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Canine β-defensin 103 (cBD103) and its common variant cBD103ΔG23 are multitasking polypeptides. As a β-defensin, cBD103 is one of many antimicrobial agents used by the innate immunity to thwart pathogenic colonization. In this study, we showed that cBD103 was expressed throughout the nasal cavity, with primary expression in the nares as well as respiratory and olfactory epithelia. In the rostral nasal concha, cBD103 was expressed in the epithelium, and to a lesser degree in the lamina propria, but was absent in goblet cells. In the main olfactory epithelium, virtually all cells in the epithelial layer and select cells associated with Bowman's glands expressed cBD103. We also showed that the ΔG23 mutation did not appreciably alter the antimicrobial activity of the peptide against several species of microorganisms tested in nutrient-rich or minimal media or minimal media with salt added. Moreover, we showed antimicrobial activity in minimal media did not necessarily predict the inhibitory action of the peptide in nutrient-rich media. Both forms of cBD103 caused ultrastructural changes (membrane blebbing, condensation of intracellular contents and cell wall lysis) in Escherichia coli and Staphylococcus aureus. As a ligand of the melanocortin receptors, we showed that cBD103ΔG23 increased ERK1/2 activation and cAMP accumulation when bound to the human or canine melanocortin-4 receptor, acting as a weak allosteric agonist., (© 2019 American Association for Anatomy.)

The colonial naked mole rat Heterocephalus glaber is a subterranean, eusocial rodent. The H. glaber vomeronasal organ neuroepithelium (VNE) displays little postnatal growth. However, the VNE remains neuronal in contrast to some mammals that possess nonfunctional vomeronasal organ remnants, for example, catarrhine primates and some bats. Here, we describe the vomeronasal organ (VNO) microanatomy in the naked mole rat and we make preliminary observations to determine if H. glaber shares its minimal postnatal VNE growth with other African mole rats. We also determine the immunoreactivity to the mitotic marker Ki67, growth-associated protein 43 (GAP43), and olfactory marker protein (OMP) in six adult and three subadult H. glaber individuals. VNE volume measurements on a small sample of Cryptomys hottentotus and Fukomys damarensis indicate that the VNE of those African mole rat species are also likely to be growth-deficient. Ki67(+) cells show that the sensory epithelium is mitotically active. GAP43 labelling indicates neurogenesis and OMP(+) cells are present though less numerous compared to GAP43(+) cells. In this respect, the VNO of H. glaber does not appear vestigial. The African mole rat VNE may be unusually variable, perhaps reflecting reduced selection pressure on the vomeronasal system. If so, African mole rats may provide a useful genetic model for understanding the morphological variability observed in the mammalian VNO. Anat Rec, 2019. © 2019 Wiley Periodicals, Inc. Anat Rec, 303:318-329, 2020. © 2019 American Association for Anatomy., (© 2019 Wiley Periodicals, Inc.)

Background : Docetaxel (DOC), or Taxotere, is an anthracycline antibiotic used to treat multiple types of cancer. It is a first-line chemotherapy treatment for patients with metastasized, hormone-resistant prostate cancer (PCa) or for patients with high-risk, localized PCa that could benefit from early chemotherapy treatment. Previously, we showed that stearidonic acid (SDA), an omega-3 fatty acid, enhances the cytotoxicity of doxorubicin (DOX) in human PCa cells. This observation suggests that PCa therapies using SDA and chemotherapeutic drugs in combination offer attractive possibilities for developing treatments that ameliorate toxic side effects of some commonly used chemotherapy drugs. Objectives: We used androgen-resistant PC3 and DU 145 cells derived from human prostate cancer to quantify the effects of combined SDA and DOC on proliferation/viability and on the production of pro-apoptotic caspases 9 and 3. We also compared the effects of SDA with those of BAY, a pharmacological inhibitor of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-ĸB), in androgen-sensitive LNCaP cells. Finally, we qualitatively and quantitatively assessed the drug combination on androgen receptor (AR) and peroxisome proliferator-activated receptor gamma (PPARγ) expression in LNCaP and PC3 cells, respectively. Methods: The half maximal inhibitory concentration (IC50) and combination indices of SDA and DOC in PC3 and DU 145 cells were determined using the MTT cell viability assay. To quantify the effects of SDA and BAY on NF-ĸB activity, we used luciferase reporter assays in LNCaP cells that were stably transduced with lentiviral vectors carrying NF-ĸB response element sequence upstream of the luciferase gene sequence. AR and PPARγ expression were assessed by western blotting and immunocytochemistry. We considered caspase 9 and 3 cleavage to be apoptosis markers and determined the drug combination effect on the extent of that cleavage by western blot analysis. Results: The cytotoxic effects of DOC were synergistically enhanced by SDA when the two were added to DU145 and PC3 cell cultures. Combination index (CI) analyses based on the Chou-Talalay method and mass action law showed synergistic interaction with CI <1. SDA suppressed TNFα-induced NF-κB activity similarly to BAY. The SDA/DOC combination down regulated testosterone (T)-induced AR and troglitazone-induced PPARγ protein expression when compared to using the drugs singly. Similarly, the SDA/DOC combination induced caspase 9 and 3 production and cleavage suggesting apoptosis induction. Like our DOX studies, this work provides proof-of-concept for using SDA and DOC in combination to reduce the dose, and therefore the toxicity, of DOC and possibly increasing the survival benefit in DOC clinical translation studies., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Adiponectin is a protein secreted by white adipocytes that plays an important role in insulin action, energy homeostasis and the development of atherosclerosis. The intracellular localization and trafficking of GLUT4 and leptin in adipocytes has been well studied, but little is known regarding the intracellular trafficking of adiponectin. Recent studies have demonstrated that constitutive adiponectin secretion is dependent on PIP2 levels and the integrity of cortical F-actin. Non-muscle myosin II is an actin-based motor that is associated with membrane vesicles and participates in vesicular trafficking in mammalian cells. Therefore, we investigated the role of myosin II in the trafficking and secretion of adiponectin in 3T3-L1 adipocytes. Confocal microscopy revealed that myosin IIA and IIB were dispersed throughout the cytoplasm of the adipocyte. Both myosin isoforms were localized in the Golgi/TGN region as evidenced by colocalization with the cis-Golgi marker, p115 and the trans-Golgi marker, γ-adaptin. Inhibition of myosin II activity by blebbistatin or actin depolymerization by latrunculin B dispersed myosin IIA and IIB towards the periphery while significantly inhibiting adiponectin secretion. Therefore, the constitutive trafficking and secretion of adiponectin in 3T3-L1 adipocytes occurs by an actin-dependent mechanism that involves the actin-based motors, myosin IIA and IIB., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Sandhoff disease (SD) is a lysosomal storage disorder characterized by the absence of hydrolytic enzyme β-N-acetylhexosaminidase (Hex), which results in storage of GM2 ganglioside in neurons and unremitting neurodegeneration. Neuron loss initially affects fine motor skills, but rapidly progresses to loss of all body faculties, a vegetative state, and death by five years of age in humans. A well-established feline model of SD allows characterization of the disease in a large animal model and provides a means to test the safety and efficacy of therapeutic interventions before initiating clinical trials. In this study, we demonstrate a robust central nervous system (CNS) inflammatory response in feline SD, primarily marked by expansion and activation of the microglial cell population. Quantification of major histocompatibility complex II (MHC-II) labeling revealed significant up-regulation throughout the CNS with areas rich in white matter most severely affected. Expression of the leukocyte chemokine macrophage inflammatory protein-1 alpha (MIP-1α) was also up-regulated in the brain. SD cats were treated with intracranial delivery of adeno-associated viral (AAV) vectors expressing feline Hex, with a study endpoint 16weeks post treatment. AAV-mediated gene delivery repressed the expansion and activation of microglia and normalized MHC-II and MIP-1α levels. These data reiterate the profound inflammatory response in SD and show that neuroinflammation is abrogated after AAV-mediated restoration of enzymatic activity., Competing Interests: On behalf of all authors, the corresponding author states that there is no conflict of interest., (Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.)

In the mammalian brain, the anatomical structure of neural circuits changes little during adulthood. As a result, adult learning and memory are thought to result from specific changes in synaptic strength. A possible exception is the olfactory bulb (OB), where activity guides interneuron turnover throughout adulthood. These adult-born granule cell (GC) interneurons form new GABAergic synapses that have little synaptic strength plasticity. In the face of persistent neuronal and synaptic turnover, how does the OB balance flexibility, as is required for adapting to changing sensory environments, with perceptual stability? Here we show that high dendritic spine turnover is a universal feature of GCs, regardless of their developmental origin and age. We find matching dynamics among postsynaptic sites on the principal neurons receiving the new synaptic inputs. We further demonstrate in silico that this coordinated structural plasticity is consistent with stable, yet flexible, decorrelated sensory representations. Together, our study reveals that persistent, coordinated synaptic structural plasticity between interneurons and principal neurons is a major mode of functional plasticity in the OB., (Copyright © 2016 Elsevier Inc. All rights reserved.)

This study investigates the vomeronasal organ in extant nocturnal strepsirhines as a model for ancestral primates. Cadaveric samples from 10 strepsirhine species, ranging from fetal to adult ages, were studied histologically. Dimensions of structures in the vomeronasal complex, such as the vomeronasal neuroepithelium (VNNE) and vomeronasal cartilage (VNC) were measured in serial sections and selected specimens were studied immunohistochemically to determine physiological aspects of the vomeronasal sensory neurons (VSNs). Osteological features corresponding to vomeronasal structures were studied histologically and related to 3-D CT reconstructions. The VNC consistently rests in a depression on the palatal portion of the maxilla, which we refer to as the vomeronasal groove (VNG). Most age comparisons indicate that in adults VNNE is about twice the length compared with perinatal animals. In VNNE volume, adults are 2- to 3-fold larger compared with perinatal specimens. Across ages, a strong linear relationship exists between VNNE dimensions and body length, mass, and midfacial length. Results indicate that the VNNE of nocturnal strepsirhines is neurogenic postnatally based on GAP43 expression. In addition, based on Olfactory Marker Protein expression, terminally differentiated VSNs are present in the VNNE. Therefore, nocturnal strepsirhines have basic similarities to rodents in growth and maturational characteristics of VSNs. These results indicate that a functional vomeronasal system is likely present in all nocturnal strepsirhines. Finally, given that osteological features such as the VNG are visible on midfacial bones, primate fossils can be assessed to determine whether primate ancestors possessed a vomeronasal complex morphologically similar to that of modern nocturnal strepsirhines., (Copyright © 2013 Wiley Periodicals, Inc.)

The melanocortin receptors (MCRs 1-5) are G protein coupled-receptors (GPCRs) that regulate food intake, inflammation, skin pigmentation, sexual function and steroidogenesis. Their peptide ligands, the melanocortins, are α-, β- and γ-melanocyte-stimulating hormone and adrenocorticotropic hormone (ACTH) all of which are secreted from the anterior pituitary gland under hypothalamic control. MC2R binds ACTH but has no affinity for the other melanocortins and is, thereby, pharmacologically different from MCRs that bind those ligands. Evidence suggests that elevated GPCRs transactivate the androgen receptor (AR), the critical mediator of prostate cell growth, and consequently promote prostate cancer cell proliferation. It may be that reduced central melanocortin signaling is coincidental with reversal of prostate cancer cachexia, but no data are available on the expression of, or the role for, MCRs in prostate cancer. Here, we show that MCR (1-5) mRNAs are expressed in androgen-dependent LNCaP and androgen-independent PC3 and DU-145 human prostate cancer cell lines. Further, MC2R, the specific target of ACTH, is expressed in LNCaP, PC3 and DU-145 cells. Among the several synthetic MCR peptide ligands that we used, only ACTH promoted concentration-dependent cell proliferation in the three cell lines as shown by MTT cell proliferation assay. In LNCaP cells, the effect was additive with testosterone stimulation and was partially blunted with SHU9119, a non-selective MCR antagonist. In the same cells, ACTH induced cAMP production and increased AR nuclear labeling in immunocytochemical assays. Our observations suggest that MC2R is involved in prostate carcinogenesis and that targeting MC2R signaling may provide a novel avenue in prostate carcinoma treatment.

Many odorants related to manufactured explosives have low volatilities and are barely detectable as odors. We previously reported that zinc metal nanoparticles increased rat olfactory epithelium responses, measured by electroolfactogram (EOG), to several odorants. Here, we report that nanomolar concentrations of zinc metal nanoparticles strongly enhanced olfactory responses to the explosives related odorants cyclohexanone, methyl benzoate, acetophenone, and eugenol. Rat olfactory epithelium was exposed to metal nanoparticles and odorant responses were quantified by EOG. Zinc nanoparticles added to explosive odorants strongly increased the odorant response in a dose-dependent manner. The enzymatic breakdown of the second messenger cyclic adenosine monophosphate (cAMP) was prevented by adding the membrane-permeable phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). This caused the olfactory cilia cAMP concentration to increase and generated EOG signals. The EOG responses generated by IBMX were not enhanced by zinc nanoparticles. Based on these observations, we conclude that zinc nanoparticles act at the receptor site and are involved in the initial events of olfaction. Our results suggest that zinc metal nanoparticles can be used to facilitate a canine detection of explosive odorants., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The study of tooth form is informative about the relationship between teeth and the material properties of foods consumed. Studies of dental functional morphology depend on precise characterization of relevant aspects of crown form; the occlusal surfaces of primate molar teeth are studied in 3-dimensional space more and more commonly today. Dental topographic analysis is becoming an increasingly popular method for studying tooth form, given its ability to characterize functionally relevant aspects of tooth form from an entire occlusal surface. This landmark-free approach has been especially valuable in studies of the effects of tooth wear on shape. Mean slope and relief, for example, have been found to be informative about the function of molar teeth in both living and extinct primates. Instructions for the use of this approach are provided here.

Although all platyrrhine primates possess a vomeronasal organ (VNO), few species have been studied in detail. Here, we revisit the microanatomy of the VNO and related features in serially sectioned samples from 41 platyrrhine cadavers (14 species) of mixed age. Procedures to identify terminally differentiated vomeronasal sensory neurons (VSNs) via immunolabeling of olfactory marker protein (OMP) were used on selected specimens. The VNO varies from an elongated epithelial tube (e.g., Ateles fusciceps) to a dorsoventrally expanded sac (e.g., Saguinus spp.). The cartilage that surrounds the VNO is J-shaped or U-shaped in most species, and articulates with a groove on the bony palate. Preliminary results indicate a significant correlation between the length of this groove and length of the VNO neuroepithelium, indicating this feature may serve as a skeletal correlate. The VNO neuroepithelium could be identified in all adult primates except Alouatta, in which poor preservation prevented determination. The VNO of Ateles, described in detail for the first time, had several rows of VSNs and nerves in the surrounding lamina propria. Patterns of OMP-reactivity in the VNO of perinatal platyrrhines indicate that few or no terminally differentiated VSNs are present at birth, thus supporting the hypothesis that some platyrrhines may have delayed maturation of the VNO. From a functional perspective, all platyrrhines studied possess structures required for chemoreception (VSNs, vomeronasal nerves). However, some microanatomical findings, such as limited reactivity to OMP in some species, indicate that some lineages of New World monkeys may have a reduced or vestigial vomeronasal system., (Copyright © 2011 Wiley Periodicals, Inc.)

Knowledge of the vomeronasal neuroepithelium (VNNE) microanatomy is disproportionately based on rodents. To broaden our knowledge, we examined olfactory marker protein (OMP) expression in a sample of twenty-three non-human primates. The density of OMP (+) vomeronasal sensory neurons (VSNs) in the VNNE was measured. Here we compared OMP (+) VSN density in five species of Saguinus (a genus of New World monkey) of different ages to a comparative primate sample that included representatives of every superfamily in which a VNO is postnatally present. In Saguinus spp., the VNNE at birth is thin, usually comprising one or two nuclear rows. At all ages studied, few VNNE cells are OMP reactive as view in coronal sections. In the comparative sample, the OMP (+) VSNs appear to be far more numerous in the spider monkey (another New World monkey) and the bushbaby (a distant relative). Other species (e.g., owl monkey) had a similar low density of OMP (+) VSNs as in Saguinus. These results expand our earlier finding that few VSNs are OMP (+) in Saguinus geoffroyi to other species of the genus. Our sample indicates that the number of OMP (+) VSNs in primates varies from ubiquitous to few with New World monkeys varying the most. The scarcity of OMP (+) cells in some primate VNOs reflects a lower number of terminally differentiated VSNs compared to a diverse range of mammals. If primates with relatively few OMP (+) VSNs have a functional vomeronasal system, OMP is not critical for stimulus detection., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Rat astrocyte function is changed by diabetes mellitus relative to the nondiabetic state and we believe that altered function contributes to the central nervous system symptoms manifested by individuals with diabetes. We report here a comparison of astrocyte glutamate uptake and GFAP expression in streptozotocin-induced type 1 diabetic rats and insulin-treated diabetic rats at 4 and 8 weeks following diabetes onset. In glial plasmalemmal vesicle (GPV) preparations from treated rats, insulin prevented the increase observed in untreated, diabetic rats of both sodium-dependent and sodium-independent glutamate uptake. We determined by immunoblotting and immunohistochemistry that insulin treatment prevented the decrease of GFAP expression detected in the cerebral cortex, hippocampus, and cerebellum of untreated, diabetic rats. These observations indicate that insulin effects on astrocyte function are significant in managing diabetes-induced central nervous system pathology.

Mammalian olfactory epithelium can withstand the external environment, undergo life-long regeneration, and respond to thousands of odorant stimuli, making it an attractive system for a variety of studies. Previously, we described a long-lived olfactory coculture of olfactory epithelium and bulb tissues and we present here the kinetic properties of that culture system. Neonatal mouse epithelial-bulbar explants were grown for periods as long as 121 days in vitro (DIV), nearly doubling the survival time of our previously longest lived cultures. Cultures at all ages responded to air-borne odorants. The youngest cultures (1-15 DIV) showed shorter half-rise and half-decay times than older cultures (21-121 DIV), and were more variable in their half-decay times. Zinc nanoparticles enhanced electro-olfactogram responses of both younger and older cultures and both groups were immunopositive for olfactory marker protein. The results show that our olfactory culture model can support mature, odorant-responsive olfactory receptor neurons that possess many of the response features of in situ olfactory receptor neurons., (Copyright © 2010 S. Karger AG, Basel.)

Zinc metal nanoparticles in picomolar concentrations strongly enhance odorant responses of olfactory sensory neurons. One- to 2-nm metallic particles contain 40-300 zinc metal atoms, which are not in an ionic state. We exposed rat olfactory epithelium to metal nanoparticles and measured odorant responses by electroolfactogram and whole-cell patch clamp. A small amount of zinc nanoparticles added to an odorant or an extracellular/intracellular particle perfusion strongly increases the odorant response in a dose-dependent manner. Zinc nanoparticles alone produce no odor effects. Copper, gold, or silver nanoparticles do not produce effects similar to those of zinc. If zinc nanoparticles are replaced by Zn(+2) ions in the same concentration range, we observed a reduction of the olfactory receptor neuron odorant response. Based on these observations, we hypothesize that zinc nanoparticles are closely located to the interface between the guanine nucleotide-binding protein and the receptor proteins and are involved in transferring signals in the initial events of olfaction. Our results suggest that zinc metal nanoparticles can be used to enhance and sustain the initial olfactory events.

Bovine viral diarrhea virus (BVDV) is a group of small enveloped viruses with a single-stranded, positive-oriented RNA genome of approximately 12.3 kb. BVDV genome directs the production of a viral polyprotein that is subsequently cleaved to release the mature viral proteins. To explore the potential of using BVDV as viral vector for stable expression of heterologous genes, eGFP2A was inserted in between N(pro) and C genes of a noncytopathic type-I BVDV strain SD1. eGFP2A was designed with eGFP protein in frame fused to the N terminus of the foot-and-mouth disease virus 2A protease. This strategy promised not only the correct processing of both viral N(pro) and C protein but also releasing of the chimeric protein from the nascent viral polyprotein. The recombinant reporter virus was successfully rescued in MDBK cells. In vitro study showed that eGFP2A protein, as expected, was expressed and processed properly from the nascent viral polyprotein. The reporter virus was similar to wt SD1 in viral RNA replication and protein expression and comparable to wt SD1 in growth kinetics except that this virus had a peak virus titer approximately 0.5 log(10) lower and a maximum yield about 4h later than wt SD1. In summary, these results indicated that BVDV is a suitable viral vector for stable expression of heterologous genes when inserted in between N(pro) and C genes.

Severe chlamydial disease typically occurs after previous infections and results from a hypersensitivity response that is also required for chlamydial elimination. Here, we quantitatively dissected the immune and disease responses to repeated Chlamydia pneumoniae lung infection by multivariate modeling with four dichotomous effects: mouse strain (A/J or C57BL/6), dietary protein content (14% protein and 0.3% L-cysteine-0.9% L-arginine, or 24% protein and 0.5% L-cysteine-2.0% L-arginine), dietary antioxidant content (90 IU alpha-tocopherol/kg body weight versus 450 IU alpha-tocopherol/kg and 0.1% g L-ascorbate), and time course (3 or 10 days postinfection). Following intranasal C. pneumoniae challenge, C57BL/6 mice on a low-protein/low-antioxidant diet, but not C57BL/6 mice on other diets or A/J mice, exhibited profoundly suppressed early lung inflammatory and pan-T-cell (CD3delta(+)) and helper T-cell (CD45) responses on day 3 but later strongly exacerbated disease on day 10. Contrast analyses characterized severe C. pneumoniae disease as being a delayed-type hypersensitivity (DTH) response with increased lung macrophage and Th1 cell marker transcripts, increased Th1:Th2 ratios, and Th1 cytokine-driven inflammation. Results from functional analyses by DTH, enzyme-linked immunospot, and immunohistofluorescence assays were consistent with the results obtained by transcript analysis. Thus, chlamydial disease after secondary infection is a temporal dysregulation of the T-cell response characterized by a profoundly delayed T-helper cell response that results in a failure to eliminate the pathogen and provokes later pathological Th1 inflammation. This delayed T-cell response is under host genetic control and nutritional influence. The mechanism that temporally and quantitatively regulates the host T-cell population is the critical determinant in chlamydial pathogenesis.

Exposure to the estrogen receptor alpha (ERalpha) ligand diethylstilbesterol (DES) between neonatal days 2 to 12 induces penile adipogenesis and adult infertility in rats. The objective of this study was to investigate the in vivo interaction between DES-activated ERalpha and the proadipogenic transcription factor peroxisome proliferator-activated receptor gamma (PPARgamma). Transcripts for PPARs alpha, beta, and gamma and gamma1a splice variant were detected in Sprague-Dawley normal rat penis with PPARgamma predominating. In addition, PPARgamma1b and PPARgamma2 were newly induced by DES. The PPARgamma transcripts were significantly upregulated with DES and reduced by antiestrogen ICI 182, 780. At the cellular level, PPARgamma protein was detected in urethral transitional epithelium and stromal, endothelial, neuronal, and smooth muscular cells. Treatment with DES activated ERalpha and induced adipocyte differentiation in corpus cavernosum penis. Those adipocytes exhibited strong nuclear PPARgamma expression. These results suggest a biological overlap between PPARgamma and ERalpha and highlight a mechanism for endocrine disruption.

The naked mole-rat (Heterocephalus glaber) is unusual in numerous life history characteristics as well as its eusocial organization. This species demonstrates widespread sexual suppression and prominent scent marking, behaviors that have been associated with pheromonal communication involving the vomeronasal organ in other rodents. Yet, previous studies indicate that urinary signals do not mediate sexual suppression in Heterocephalus. Surprisingly, no previous studies have examined the vomeronasal organ in this species. Here, we show that Heterocephalus is unique among rodents in showing no evidence of postnatal volumetric growth in the vomeronasal neuroepithelium. Subadults from birth to weaning fell within the same volume range as adults regardless of breeding/non-breeding status of the latter. A comparison of existing ontogenetic data on other mammals suggests that the proportionally small VNOs of Heterocephalus may be explained by a deficiency in VNNE growth. Growth deficiency of the vomeronasal organ in Heterocephalus may relate to a diminished role that pheromones play in certain social interactions for this species, such as breeding suppression. In light of the unique aspects of the vomeronasal organ in Heterocephalus, comparative studies of rodents may provide a model for understanding variation of this sensory system in other mammalian orders including primates, an order which shows a range from vestigial to demonstrably functional vomeronasal organs.

Explanations for the chemosensory abilities of newborn mammals focus primarily on food (milk) acquisition and communication (e.g., maternal-infant bonding). However, the relative importance of the main and accessory (vomeronasal) olfactory systems is hypothesized to differ at birth between altricial and precocial mammals. Strepsirrhines (lemurs and lorises) possess main and accessory olfactory systems, and vary in life-history traits related to infant dependency and maturation. Accordingly, this study examines the size and maturational characteristics of vomeronasal (VNNE) and olfactory (OE) neuroepithelia in strepsirrhines. Serially sectioned heads of 18 infant cadavers were examined microscopically for neuroepithelial distribution. Measurements were taken on the length of the nasal fossa on one side that was occupied by VNNE and OE. The data were corrected for body size using the cranial length or body mass, and were then examined for correlation with several life-history variables, as well as activity pattern. In addition, immunohistochemistry was used to identify cells in the VNNE and OE that express olfactory marker protein (OMP), a marker of mature olfactory neurons. Relative OE extent was not significantly correlated with any of the life-history variables. Relative VNNE length was negatively correlated with relative gestation length and relative neonatal mass (P<0.05). However, when we corrected for phylogenetic relationships, we found no significant correlations between either of the neuroepithelial measurements and life-history variables. Immunohistochemical findings suggest that OE has more OMP-reactive cells than VNNE in all species. OMP-reactive cells appear to be less numerous in diurnal species compared to most nocturnal species. These results indicate that the VNNE may be relatively longer at birth in altricial species. However, it remains uncertain how phylogeny and/or ontogeny may explain these findings.

Adiponectin is an adipokine with profound insulin-sensitizing, anti-inflammatory, and anti-atherogenic properties. Plasma levels of adiponectin are reduced in insulin resistant states such as obesity, type 2 diabetes and cardiovascular disease. However, the mechanism(s) by which adiponectin concentrations are decreased during disease development is unclear. Studies have shown that endothelin-1 (ET-1), a vasoconstrictor peptide, affects adipocyte glucose metabolism and secretion of adipokines such as leptin, resistin, and adiponectin. The goal of our study was to determine the mechanism by which ET-1 decreases adiponectin secretion. 3T3-L1 adipocytes were treated for 24h with ET-1 (10nM) and then stimulated with vehicle or insulin (100 nM) for a period of 1-2h. Chronic ET-1 (24h) treatment significantly decreased basal and insulin-stimulated adiponectin secretion by 66% and 47%, respectively. Inhibition of phosphatidylinositol 4,5-bisphosphate (PIP(2)) hydrolysis by the PLCbeta inhibitor, U73122, or exogenous addition of PIP(2):histone carrier complex (1.25:0.625 microM) ameliorated the decrease in basal and insulin-stimulated adiponectin secretion observed with ET-1. However, treatment with exogenous PIP(2):histone carrier complex and the actin depolymerizing agent latrunculin B (20 microM) did not reverse the ET-1-mediated decrease in adiponectin secretion. In conclusion, we demonstrate that ET-1 inhibits basal and insulin-stimulated adiponectin secretion through PIP(2) modulation of the actin cytoskeleton.

The present study examined interspecies, intersexual, and age-related changes in size of the vomeronasal neuroepithelium (VNNE) of two species of greater bushbabies (genus Otolemur, Infraorder Lorisiformes, Suborder Strepsirrhini). Tissue blocks containing the vomeronasal organs of nine O. crassicaudatus (8 adults, 1 neonate) and ten O. garnettii (9 adults, 1 neonate) were studied by means of serial paraffin sectioning and computer-based reconstruction of VNNE volume. In addition, the immunoreactivity of the VNNE to two neuronal markers, neuron-specific beta tubulin (BT) and olfactory marker protein (OMP) was compared between species, sexes, and ages. Results indicated that a clear VNNE is present at birth in both species, and OMP immunoreactivity was verified in O. garnettii at birth. Male and female adults of both species showed OMP-immunoreactive and BT-immunoreactive neurons in the VNNE. Immunohistochemical findings indicated that all males and the youngest females had the thickest VNNE, especially at the marginal junctions with the receptor-free epithelium. Results of a 2-way Analysis of Variance (ANOVA, species x sex) revealed no significant differences in VNNE length or volume between species, but O. crassicaudatus had significantly (p < 0.05) greater palatal length. Significant (p < 0.05) differences also were found between sexes in VNNE volume, but no significant differences in palatal length or VNNE length. The distribution of VNNE volume against age indicated that the sex differences were more pronounced in O. crassicaudatus than O. garnettii. For both species and sexes, distribution of VNNE volume against age suggested an age-related reduction in volume. These findings demonstrate postnatal plasticity in VNNE size in Otolemur that is reminiscent of that found for olfactory structures in some rodents. Bushbabies or other strepsirrhine primates may offer an opportunity for further understanding of behavioral correlates of VNNE postnatal plasticity, which may represent primitive functional characteristics of the order Primates.

Many diabetic individuals develop anosmia but the mechanism(s) causing the dysfunction in the olfactory system is (are) unknown. Glial fibrillary acidic protein expression is reduced in diabetic retinopathy and is also reduced, with unknown consequences, in other brain regions of diabetic rats. We used immunohistochemistry and immunoblotting from untreated control and streptozotocin-induced type 1 (insulin dependent) diabetic rats to investigate main olfactory epithelial mitotic rate and glial fibrillary acidic protein expression in the lamina propria of the sensory epithelium and in the olfactory bulb. Numbers of bromodeoxyuridine-positive cells were significantly lower in the diabetic sensory epithelium compared to non-diabetic controls. Immunohistochemical observations suggested a qualitative difference in glial fibrillary acidic protein expression in both regions examined especially in the olfactory bulb external plexiform layer and the lamina propria. Immunoblot analysis confirmed that the diabetic olfactory bulb and lamina propria expressed less glial fibrillary acidic protein compared to the non-diabetic control group. The lower expression levels in the olfactory bulb external plexiform layer suggested by immunohistochemistry do not reflect a change in the number of astrocytes since the numbers of S100B(+) cells were not different between the two groups.

Vomeronasal organ (VNO) morphology varies markedly across primate taxa. Old World monkeys display no postnatal VNO. Humans and at least some apes retain a vestigial VNO during postnatal life, whereas the strepsirrhines and New World Monkeys present a morphologically well-defined VNO that, in many species, is presumed to function as an olfactory organ. Available microanatomical and behavioral studies suggest that VNO function in these species does not precisely duplicate that described in other mammalian taxa. The questions of which species retain a functional VNO and what functions they serve require inquiry along diverse lines but, to be functional, the vomeronasal epithelium must be neuronal and olfactory. We used immunohistochemistry to establish these criteria in six primate species. We compared the expression of two neuronal markers, neuron-specific beta-tubulin (BT) and protein gene product 9.5, and olfactory marker protein (OMP), a marker of mature olfactory sensory neurons, in paraffin-embedded VNO sections from two strepsirrhine and four haplorhine species, all of which retain morphologically well-defined VNOs during postnatal life. The infant Eulemur mongoz, adult Otolemur crassicaudatus, neonatal Leontopithicus rosalia, and adult Callithrix jacchus express all three proteins in their well-defined vomeronasal neuroepithelia. The infant Tarsius syrichta showed some BT and OMP immunoreactivity. We establish that two strepsirrhine species and at least some New World haplorhines have mature sensory neurons in the VNO. In contrast, at all ages examined, Saguinus geoffroyi VNO expresses these markers in only a few cells., ((c) 2004 Wiley-Liss, Inc.)

Paleoprimatologists depend on relationships between form and function of teeth to reconstruct the diets of fossil species. Most of this work has been limited to studies of unworn teeth. A new approach, dental topographic analysis, allows the characterization and comparison of worn primate teeth. Variably worn museum specimens have been used to construct species-specific wear sequences so that measurements can be compared by wear stage among taxa with known differences in diet. This assumes that individuals in a species tend to wear their molar teeth in similar ways, a supposition that has yet to be tested. Here we evaluate this assumption with a longitudinal study of changes in tooth form over time in primates. Fourteen individual mantled howling monkeys (Alouatta palliata) were captured and then recaptured after 2, 4, and 7 years when possible at Hacienda La Pacifica in Costa Rica between 1989-1999. Dental impressions were taken each time, and molar casts were produced and analyzed using dental topographic analysis. Results showed consistent decreases in crown slope and occlusal relief. In contrast, crown angularity, a measure of surface jaggedness, remained fairly constant except with extreme wear. There were no evident differences between specimens collected in different microhabitats. These results suggest that different individual mantled howling monkeys wear their teeth down in similar ways, evidently following a species-specific wear sequence. Dental topographic analysis may therefore be used to compare morphology among similarly worn individuals from different species.

Callitrichid primates (tamarins, marmosets) have extreme variation in the vomeronasal organ (VNO), including ontogenetic differences in the neuroepithelium and vomeronasal duct (VND) patency at birth. Such differences render the timing and extent of VNO maturation debatable in callitrichids, but no studies have used neuron-specific immunohistochemical markers to address this question. The present study compared the number of VNO epithelial cells that express immunoreactivity to neuron-specific beta-tubulin III (BT), VNO length, and VNO cross-sectional area between two species of tamarins (Leontopithecus rosalia and Saguinus geoffroyi) that differed in perinatal VND patency. Neonatal lemurs and adult marmosets and bushbabies were also examined for a comparison to species previously shown to have a relatively large amount of VNO neuroepithelium and patent VNDs. The head of each specimen was serially sectioned in the coronal plane. Based on known rostrocaudal start/stop points of the VNO, selected unstained sections were used for BT protocols and area measurement at three percentiles (25th, 50th, 75th) in each specimen. Each section was photographed and enlarged for cell counts and measurement of cross-sectional epithelial area. In each specimen, the number of BT(+) cells in the VNO was counted at each percentile and expressed as a number per mm(2). Results indicated that lemur VNOs had a dense population of BT(+) cells at birth, but the VNO was more varied in the tamarin species. S. geoffroyi had few or no BT(+) cells in VNOs of neonates, which had fused VNDs, but had an increased BT(+) population by 1 and 2 months postnatal age, when the VND was patent. Of the species with patent VNDs at birth, neonatal L. rosalia had a denser population of BT(+) cells compared to S. geoffroyi, though not to the degree seen in neonatal lemurs or adult marmosets and bushbabies. These findings show that BT immunohistochemistry is a useful comparative method for the study of VNOs in subadult primates. Since the quantity of nonsensory VNO epithelium varies substantially between species, epithelial area measurements may be misleading, and BT(+) cell counts appeared to be the best quantitative method for comparing receptor neuron numbers among primates. It is suggested that the greater BT(+) cell population in L. rosalia at all subadult stages examined reveals an earlier maturation of the neuroepithelium compared to S. geoffroyi. Further investigation should consider whether this may relate to a comparatively brief subadult ontogeny and early onset of adult behaviors in L. rosalia compared to other tamarins studied to date., (Copyright 2004 Wiley-Liss, Inc.)

The canine's olfactory acuity is legendary, but neither its main olfactory system nor its vomeronasal system has been described in much detail. We used immunohistochemistry on paraffin-embedded sections of male and female adult dog vomeronasal organ (VNO) to characterize the expression of proteins known to be expressed in the VNO of several other mammals. Basal cell bodies were more apparent in each section than in rodent VNO and expressed immunoreactivity to anticytokeratin and antiepidermal growth factor receptor antibodies. The thin layer of neurone cell bodies in the sensory epithelium and axon fascicles in the lamina propria expressed immunoreactivity to neurone cell adhesion molecule, neurone-specific beta tubulin and protein gene product 9.5. Some neurones expressed growth-associated protein 43 (GAP43): and a number of those also expressed neurone-specific beta tubulin-immunoreactivity. Some axon fascicles were double labelled for those two proteins. The G-protein alpha subunits Gi and Go, involved in the signal transduction pathway, showed immunoreactivity in the sensory cell layer. Our results demonstrate that the canine vomeronasal organ contains a population of cells that expresses several neuronal markers. Furthermore, GAP43 immunoreactivity suggests that the sensory epithelium is neurogenic in adult dogs.

The vomeronasal organ (VNO) is the receptor portion of the accessory olfactory system and transduces chemical cues that identify social hierarchy, reproductive status, conspecifics and prey. Signal transduction in VNO neurons is apparently accomplished via an inositol 1,4,5-trisphosphate (IP3)-activated calcium conductance that includes a different set of G proteins than those identified in vertebrate olfactory sensory neurons. We used immunohistochemical (IHC) and SDS-PAGE/western analysis to localize three IP3 receptors (IP3R) in the rat VNO epithelium. Type-I IP3R expression was weak or absent. Antisera for type-II and -III IP3R recognized appropriate molecular weight proteins by SDS-PAGE, and labeled protein could be abolished by pre-adsorption of the respective antibody with antigenic peptide. In tissue sections, type-II IP3R immunoreactivity was present in the supporting cell zone but not in the sensory cell zone. Type-III IP3R immunoreactivity was present throughout the sensory zone and overlapped that of transient receptor potential channel 2 (TRPC2) in the microvillar layer of sensory epithelium. Co-immunoprecipitation of type-III IP3R and TRPC2 from VNO lysates confirmed the overlapping immunoreactivity patterns. The protein-protein interaction complex between type-III IP3R and TRPC2 could initiate calcium signaling leading to electrical signal production in VNO neurons.

The heterotrimeric G-protein Gs couples cell-surface receptors to the activation of adenylyl cyclases and cyclic AMP production (reviewed in refs 1, 2). RGS proteins, which act as GTPase-activating proteins (GAPs) for the G-protein alpha-subunits alpha(i) and alpha(q), lack such activity for alpha(s) (refs 3-6). But several RGS proteins inhibit cAMP production by Gs-linked receptors. Here we report that RGS2 reduces cAMP production by odorant-stimulated olfactory epithelium membranes, in which the alpha(s) family member alpha(olf) links odorant receptors to adenylyl cyclase activation. Unexpectedly, RGS2 reduces odorant-elicited cAMP production, not by acting on alpha(olf) but by inhibiting the activity of adenylyl cyclase type III, the predominant adenylyl cyclase isoform in olfactory neurons. Furthermore, whole-cell voltage clamp recordings of odorant-stimulated olfactory neurons indicate that endogenous RGS2 negatively regulates odorant-evoked intracellular signalling. These results reveal a mechanism for controlling the activities of adenylyl cyclases, which probably contributes to the ability of olfactory neurons to discriminate odours.

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To determine the morphological correlates of vitellogenin uptake and distribution, vitellogenic ovarian follicles of Acheta domesticus were examined by light and transmission electron microscopy. Yolk deposition was usually restricted to the terminal follicle of each panoistic ovariole, but if the terminal follicle had completed vitellogenesis and had not yet been ovulated, the penultimate follicle was often vitellogenic. Vitellogenesis occurred only in the ovaries of adults, with follicles representing all stages of vitellogenic development occurring simultaneously within a single ovary. Vitellogenic follicles were 0.75 to >2.0 mm long. The single irregularly shaped nucleus of each follicle cell contained two to three nucleoli. During vitellogenesis the epithelium of individual follicles progressed from columnar to cuboidal to squamous. The distribution of cytoplasmic organelles within individual follicle cells was polarized during early and middle stages of vitellogenesis. Near the end of vitellogenesis, polarity disappeared, the follicular epithelium became squamous, and the frequency of vesicular and multivesicular bodies in the follicle cell cytoplasm increased. A well-developed capability for follicle cell protein synthesis and secretion throughout vitellogenesis was indicated by abundant rough endoplasmic reticulum and Golgi bodies. Receptor-mediated endocytotic activity by the follicle cells during all stages of vitellogenesis was suggested by omnipresent coated pits on all surfaces. Septate junctions, gap-like junctions, and desmosomes all occurred in the lateral membranes, while gap-like junctions predominated at the oocyte-follicle cell interface. In follicles longer than 1 mm, adjacent follicle cells were separated by intercellular channels that widened to as much as 10 m̈m by the completion of vitellogenesis. The channels contained a flocculent material, and a similar-appearing material filled the space between the oolemma and follicle cell apical membranes. The oolemma contained a profusion of coated pits throughout vitellogenesis., (Copyright © 1989 Wiley-Liss, Inc.)