for 28days. During the exposure period, growth and condition of the fish were assessed based on biometric measurements. Additionally, selected biochemical markers were analysed in the fishes' blood, and their livers were carefully examined for morphological, ultrastructural, and molecular changes. The higher dose of MC-LR (100μg·kg -1 ) caused severe liver injury at the beginning of the exposure period, whereas the lower dose (10μg·kg -1 ) caused less, probably reversible injury, and its effects began to be observed later in the exposure period. These marked changes were accompanied by substantial MC-LR uptake by the liver. However, starting on the 7th day of exposure, cell debris began to be removed by phagocytes, then by 14th day, proliferation of liver cells had markedly increased, which led to reconstruction of the liver parenchyma at the end of the treatment. Surprisingly, despite weekly-repeated intraperitoneal injections, MC-LR did not accumulate over time of exposure which suggests its limited uptake in the later phase of exposure. In support, mRNA expression of the membrane transport protein oatp1d was decreased at the same time as the regenerative processes were observed. Our study shows that closing of active membrane transport may serve as one defence mechanism against further MC-LR intoxication., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The aim of the present study was to characterize the daily profiles of melatonin synthesis-related indoles in the pineal glands of male Hy-Line chickens hatched in the winter and reared under controlled light (L:D 12:12) conditions. The pineal glands were isolated from 16-day-old birds immediately after decapitation every 2h over a 24-h period. The indole contents were measured using HPLC with fluorescence detection. According to the obtained data, in chicken pineal glands tryptophan occurred at the highest level among the investigated compounds, showing rhythmical, daily changes with decreased levels during the scotophase. Rhythmical fluctuations were also observed for all studied tryptophan derivatives, except serotonin, 5-hydroxyindole acetic acid and 5-hydroxytryptophol. The highest levels of 5-hydroxytryptophan, N-acetylserotonin and melatonin were observed during the night, whereas the highest levels of 5-methoxytryptophol and 5-methoxyindoleacetic acid were observed during the day. The most interesting results concerned serotonin and the associated oxidative deamination products. The serotonin content did not exceed 5% of all investigated indoles, suggesting that there is no reserve pool of this indoleamine in the chicken pineal gland and that the activity of aromatic L-amino acid decarboxylase might be an important factor of melatonin synthesis regulation. In contrast to serotonin, 5-hydroxyindole acetic acid occurred at high levels in the investigated pineal gland, suggesting intensive oxidative deamination and explaining the observed low content of this indoleamine., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Deoxynivalenol (DON) and zearalenone (ZEN), produced by microfungi of the Fusarium family, are among the most commonly occurring mycotoxins. They are considered important factors affecting human and animal health as well as livestock productivity. The aim of this study was to determine the effect of low doses of these mycotoxins on the histological structure of the pig duodenum. The study was performed on 72 gilts, with initial weights of approximately 25kg, divided into 4 equal groups. Group I received per os ZEN (40μg/kg BW), group II-DON (12μg/kg BW), group III-ZEN (40μg/kg BW) and DON (12μg/kg BW), and group IV-vehicle. The pigs were killed after 1, 2, 3, 4, 5 and 6 weeks of the treatment, and the duodenum samples were prepared for histological investigations. The slides were digitalized and subjected to morphometrical analysis. The treatment with DON and ZEN did not change the architecture of the mucosa or the ratio between goblet and adsorptive cells in the epithelium. The administration of DON induced an increase in the number of lymphocytes in the mucosal epithelium. Both mycotoxins, administered alone or together, increased the quantity of lymphocytes, plasma cells and macrophages with black-brown granules in the lamina propria. The time-courses of changes in the number of defense system cells evoked by DON and ZEN were different. In conclusion, dietary exposure to low doses of Fusarium mycotoxins should be considered an important risk factor for subclinical inflammation in the small intestine., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

Immature gilts were administered per os with zearalenone (ZEN) at 40 μg/kg BW (group Z, n = 9), deoxynivalenol (DON) at 12 μg/kg BW (group D, n = 9), a mixture of ZEN and DON (group M, n = 9) or a placebo (group C, n = 9) over a period of six weeks. The pigs were sacrificed after one, three, or six weeks of the treatment (12 pigs per each time-point). Histological investigations revealed an increase in the mucosal thickness and the crypt depth as well as a decrease in the ratio of the villus height to the crypt depth in groups D and M after six weeks of exposure to the mycotoxins. The number of goblet cells in the villus epithelium was elevated in groups Z and M after one week and in group D after three weeks. The administration of ZEN increased the lymphocyte number in the villus epithelium after 1 week and the plasma cell quantity in the lamina propria after one, three, and six weeks of the experiment. DON treatment resulted in an increase in the lymphocyte number in the villus epithelium and the lamina propria after six weeks, and in the plasma cell quantity in the lamina propria after one, three, and six weeks of exposure. In group M, lymphocyte counts in the epithelium and the lamina propria increased significantly after six weeks. Neither mycotoxin induced significant adverse changes in the ultrastructure of the mucosal epithelium and the lamina propria or in the intestinal barrier permeability. Our results indicate that immune cells are the principal target of low doses of ZEN and DON.

Current technologies have become a source of omnipresent electromagnetic pollution from generated electromagnetic fields and resulting electromagnetic radiation. In many cases this pollution is much stronger than any natural sources of electromagnetic fields or radiation. The harm caused by this pollution is still open to question since there is no clear and definitive evidence of its negative influence on humans. This is despite the fact that extremely low frequency electromagnetic fields were classified as potentially carcinogenic. For these reasons, in recent decades a significant growth can be observed in scientific research in order to understand the influence of electromagnetic radiation on living organisms. However, for this type of research the appropriate selection of relevant model organisms is of great importance. It should be noted here that the great majority of scientific research papers published in this field concerned various tests performed on mammals, practically neglecting lower organisms. In that context the objective of this paper is to systematise our knowledge in this area, in which the influence of electromagnetic radiation on lower organisms was investigated, including bacteria, E. coli and B. subtilis, nematode, Caenorhabditis elegans, land snail, Helix pomatia, common fruit fly, Drosophila melanogaster, and clawed frog, Xenopus laevis.

This study characterizes the diurnal profiles of ten melatonin synthesis-related indoles, the quantitative relations between these compounds, and daily variations in the contents of catecholamines and their metabolites in the domestic duck pineal organ. Fourteen-week-old birds, which were reared under a 12L:12D cycle, were killed at two-hour intervals. The indole contents were measured using HPLC with fluorescence detection, whereas the levels of catecholamines and their metabolites were measured using HPLC with electrochemical detection. All indole contents, except for tryptophan, showed significant diurnal variations. The 5-hydroxytryptophan level was approximately two-fold higher during the scotophase than during the photophase. The serotonin content increased during the first half of the photophase, remained elevated for approximately 10 h and then rapidly decreased in the middle of the scotophase. N-acetylserotonin showed the most prominent changes, with a more than 15-fold increase at night. The melatonin cycle demonstrated only an approximately 5-fold difference between the peak and nadir. The 5-methoxytryptamine content was markedly elevated during the scotophase. The 5-hydroxyindole acetic acid, 5-hydroxytryptophol, 5-methoxyindole acetic acid and 5-methoxytryptophol profiles were analogous to the serotonin rhythm. The norepinephrine and dopamine contents showed no significant changes. The DOPA, DOPAC and homovanillic acid levels were higher during the scotophase than during the photophase. Vanillylmandelic acid showed the opposite rhythm, with an elevated level during the daytime.

Anatomical, histological, and ultrastructural studies of the European beaver stomach revealed several unique morphological features. The prominent attribute of its gross morphology was the cardiogastric gland (CGG), located near the oesophageal entrance. Light microscopy showed that the CGG was formed by invaginations of the mucosa into the submucosa, which contained densely packed proper gastric glands comprised primarily of parietal and chief cells. Mucous neck cells represented <0.1% of cells in the CGG gastric glands and 22-32% of cells in the proper gastric glands of the mucosa lining the stomach lumen. These data suggest that chief cells in the CGG develop from undifferentiated cells that migrate through the gastric gland neck rather than from mucous neck cells. Classical chief cell formation (i.e., arising from mucous neck cells) occurred in the mucosa lining the stomach lumen, however. The muscularis around the CGG consisted primarily of skeletal muscle tissue. The cardiac region was rudimentary while the fundus/corpus and pyloric regions were equally developed. Another unusual feature of the beaver stomach was the presence of specific mucus with a thickness up to 950 µm (in frozen, unfixed sections) that coated the mucosa. Our observations suggest that the formation of this mucus is complex and includes the secretory granule accumulation in the cytoplasm of pit cells, the granule aggregation inside cells, and the incorporation of degenerating cells into the mucus.

The ovine pineal is generally considered as an interesting model for the study on adrenergic regulation of melatonin secretion due to some functional similarities with this gland in the human. The present investigations, performed in the superfusion culture of pineal explants, demonstrated that the norepinephrine-induced elevation of melatonin secretion in ovine pinealocytes comprised of two subsequent periods: a rapid increase phase and a slow increase phase. The first one included the quick rise in release of N-acetylserotonin and melatonin, occurring parallel to elevation of NE concentration in the medium surrounding explants. This rapid increase phase was not affected by inhibition of translation. The second, slow increase phase began after NE level had reached the maximum concentration in the culture medium and lasted about two hours. It was completely abolished by the treatment with translation inhibitors. The obtained results showed for the first time that the regulation of N-acetylserotonin synthesis in pinealocytes of some species like the sheep involves the on/off mechanism, which is completely independent of protein synthesis and works very fast. They provided strong evidence pointing to the need of revision of the current opinion that arylalkylamines N-acetyltransferase activity in pinealocytes is controlled exclusively by changes in enzyme abundance.

One of the side effects of each electrical device work is the electromagnetic field generated near its workplace. All organisms, including humans, are exposed daily to the influence of different types of this field, characterized by various physical parameters. Therefore, it is important to accurately determine the effects of an electromagnetic field on the physiological and pathological processes occurring in cells, tissues, and organs. Numerous epidemiological and experimental data suggest that the extremely low frequency magnetic field generated by electrical transmission lines and electrically powered devices and the high frequencies electromagnetic radiation emitted by electronic devices have a potentially negative impact on the circadian system. On the other hand, several studies have found no influence of these fields on chronobiological parameters. According to the current state of knowledge, some previously proposed hypotheses, including one concerning the key role of melatonin secretion disruption in pathogenesis of electromagnetic field induced diseases, need to be revised. This paper reviews the data on the effect of electric, magnetic, and electromagnetic fields on melatonin and cortisol rhythms-two major markers of the circadian system as well as on sleep. It also provides the basic information about the nature, classification, parameters, and sources of these fields.

Innervation of the mammalian pineal gland during prenatal development is poorly recognized. Therefore, immunofluorescence studies of the pineals of 70- and 90-day-old foetuses of the domestic pig were performed using antibodies against tyrosine hydroxylase (TH), dopamine-β-hydroxylase (DβH), neuropeptide Y (NPY) and C-terminal flanking peptide of NPY (CPON). The investigated glands were supplied by numerous nerve fibres containing TH and DβH. The density of these fibres was higher in the distal and middle parts of the gland than in the proximal one. NPY and CPON were identified in the majority of DβH-positive fibres as well as in a small population of DβH-negative fibres localized mainly in the proximal part of the pineal. The immunoreactive fibres were more numerous in 90-day-old foetuses than in 70-day-old ones. The effect of norepinephrine on melatonin secretion by the foetal pineals in the short-term organ culture was studied to determine the role of DβH-positive fibres during prenatal life. For the same purpose melatonin was measured in the blood in the umbilical cords and in the jugular vein of the mother. The pineals of both groups of foetuses did not secrete melatonin in the organ culture, independently of the presence or absence of norepinephrine in the medium. Melatonin concentrations in the blood in the umbilical cords of foetuses from the same litter and in the jugular vein of their mother were similar. The presence of adrenergic nerve fibres in the pig pineal during gestation does not seem to be associated with the control of melatonin secretion.

Implementation of the swine umbilical vein endothelial cells (SUVECs) model in vitro can be instrumental in determining the biology of endothelial cells. We have generated an immortalized endothelial cell line, G-1410, using Simian virus 40 T-antigen (SV40 T-ag) primarily to overcome the short life span before the onset of senescence and high variability among enzymatically isolated cells of primary cultures. Fast proliferating cells were selected from cultures and, after a fifth passage, examined for the presence of the SV40 T-ag by PCR and immunocytochemistry. Phase contrast and transmission electron microscopy revealed that G-1410 cells did not differ morphologically from SUVECs. The G-1410 cells exhibited positive staining for vascular endothelial (VE)-cadherin and von Willebrand factor (vWF), and formed capillary-like tube structures on Matrigel. Despite the strong oncogenic signal provided by SV40 T-ag, these transformed G-1410 cells have remained karyotypically normal and non-tumorigenic. G-1410 cells also responded to stimulation with VEGF, FGF-2, and newborn calf serum. Moreover, G-1410 cells showed elevated expression of VEGF120, VEGF164 (VEGF-A), and FGF-2 at both mRNA and protein levels. In conclusion, based on the cytological and functional evaluation of the newly obtained immortalized cell line, it can be concluded that G-1410 cells provide a useful tool for studying the effects of VEGF and FGF systems, and other signal transduction pathways related to angiogenesis., (Copyright © 2011 Wiley-Liss, Inc.)

The presence of melatonin and the enzymes (transcripts and activities) involved in its synthesis, i.e. arylalkylamine N-acetyltransferase (AA-NAT) and hydroxyindole-O-methyltransferase (HIOMT), was investigated in the eggs and early embryos of Japanese quail at Hamburger-Hamilton stages 1-10. Melatonin was present in the egg yolk (approximately 70 pg/g) and albumen (approximately 20 pg/g). The average content of melatonin was approximately 416 pg/egg. AA-NAT and HIOMT transcripts were present in the oocytes, blastoderms, and ovarian follicles. AA-NAT-like and HIOMT activities were detected in quail egg yolk. The activity of AA-NAT in yolk was comparable with that found in the pineal gland when calculated per milligram of yolk or pineal gland, but was significantly lower when re-calculated per milligram of protein in the yolk or pineal gland. AA-NAT-like activity was also identified in the ovarian follicles. Low HIOMT activity was detected in yolk, but not in the ovarian follicle. Both enzymes were essentially absent from early embryos although some residual activities, probably of yolk origin, were present in the stage 1 embryo. Melatonin and all the constituents needed for its synthesis (serotonin, AA-NAT and HIOMT activities) are contained within the avian yolk and could be used by the embryo from the very beginning of its development. The role of extrapineal melatonin in early avian development may be in protecting the embryo from the action of free radicals formed during intensive embryonic metabolism and/or it may participate (together with serotonin) in a 'diffuse neuroendocrine system' acting at early developmental stages, before differentiation of the nervous system.

Objectives: Cell-permeable and specific inhibitors of melatonin secretion are sill lacking among tools of the pineal research. Recently, a large effort has been made in the development of arylalkylamine N-acetyltransferase inhibitors, but in most cases the new drugs were tested exclusively using cell-free assays or non-pineal cells. The aim of the present study was to characterize the effect of N-bromoacetyltryptamine (BAT), the first synthesized cell-permeable inhibitor of arylalkylamine N - acetyltransferase, on melatonin secretion from rat and pig pineal glands., Methods: The studies were performed in the superfusion cultures of rat and pig pineal explants. Melatonin secretion was determined by radioimmunoassay (RIA)., Results: BAT strongly inhibited the non-stimulated and norepinephrine - stimulated melatonin secretion from the pig and rat pineal explants, with ED50 0.3 - 0.7 microM. The adrenergic stimulation did not modify significantly the inhibitory potency of BAT on the melatonin release. The decline in melatonin secretion induced by the BAT - treatment was biphasic in both rat and pig pinealocytes, with an initial rapid phase followed by a slow one. The half-time of BAT-induced decline in the non - stimulated and norepinephrine-stimulated melatonin secretion was ca. 25 - 35 minutes. The inhibitory effect of BAT was reversible in pinealocytes of both investigated mammals., Conclusions: The results show that BAT is a potent and reversible inhibitor of the melatonin secretion in the mammalian pineal gland and open the way for the use of this inhibitor in investigations on the pinealocyte physiology performed in vitro.

Objective: The study was performed to analyze structural changes of the turkey pineal during the post-hatching development., Material and Methods: The pineals were collected from male turkeys at the age of 1 day, 2, 8, 22, 56 weeks and subjected to histological investigations including morphometrical analyses. The pinealocytes were identified immunohistochemically using antiserum against hydroxyinolo-O-methyltransferase (HIOMT)., Results and Conclusions: Independently of age, the pineal consisted of a narrow proximal part and a club-shaped top. The narrow part extended into the stalk attached to the diencephalon. The pineal parenchyma was formed by the follicles, surrounded by the connective tissue. The caudal part of the organ contained the pineal lumen, which prolonged into the stalk lumen. Up to the age of two weeks the stalk lumen was open to the third ventricle, later--closed. The proximal part of the stalk showed age-dependent reduction. During the investigated period of life, the pineal increased in size due to creation of new follicles, enlargement of the follicles and development of the stroma. In immature turkeys, the follicular wall was formed by elongated cells bordering the lumen and sparse, peripherally localized, round cells. This pseudostratified organization was transformed during ontogenesis into thick, multilayer structure (characteristic for 22- and 56-week-old turkeys) composed by the layer of elongated cells and several layers of round cells, located peripherally. The rudimentary-receptor and secretory pinealocytes were demonstrated based on HIOMT-immunoreactivity. The secretory pinealocytes were sparse in young birds and predominated in mature turkeys. Intra-pineal calcified concrements occurred in 56-week-old turkeys.

Objective: The study was performed to analyze the ultrastructure of ovine pinealocytes during the period of postnatal development., Material and Methods: The pineals of newborn, 10-week and one-year old females of the domestic sheep were prepared for ultrastructural investigations. The point count analysis was used in quantitative studies of the pinealocyte substructures., Results: The prominent feature of pinealocytes in the newborns was the presence of well developed rough endoplasmic reticulum and numerous polysomes. The pinealocyte cytoplasm contained also smooth endoplasmic reticulum, mitochondria, Golgi apparatus, lysosomes, dense core vesicles, multivesicular bodies and lipid droplets. Pinealocytes of the 10-week and 1-year old sheep were characterized by the occurrence of numerous vesicles and short cisterns of smooth endoplasmic reticulum, abundant microtubules and lipid droplets. Pinealocytes of the adult sheep were distinguished by well developed Golgi apparatus, numerous dense core vesicles and multivesicular bodies. The relative volume of rough endoplasmic reticulum in pinealocytes was significantly higher in the newborn sheep than in two other groups. The relative volume of mitochondria was significantly higher in pinealocytes of the 10-week old sheep than the newborns and one-year old animals. The relative volume of Golgi apparatus was significantly higher in the one-year old animals than in two other groups. No differences concern lysosomes. The relative volume of lipid droplets as well as the numerical density of dense core vesicles and multivesicular bodies increased significantly with age., Conclusion: The ultrastructure of ovine pinealocytes undergoes the marked changes during postnatal development. The changes concern mainly substructures involved in secretory activity.

The study was conducted to investigate diurnal changes in pinealocyte ultrastructure, pineal serotonin content and plasma melatonin concentration in the domestic pig. The immature pigs (n=24) were kept under a cycle of 12 h light : 12 h dark, with a photophase between 0800 and 2000. During the photophase the animals were exposed to direct sunlight. After four weeks the gilts were slaughtered at 0900, 1400, 2100 and 0200. The pineals were removed and divided into two parts - one for quantitative ultrastructural study (by a point count method) and one for serotonin assay. Simultaneously, blood samples were taken for melatonin assay. The relative volume of mitochondria in pinealocyte perikarya was significantly higher at 1400 than at 0200 and 0900 as well as at 2100 than at 0200. The relative volume of Golgi apparatus was higher at 0900 and 1400 than at 0200. The relative volume of dense bodies of the MBB-1 type in pinealocyte perikarya was significantly lower at 1400 and 2100 than at 0900. In contrast, the relative volume of MBB-2 was higher at 1400 than at 0900 and 0200. The numerical density of DCV in perikarya was significantly higher at 0200 than at 1400. No significant differences were found in rough endoplasmic reticulum, lysosomes and multivesicular bodies. The pineal serotonin content showed a prominent rhythm with the maximum at 1400. The plasma melatonin concentration was significantly higher at 0200 than at 0900, 1400 and 2100. The obtained results demonstrate that both pinealocyte ultrastructure and pineal biochemistry in the pig undergo significant changes in the course of the diurnal rhythm.

The pineal functions are modulated by some neuropeptides including PHI and VIP. The presence of PHI-immunoreactive and VIP-immunoreactive nerve fibers in the pineal gland has been shown in several mammalian species. Both peptides influence the pineal serotonin N-acetyltransferase activity and melatonin synthesis. The aim of the present study was to examine the localization of PHI- and VIP-immunoreactive nerve fibers in the pig pineal gland. Four three-month old female pigs housed in natural light conditions, with free access to food and water, were used in the study. The pineals were fixed by perfusion with 4% paraformaldehyde in 0.1 M phosphate buffer. An immunohistochemical ABC streptavidin-biotin-complex method was used for the demonstration of PHI and VIP. PHI- and VIP-immunopositive nerve fibers were found in the pineal gland as well as in the habenular and posterior commissural areas. In the pineal gland, the density of PHI-immunoreactive nerve fibers was considerably higher than that of the fibers containing VIP. PHI- and VIP-immunopositive nerve fibers were more abundant in the cortical than in the medullary part of the gland. The nerve fibers formed bundles in the pineal capsule, from where they penetrated to the connective tissue septa and formed a dense meshwork surrounding blood vessels. In the parenchyma, PHI- and VIP-immunoreactive nerve terminals created baskets around clusters of pinealocytes. No PHI- or VIP-immunopositive cells were found in the pig pineal gland.

The sympathetic nerve fibers originating from the superior cervical ganglia and supplying the pineal gland play the most important role in the control of the pineal activity in mammals. NPY and CPON are also present in the majority of the pinealopetal sympathetic neurons. In this study, immunohistochemical techniques were used to demonstrate the existence and coexistence of tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DbetaH) as well as NPY and CPON in the nerve fibers supplying the chinchilla pineal gland. Ten two-year-old female chinchillas housed in natural light conditions were used in the study. The pineals were fixed by perfusion. ABC immunohistochemical technique and immunofluorescence labelling method were employed. TH-immunoreactive (TH-IR) varicose nerve fibers were observed in the pineal gland as well as in the posterior commissural area. Within the chinchilla pineal gland, TH-IR nerve fibers were located in the capsule and connective tissue septa. Numerous varicose TH-IR branches penetrated into the parenchyma and formed a network showing the highest density in the proximal region of the gland. In the central and distal parts of the pineal parenchyma, a subtle network, composed of thin varicose nerve branches, was observed. Double immunostaining revealed that the majority of TH-IR nerve fibers was positive for DbetaH or NPY. TH- and DbetaH-positive neuron-like cells were observed in the proximal region of the gland. The pattern of pineal innervation immunoreactive to CPON was similar to the innervation containing NPY, TH and DbetaH. The chinchilla intrapineal innervation containing TH, DbetaH, NPY and CPON is characterized by the higher density in the proximal part of the gland than in the middle and distal ones. The specific feature of the chinchilla pineal is also the presence of single TH/DbetaH-immunoreactive neuron-like cells in the proximal part of the gland.

Background: Norepinephrine is the main neurotransmitter controlling melatonin secretion in the mammalian pineal gland. Presynaptic autoregulation of norepinephrine release from the sympathetic nerve fibers in the pineal gland is poorly known., Methods: Uptake and depolarization-evoked release of (3)H-norepinephrine were investigated in vitro using pieces of the pig pineals. Specific antagonists and agonists of alpha(2)-adrenoceptors were employed for the characterization of alpha(2)-adrenoceptors involved in the autoregulation of depolarization-evoked release of norepinephrine in the pig pineal., Results: The level of neuronal uptake of norepinephrine in the pig pineal was 3.5+/-0.9 pmol/h/mg of wet tissue and represented about 77% of the total tissue radioactivity. Potassium ions at concentration of 60 mM significantly evoked tritium release. This effect was abolished in the absence of extracellular Ca(2+) and was diminished in the presence of Cd(2+). Antagonists of alpha(2)-adrenoceptors increased depolarization evoked tritium release. The order of its potency (based on pED(30)) was rauwolscine > phentolamine > BRL 44408 > WB 4101> RS 79948 = yohimbine >> prazosin >> imiloxan. alpha(2)-agonists decreased K(+) evoked release of tritium with the order of potency: UK14,304 > norepinephrine = guanfacine > oxymetazoline., Conclusion: The processes of neuronal uptake and depolarization-evoked release of norepinephrine from the sympathetic nerve endings in the pig pineal gland have been demonstrated. The studies with the use of adrenergic antagonists and agonists indicate that the sympathetic nerve fibers in the pig pineal gland possess functional presynaptic alpha(2)-adrenoceptors, which are involved in norepinephrine release inhibition. Due to the pharmacological properties these receptors closely resemble the subtype alpha(2A).

Vasoactive intestinal peptide (VIP) is one of neuropeptides involved in the regulation of the pineal gland function. The acute treatment of rat pinealocytes with VIP caused changes in their biochemical parameters. The present study concerns the effects of the chronic treatment with VIP on ultrastructure and function of the rat pinealocytes in organ culture. The pineals of adult male rats were assigned to one of three groups and placed in organ culture for four consecutive days. The pineals of the first group were incubated in the control medium, the pineals of the second group--12 hrs in control medium and 12 hrs in medium with 1 microM VIP (between 20.00 and 8.00) during each day, the pineals of the third group--24 hrs per day in medium with 1 microM VIP. The melatonin concentration was measured using RIA and activity of enzymes using radiochemical methods. Point count method was used in quantitative ultrastructural analysis. Both modes of chronic treatment with VIP increased significantly the level of melatonin secretion during four days of the culture and the content of this hormone in the pineal explants at the end of the experiment. Treatment with the neuropeptide for 12 hrs and 24 hrs per day elevated also the activity of arylalkylamine N-acetyltransferase and hydroxyindole-O-methyltransferase. On the other hand, VIP had no effect on the activity of arylamine-N-acetyltransferase. VIP increased the relative volume of rough endoplasmic reticulum, Golgi apparatus and mitochondria and did not influence the relative volume of lysosomes and lipid droplets as well as the numerical density of dense core vesicles in the examined rat pinealocytes. The obtained results indicate stimulatory effect of chronic treatment with VIP on the synthesis and secretion of melatonin in the rat pinealocytes in vitro. The results of morphological study are in agreement with the obtained biochemical data and point to the increase in secretory and metabolic activity of the rat pinealocytes in response to VIP.

An immunohistochemical study of the pineal gland of the domestic pig was carried out using the antisera raised against vasopressin (VP). The pineal glands were taken from the newborn, 21-day- and 7-month old female pigs. The pig pineal gland is moderately innervated by VP-immunoreactive nerve fibers. They run from the habenular commissure into the connective tissue septa and further into the pineal parenchyma. In the subependymal tissue as well as in the connective tissue septa, the fibers are smooth or with small varicosities and in the parenchyma with large ones. The obtained results point to extrapineal and extraepithalamic source of the fibers. The density of VP-immunoreactive fibers in the pineal gland of 7-month old pigs is higher than in the younger animals.

OBJECTIVES: The characteristic feature of the pig pinealocytes is the presence of numerous membrane bounded bodies (MBB), which according to our previous results may be involved in the secretory activity. The present study was undertaken to analyze the effect of continuous darkness and illumination on the ultrastructure of the pig pinealocytes. MATERIAL AND METHODS: The study was performed on three groups of gilts. The first group (control) was kept under a cycle of 14hrs light (500 lx) and 10hrs dark per day. The second group was exposed to continuous illumination (500 lx). The third group was kept under red light with intensity less than 1 lx, which was considered as darkness. The pigs were kept for 14 days under above reported conditions and then slaughtered at 08:00. The point count analysis was used in quantitative studies of pinealocyte substructures. RESULTS: The exposition of pigs to continuous illumination resulted in the decrease in the relative volume of mitochondria and in the numerical density of multivesicular bodies as well as in the increase in the relative volume of MBB in pinealocyte cell bodies. The exposition to continuous darkness led to the increase in the relative volume of mitochondria and the numerical density of dense core vesicles as well as induced some changes in smooth endoplasmic reticulum in pinealocyte cell bodies. CONCLUSIONS: The obtained results point to mitochondria, MBB, multivesicular bodies, dense core vesicles and smooth endoplasmic reticulum as the structures of the pig pinealocyte, which are controlled by environmental light conditions.

OBJECTIVES: Results of the majority of studies have revealed that diurnal changes in circulating melatonin level in the domestic pig differ from the typical patterns observed in other species. The aim of the present investigation was to study the effect of continuous darkness and continuous illumination on plasma melatonin in the pig. MATERIAL AND METHODS: The study was performed on three groups of immature gilts. The first group was kept under 14hrs light:10hrs dark cycle (500 lx of fluorescent light during photophase), the second group-under continuous illumination (500 lx of fluorescent light) and the third group-under red light with intensity below 1 lx, which was considered as darkness. The pigs were maintained nine days under above reported conditions and then plasma melatonin was monitored during five consecutive days. RESULTS: The diurnal changes in plasma melatonin concentration with increased levels during scotophases were observed in gilts kept under light:dark cycle, but these changes were characterized by low regularity and repeatability. In pigs kept under continuous darkness the circadian changes in plasma melatonin with the highest levels during natural nights were found. No significant circadian changes in plasma melatonin were noted in gilts exposed to continuous illumination. CONCLUSIONS: The obtained results suggest that the diurnal rhythm of melatonin secretion in the domestic pig is generated endogenously and entrained by environmental light. The present results supported also the previously reported observations showing low regularity of diurnal changes in circulating melatonin in the pig.

Up till now the results of performed investigations suggest the involvement of opioids in the regulation of the pineal gland activity in mammals. On the other hand, they show the existence of large interspecies differences in the presence of opioid peptides in the pineal gland and in the effects of opiates on the melatonin secretion. The aim of the present work was to study the influence of morphine on the melatonin secretion in the domestic pig. Morphine (about 2.5 mg/kg) was given intravenously to immature gilts during the day, during the night or during the night with turned on fluorescent illumination (intensity 500 lx at the level of the animal heads) and plasma melatonin level was measured. The effect of various concentrations of morphine on basal or norepinephrine-stimulated melatonin secretion was also investigated using perfusion culture of pineal glands of immature female pigs. Morphine did not change plasma melatonin concentration in the domestic pig when administered in a single dose at the beginning of the light or the dark phase of the diurnal light-dark cycle. However, morphine administration at the beginning of the night resulted in significantly decreased the plasma melatonin level in animals exposed to light, with intensity 500 lx, which was insufficient to block nocturnal rise in plasma concentration of this pineal hormone in the untreated pigs. Morphine had also no effect on the level of basal and norepinephrine-stimulated melatonin secretion in vitro. The obtained results suggest that in immature pigs morphine does not influence directly the pineal activity, but may modulate the melatonin secretion indirectly, increasing the sensitivity of the system generating melatonin synthesis and secretion to the light.