Search

The increased rates of nicotine exposure by electronic nicotine delivery systems (vaping), ingestion, or patches during pregnancy as an alternative to the smoking of tobacco arise concerns about the neurodevelopmental, cognitive, and behavioral long-term consequences in the juvenile offspring. Nowadays, the use of electronic cigarettes as supposed a safer smoking alternative has been increased mainly in young females at reproductive age, due to the "safety" misconception. However, previous studies suggest that exposure to nicotine during pregnancy and prenatal development may lead to detrimental effects in the postnatal lifespan. Nicotine, as an alkaloid, alters the reward system acting as acetylcholine (ACh) agonist on nicotinic cholinergic receptors (nAChRs). In early brain development, the cholinergic system is also involved in neurite outgrowth, cell survival, proliferation, differentiation, neurogenesis, and many other critical processes being considered as a developmental signal marker. The nicotine noxious effect at those early stages may impact the system programming and plasticity in the long-term postnatal life. In this study, we analyze the circadian locomotor activity and learning efficiency rhythms in the juvenile male offspring of mice exposed to nicotine through pregnancy and lactation. Attenuated rhythm amplitude and relative power of the circadian component were found in the nicotine exposed offspring (pN). The acrophase (the best performance during a 24-h cycle) of learning efficiency was delayed and the long-term memory consolidation task failed after 8 days of learning experience. The aforementioned results suggest nicotine exposure in uterus modifies the circadian modulation related to the memory consolidation and locomotor systems as well as its environmental temporal synchronization.

Mechanosensitive ion channels rely on membrane composition to transduce physical stimuli into electrical signals. The Piezo1 channel mediates mechanoelectrical transduction and regulates crucial physiological processes, including vascular architecture and remodeling, cell migration, and erythrocyte volume. The identity of the membrane components that modulate Piezo1 function remain largely unknown. Using lipid profiling analyses, we here identify dietary fatty acids that tune Piezo1 mechanical response. We find that margaric acid, a saturated fatty acid present in dairy products and fish, inhibits Piezo1 activation and polyunsaturated fatty acids (PUFAs), present in fish oils, modulate channel inactivation. Force measurements reveal that margaric acid increases membrane bending stiffness, whereas PUFAs decrease it. We use fatty acid supplementation to abrogate the phenotype of gain-of-function Piezo1 mutations causing human dehydrated hereditary stomatocytosis. Beyond Piezo1, our findings demonstrate that cell-intrinsic lipid profile and changes in the fatty acid metabolism can dictate the cell’s response to mechanical cues., Piezo channels are mechanosensitive and rely on membrane composition to transduce physical stimuli into electrical signals. Here authors analyse the membrane components that modulate Piezo1 function using lipid profiling which shows that fatty acid metabolism can modulate ion channel activity.

Dietary consumption of ω-3 polyunsaturated fatty acids (PUFAs), present in fish oils, is known to improve the vascular response, but their molecular targets remain largely unknown. Activation of the TRPV4 channel has been implicated in endothelium-dependent vasorelaxation. Here, we studied the contribution of ω-3 PUFAs to TRPV4 function by precisely manipulating the fatty acid content in Caenorhabditis elegans. By genetically depriving the worms of PUFAs, we determined that the metabolism of ω-3 fatty acids is required for TRPV4 activity. Functional, lipid metabolome, and biophysical analyses demonstrated that ω-3 PUFAs enhance TRPV4 function in human endothelial cells and support the hypothesis that lipid metabolism and membrane remodeling regulate cell reactivity. We propose a model whereby the eicosanoid’s epoxide group location increases membrane fluidity and influences the endothelial cell response by increasing TRPV4 channel activity. ω-3 PUFA-like molecules might be viable antihypertensive agents for targeting TRPV4 to reduce systemic blood pressure.

The transient receptor potential vanilloid 1 (TRPV1) channel is an essential component of the cellular mechanism through which noxious stimuli evoke pain. Functional and structural characterizations of TRPV1 shed light on vanilloid activation, yet the mechanisms for temperature and proton gating remain largely unknown. Spectroscopic approaches are needed to understand the mechanisms by which TRPV1 translates diverse stimuli into channel opening. Here, we have engineered a minimal cysteine-less rat TRPV1 construct (eTRPV1) that can be stably purified and reconstituted for spectroscopic studies. Biophysical analyses of TRPV1 constructs reveal that the S5-pore helix loop influences protein stability and vanilloid and proton responses, but not thermal sensitivity. Cysteine mutants retain function and stability for double electron-electron resonance (DEER) and electron paramagnetic resonance (EPR) spectroscopies. DEER measurements in the closed state demonstrate that eTRPV1 reports distances in the extracellular vestibule, equivalent to those observed in the apo TRPV1 structure. EPR measurements show a distinct pattern of mobilities and spectral features, in detergent and liposomes, for residues at the pore domain that agree with their location in the TRPV1 structure. Our results set the stage for a systematic characterization of TRPV1 using spectroscopic approaches to reveal conformational changes compatible with thermal- and ligand-dependent gating.

The kidney maintains the internal milieu by regulating the retention and excretion of proteins, ions, and small molecules. The glomerular podocyte forms the slit diaphragm of the ultrafiltration filter, whose damage leads to progressive kidney failure and focal segmental glomerulosclerosis (FSGS). The canonical transient receptor potential 6 (TRPC6) ion channel is expressed in the podocyte, and mutations in its cytoplasmic domain cause FSGS in humans. In vitro evaluation of disease-causing mutations in TRPC6 has revealed that these genetic alterations result in abnormal ion channel gating. However, the mechanism whereby the cytoplasmic domain modulates TRPC6 function is largely unknown. Here, we report a cryo-EM structure of the cytoplasmic domain of murine TRPC6 at 3.8 A resolution. The cytoplasmic fold of TRPC6 is characterized by an inverted dome-like chamber pierced by four radial horizontal helices that converge into a vertical coiled-coil at the central axis. Unlike other TRP channels, TRPC6 displays a unique domain swap that occurs at the junction of the horizontal helices and coiled-coil. Multiple FSGS mutations converge at the buried interface between the vertical coiled-coil and the ankyrin repeats, which form the dome, suggesting these regions are critical for allosteric gating modulation. This functionally critical interface is a potential target for drug design. Importantly, dysfunction in other family members leads to learning deficits (TRPC1/4/5) and ataxia (TRPC3). Our data provide a structural framework for the mechanistic investigation of the TRPC family.

Acanthospermal B (AcB), the major sesquiterpene lactone (SL) of Acanthospermum hispidum, an herb widely spread in Argentina, is a selective antibacterial agent against Enterococcus faecalis and Staphylococcus aureus, but inactive on Gram-negative and Lactobacillus. Methicillin-resistant Staphylococcus aureus (MRSA) is one of the main microorganisms involved in human chronic infection. A balb\c mouse skin infection model was developed to reproduce the lesions caused by acute and chronic infections produced by MRSA. After determination of the maximum concentration of AcB unable to produce tissular injury after intradermal injection, the anti-MRSA effect of AcB was evaluated on skin, liver and spleen tissues of infected mice. AcB, at doses of 2.5 mg/kg, produced a ten times decrease of MRSA growth in skin infection. In addition, the same dose prevented the dissemination to liver and/or spleen. AcB also displayed a bacteriostatic effect, in vitro, on MRSA cultures at 50 µg/mL that seems to be caused by partial denaturation of total bacterial DNA and/or inhibition of the PCR reaction in not denaturized DNA. Finally, total MRSA cell wall lysis occurred at a concentration of 100 µg/mL of AcB after 2 h of exposure. Copyright © 2010 John Wiley & Sons, Ltd.

We study the differences between Pseudomonas aeruginosa from an infected wound (clinical strain) and a colony isolated from it. We assessed the in vitro inhibition of these P. aeruginosa biofilms by DNase and filtrate of Lactobacillus plantarum cultures (acid = AF and neutralize = NF) with crystal violet technique. Inhibition by AF was greatest than DNase for clinical and isolated strain (p

Recent studies have shown that anesthetic agents alter the physical properties of lipid rafts on model membranes. However, if this destabilization occurs in brain membranes, altering the lipid raft-protein interaction, remains unknown. We analyzed the effects produced by pentobarbital (PB) on brain plasma membranes and lipid rafts in vivo. We characterized for the first time the thermotropic behavior of plasma membranes, synaptosomes, and lipid rafts from rat brain. We found that the transition temperature from the ordered gel to disordered liquid phase of lipids is close to physiological temperature. We then studied the effect of PB on protein composition of lipid rafts. Our results show a reduction of the total protein associated to rafts, with a higher reduction of the NMDAR compared to the GABAA receptor. Both receptors are considered the main targets of PB. In general, our results suggest that lipid rafts could be plausible mediators in anesthetic action.

Brevipalpus phoenicis s.l. is an economically important vector of the Citrus leprosis virus-C (CiLV-C), one of the most severe diseases attacking citrus orchards worldwide. Effective control strategies for this mite should be designed based on basic information including its population structure, and particularly the factors that influence its dynamics. We sampled sweet orange orchards extensively in eight locations in Brazil and 12 in Mexico. Population genetic structure and genetic variation between both countries, among locations and among sampling sites within locations were evaluated by analysing nucleotide sequence data from fragments of the mitochondrial cytochrome oxidase subunit I (COI). In both countries, B. yothersi was the most common species and was found in almost all locations. Individuals from B. papayensis were found in two locations in Brazil. Brevipalpus yothersi populations collected in Brazil were more genetically diverse (14 haplotypes) than Mexican populations (four haplotypes). Although geographical origin had a low but significant effect (ca. 25%) on the population structure, the greatest effect was from the within location comparison (37.02 %). Potential factors driving our results were discussed.

Health claims of lactic acid bacteria (LAB) used in functional foods and pharmaceutical preparations are based on the capacity of these microorganisms to stimulate the host immune system. In this study, the antigenic effect of LAB (Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus) on the gut immune system of BALB/c mice was evaluated. A dose-dependent increase of the Bcl2 protein was observed with all LAB assayed. Furthermore, the analysis of cytokine-producing cells in the lamina propria of gut showed that TNFalpha and INFgamma values, determined in macrophages cultured from Peyer patches, were enhanced for all the LAB assayed. An important increase of interleukins IL-10 and IL-4 was observed mainly in mice fed with Lactobacillus delbrueckii ssp. bulgaricus or Lactobacillus casei, while a significant induction of IL-2 and IL-12 was only observed with L. acidophilus (P

We report an experimental study of mouse sperm motility that shows chief aspects characteristic of neurons: the anesthetic (produced by tetracaine) and excitatory (produced by either caffeine or calcium) effects and their antagonic action. While tetracaine inhibits sperm motility and caffeine has an excitatory action, the combination of these two substances balance the effects, producing a motility quite similar to that of control cells. We also study the effects of these agents (anesthetic and excitatory) on the melting points of pure lipid liposomes constituted by 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and dipalmitoyl phosphatidic acid (DPPA). Tetracaine induces a large fluidization of the membrane, shifting the liposomes melting transition temperature to much lower values. The effect of caffeine is null, but its addition to tetracaine-doped liposomes greatly screen the fluidization effect. A high calcium concentration stiffens pure lipid membranes and strongly reduces the effect of tetracaine. Molecular Dynamics Simulations are performed to further understand our experimental findings at the molecular level. We find a strong correlation between the effect of antagonic molecules that could explain how the mechanical properties suitable for normal cell functioning are affected and recovered.

Regional ecological patterns, distribution and population structure of Prionace glauca were analyzed based on samples collected on-board two long-line fleets operating in oceanic waters (1994-96/2000-02) and in coastal oceanic waters (2003-2009) of the eastern tropical Pacific off México. Generalized additive models were applied to catch per unit of effort data to evaluate the effect of spatial, temporal and environmental factors on the horizontal distribution of the life stages (juvenile, adult) and the sexes at the estimated depth of catch. The presence of breeding areas was explored. The population structure was characterized by the presence of juveniles' aggregations and pregnant females towards coastal waters and the presence of adult males' aggregations towards oceanic waters. The species exhibited horizontal segregation by sex-size and vertical segregation by sex. Distribution of the sex-size groups at oceanic waters was seasonally affected by the latitude; however, at coastal oceanic waters mainly females were influenced by the longitude. Latitudinal changes on the horizontal distribution were coupled to the seasonal forward and backward of water masses through the study area. Adult males showed positive relationship with high temperatures and high-salinities waters (17.0°-20.0 °C; 34.2-34.4) although they were also detected in low-salinities waters. The distribution of juvenile males mainly occurred beyond low temperatures and low-salinities waters (14.0°-15.0 °C; 33.6-34.1), suggesting a wide tolerance of adult males to explore subartic and subtropical waters. At oceanic areas, adult females were aggregated towards latitudes25.0°N, mainly associated to subtropical waters during summer. The distribution of juvenile females indicated its preference by lower temperatures and more saline waters. Presence of pregnant females suggests that the eastern tropical Pacific off México represents an ecological key region to the reproductive cycle of P. glauca.

In a previous study we determined that by-products of Lactobacillus plantarum inhibited pathogenicity of Pseudomonas aeruginosa and is effective in the treatment of infected wounds. This study assesses the cytotoxic activity of acetic acid (AA), supernatants of L. plantarum and P. aeruginosa, with and without signal acyl-homoserine-lactones (AHL), and mixtures of both bacterial supernatants on human neutrophils. Cytotoxicity was determined through viability using trypan blue, apoptosis by Annexin V, necrosis by propidium iodide and intracellular pH by SNARF-1. We found that supernatants of L. plantarum caused less cytotoxicity than AA at the same extracellular pH (p < 0.05). P. aeruginosa induced a remarkable drop in intracellular pH, which was independent of extracellular pH. This intracellular acidity was correlated with a significant decrease in viability and was higher than supernatants of AHL producing P. aeruginosa (p < 0.05). When supernatants were mixed, the quantity of AHL diminished (p < 0.001) and the cytotoxic effect induced by P. aeruginosa was ameliorated by L. plantarum supernatant (p < 0.001 vs p < 0.01). These results are in agreement with the inflammatory in vivo assays determined by intradermal inoculations in Balb/c mice. Our findings will be useful for the formulation of effective and inexpensive products to resolve infected chronic wounds in our hospitals. © 2009 Elsevier B.V. All rights reserved. Fil: Ramos Vernieri, Alberto Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Tucuman. Facultad de Bioquimica, Quimica y Farmacia. Instituto de Microbiologia. Cátedra de Inmunología; Argentina Fil: Gobbato, Nadia Margarita. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Tucuman. Facultad de Bioquimica, Quimica y Farmacia. Instituto de Microbiologia. Cátedra de Inmunología; Argentina Fil: Rachid, Mirta Maria. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Tucuman. Facultad de Bioquimica, Quimica y Farmacia. Instituto de Microbiologia. Cátedra de Inmunología; Argentina Fil: González, L.. Universidad Nacional de Tucuman. Rectorado. Unidad de Citometria de Flujo; Argentina Fil: Yantorno, Osvaldo Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Fermentaciones Industriales. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Fermentaciones Industriales; Argentina Fil: Valdéz, Juan Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Tucuman. Facultad de Bioquimica, Quimica y Farmacia. Instituto de Microbiologia. Cátedra de Inmunología; Argentina. Universidad Nacional de Tucuman. Rectorado. Unidad de Citometria de Flujo; Argentina

Balb/c mice bearing a methylcholanthrene-induced fibrosarcoma were used to compare the activation levels of tumor-associated and peritoneal macrophages. Two stages of tumor growth were examined, namely "small" and "large" tumors, with average diameters of 10 and 30 mm, respectively. The activation state, determined by measurement of both phagocytic index and beta-glucuronidase content, was found to be markedly higher in tumor-associated macrophages than in their peritoneal counterparts and it was, in addition, independent of tumor progression. The percentage of tumor-associated macrophages, which were detected on the basis of Fc receptor expression, remained constant in the growing neoplasm, at approximately 23% of total cell population. None of these parameters were affected by inoculation with an immunopotentiating dose of heat-killed Candida albicans which, on the other hand, seemed not to alter the course of the tumor. These data suggest that within the tumor microenvironment macrophages would somehow be maintained at a constant proportion and at a highly activated state, while outside the tumor they would be at a lower activation level. Our results also suggest that TAM would not possess antitumor activity in vivo, although we have found this activity in vitro.

The effect of peptides released during the fermentation of milk on the humoral immune system and on fibrosarcoma growth was studied. Lactobacillus helveticus was able to release peptidic compounds during milk fermentation due to its high proteolytic activity, as was shown by the degree of proteolysis and size-exclusion HPLC elution profiles. Three fractions of these compounds were separated and fed to mice during different periods (2, 5, and 7 d). The humoral immune response was assessed by following the number of IgA-secreting cells, and the antitumor activity was monitored by studying the regression of subcutaneously implanted fibrosarcomas. Feeding during 2 and 7 d with the medium-sized fraction (Fraction II) significantly increased the IgA-producing cells in the intestines, whereas feeding with the large compound fraction (Fraction I) during 5 d and the small compound fraction (Fraction III) during all three feeding periods provided similar increases. A double dose of Fraction II showed the highest IgA-producing cell count. The increase by Fraction III was shown to be caused by the presence of L-Tryptophan. Fraction II significantly decreased the size of fibrosarcoma when previously fed during 7 d, and feeding with Fraction I during 5 d decreased significantly its size after 35 d of growth. Although the mechanisms by which lactic acid bacteria enhance the immune system are not clear, this study clearly shows that bioactive compounds released in fermented milks contribute to the immunoenhancing and antitumor properties of these products. The release of bioactive peptides by lactic acid bacteria can have important implications on the modulation of the cellular immune response. Fil: Leblanc, Jean Guy Joseph. Université de Moncton; Canadá. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Matar, Chantal. Université de Moncton; Canadá Fil: Valdez Marín, Juan Carlos. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia; Argentina Fil: de Moreno, Maria Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Université de Moncton; Canadá Fil: Perdigon, Gabriela del Valle. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia; Argentina

The beneficial effect of yoghurt consumption on health and on the improvement of the mucosal immune system is well established, as is the diet-associated risk of colon cancer. In an experimental model in BALB/c mice we demonstrated that yoghurt added to the diet for 10 consecutive days, with the procedure repeated each 10 days for 6 months, inhibited the development of a colorectal carcinoma induced by 1,2 dimethylhydrazine (DMH). The immunoregulatory mechanisms involved in the inhibition of tumour growth by yoghurt were also examined in these studies. We determined B lymphocytes IgA(+) and IgG(+), as well as CD4(+) and CD8(+) T cells in the large intestine. We measured cellular apoptosis and the cytokines TNF-alpha, IFN-gamma and IL-10. An increase in the number of IgA(+) (P

The effect of milks fermented by Lactobacillus helveticus and its non-proteolytic variant on mucosal and tumoral immunity was studied. Milks fermented by Lb. helveticus wild type or its non-proteolytic variant were administered orally to mice for different periods (3, 5 and 7 d). The immune response was assessed by analysing the activity of the peritoneal macrophages, the number of cells secreting IgA associated with the gut-associated lymphoid tissue and with the bronchial-associated lymphoid tissue. The number of cells was determined by direct immunofluorescence. The antitumour activity was monitored by studying the regression of the subcutaneously implanted fibrosarcomas. After 3 d feeding of milk fermented by Lb. helveticus wild type, the number of sIgA increased significantly at both the intestinal and bronchial levels, indicating that a cellular migration had occurred. This effect was not noticeable when milk fermented by Lb. helveticus Protease (−) was orally administered. Both fermented milks (wild type or its variant) exhibited an effect on the activity of the peritoneal macrophages, which might be indirectly correlated to the regression of the fibrosarcoma. Although the mechanism by which the lactic acid bacteria enhance the immune system is not clear, this study clearly suggests that the bioactive compounds released during milk fermentation might contribute to the immunoenhancing properties of these products. By releasing biopeptide, lactic acid bacteria have important implications in modulation of the host's immune response, more specifically its cellular immune response.

In order to grow, solid tumors need to develop new blood vessels. Neoplastic cells secrete growth factors that stimulate angiogenesis and tumor growth. Since Carrageenan acts as in vitro blocking agent which interferes with growth factor-receptor binding, we tested its action in vivo in order to analyze its growth inhibition capability in an experimental murine fibrosarcoma model. Indomethacin was used as a non-steroidal anti-inflammatory agent to neutralize the inflammatory action of Carrageenan. A murine fibrosarcoma was induced with methylcholanthrene in Balb/c mice and maintained by serial passage of tumor cells in mice of the same strain. Tumor volume was evaluated measuring two dimensions and applying the formula V = 0.4 x d2 x D. The mice with tumors were separated into groups; one of them was used as control and the other ones were treated with Indomethacin, Carrageenan and Carrageenan-Indomethacin. Tumor volume was compared between groups using the Student t Test. We demonstrated that Carrageenan and Indomethacin inhibit tumor growth. The inhibitory action of Carrageenan is significantly higher than the antitumoral effect of either Indomethacin or Carrageenan-Indomethacin.

The effect of yogurt on the inhibition of colon tumours induced by 1,2-dimethylhydrazine in BALB/c mice has been studied, and the hypothesis examined that yogurt induces a great reduction in the inflammatory immune response and inhibits tumour growth. Mice were assigned to five experimental groups: a control group fed with a conventional balanced diet, and four other test groups that received yogurt supplements for 2, 5, 7 or 10 consecutive days. At the end of each feeding period, mice were given subcutaneous injections of 1,2-dimethylhydrazine (20 mg/kg) once a week for 8 weeks. After tumour induction, yogurt was given again for 2, 5, 7 or 10 consecutive days each 10 d for 20 weeks. By week 20, 70% of the animals in the control group had developed colorectal tumours. From week 8, there was a considerable infiltration of mononuclear cells into the lamina propria of the large intestine. There was an increase in the number of IgG-producing cells and a slight increase in the IgA-secreting cells, and of CD8+ but not CD4+ T lymphocytes, a high level of β-glucuronidase activity in the intestinal fluid and leucocytosis with neutrophilia in the blood. However, in the test groups given yogurt tumour growth was inhibited, the effect being more evident with 7 or 10 d treatment. The inflammatory immune response as measured by the characteristics we assessed was also reduced, with an increase in the IgA-secreting cells and in CD4+ T lymphocytes. The blood count was similar to that of normal animals and no colorectal tumours were observed in week 20. We suggest that one of the mechanisms by which yogurt exerts antitumour activity is through its immunomodulator activity, by reducing the inflammatory immune response, which was markedly increased when the carcinogen was administered.

We investigated the time-dependent effects of 8 Gy of gamma radiation on the activities of cyclin-dependent kinases (Cdk's) and the incorporation of the thymidine analog bromodeoxyuridine (BrdU) throughout the S phase in Chinese hamster ovary (CHO) cells. The in vitro Cdk activities of immunoprecipitated cyclin E, cyclin A and Cdk2 were reduced about 30% per cell within 0.5-1 h after irradiation, but they recovered at different rates. The kinase activity of the cyclin E-Cdk2 complex recovered first and exceeded the control values by 1.5-2 h after irradiation. Cyclin A-Cdk activities began to recover at 3-4 h after irradiation, and cyclin E/A-Cdk2 activities recovered at intermediate rates. The super-recovery of cyclin E-Cdk2 coincided with the appearance of a small synchronous population of cells entering into S phase, consistent with transient G1-phase delay/recovery regulated by cyclin E-Cdk2, whereas the activities of cyclin A-Cdk's (75% cyclin A-Cdk2; 25% cyclin A-Cdc2 when inhibition was maximal) were correlated with rates of total DNA synthesis. Multivariate flow cytometry analyses of BrdU incorporation demonstrated that radiation-induced inhibition of DNA synthesis occurred predominantly within the last quarter of S phase and that the majority of the irradiated cells failed to enter G2 phase for 4-5 h. The recovery of cyclin A-Cdk activities coincided with increased levels of total DNA synthesis and BrdU incorporation into cells within the last quarter of S phase. Western blot analysis demonstrated that levels of Waf1/p21 did not increase during inhibition of cyclin A-Cdk's and DNA synthesis in the irradiated p53-mutated CHO cells; however, Cdc2 and Cdk2 exhibited increased levels of phosphotyrosine. The results (1) indicate that the transient G1-phase delay or G1/S-phase checkpoint (Lee et al., Proc. Natl. Acad. Sci. USA 94, 526-531, 1997) is mediated by inhibition of cyclin E-Cdk2 and (2) point to the existence of a radiation-induced S-phase checkpoint located about 75% into S phase involving the inhibition of cyclin A-Cdk's by a p53/Waf1-independent pathway in CHO cells.

Deuterium oxide (D2O) has been shown in previous studies to increase both the fluorescence lifetime and fluorescence intensity of propidium iodide (PI) and ethidium bromide (EB) when bound to nucleic acid structures. We have used spectroscopic analysis and conventional and phase-sensitive flow cytometry to compare changes in PI and EB fluorescence intensity and lifetime bound to DNA and fixed Chinese hamster ovary (CHO) cells in the presence of D2O vs. phosphate-buffered saline (PBS). Spectroscopic and flow cytometric studies showed a twofold enhancement of fluorescence intensity of PI and EB bound to fixed CHO cells in D2O and a 5 ns increase in PI and EB fluorescence lifetimes in D2O. The fluorescence lifetime of HL-60 cells stained with PI or EB was found to be 1-2 ns different from that of CHO cells, indicating that the lifetime of these fluorochromes is sensitive to chromatin configuration in different cells types. Apoptotic subpopulations of HL-60 cells had a significantly reduced fluorescence lifetime compared to nonapoptotic subpopulations. Results indicate that different chromatin states, or differences in the structures of PI and EB, lead to alterations in the fluorescence intensity and fluorescence lifetime of these intercalating probes.

32P-Labeled histone H1 was isolated from synchronized Chinese hamster (line CHO) cells, subjected to tryptic digestion, and fractionated into 15 phosphopeptides by high performance liquid chromatography. These phosphopeptides were grouped into five classes having different cell cycle phosphorylation kinetics: 1) peptides reaching a maximum phosphorylation rate in S and then declining in G2 and M, 2) peptides reaching a maximum phosphorylation rate in G2 and then remaining constant or declining in M, 3) peptides with increasing phosphorylation throughout S and G2 and reaching a maximum in M, 4) one peptide that was phosphorylated only in M, and 5) peptides that had low levels of phosphorylation that remained constant throughout the cell cycle. Amino acid analysis and sequencing demonstrated that the mitotic specific H1 phosphopeptide was the 16-amino acid, N-terminal, tryptic peptide Ac-SETAPAAPAAAPPAEK of the H1-1 class. This peptide, which is phosphorylated on both the Ser and Thr, does not contain the consensus sequence (S/T)PXZ (where X is any amino acid and Z is a basic amino acid). This sequence is thought to be required by the p34cdc2/cyclin B kinase that has maximum phosphorylating activity in mitosis. These data indicate that this kinase either does not have an obligatory requirement for the consensus sequence in vivo as generally believed or that it is not the enzyme responsible for the mitotic specific H1 phosphorylation.

The study of motor properties of cells under appropriate physical-chemical conditions is a significant problem nowadays. The standard techniques presently used do not allow to evaluate neither large samples nor to control their thermodynamic conditions. In this work, we report a cell motility sensor based on an optical technique with a time-resolved correlation, adapted in a system able to study several samples simultaneously. Image correlation analysis is used to follow their temporal behavior. A wide variety of motile cells, such as archaea, bacteria, spermatozoa, and even contractile cells, can be studied using this technique. Here, we tested our technique with the study of sperm motility. In particular, both the sperm motility and its prevalence are studied under a temperature range from 0 to 37 °C. We found that incubation at 10 °C presents the lengthiest prevalence in motility and observed, for the first time, an interesting thermal reversibility behavior.

SummaryThe effect of giving yogurt supplements to Balb/c mice on the various gut-associated lymphoid cells was studied. Animals were fed for 2, 5, 7 and 10 consecutive days. The different lymphoid cell types were identified and counted by haematoxylin–eosin staining of histological slices. The numbers of cells secreting IgA, IgG and IgM and the numbers of T lymphocytes were determined by direct immunofluorescence. The degree of activation of the intestinal macrophages in the small intestine was assessed by measuring the β-glucuronidase (EC 3.2.1.31) released into the intestinal fluid, and also by a histochemical method. Throughout the feeding period, there were no histological alterations in the gut, but there was marked cell infiltration, mainly of plasma cells and lymphocytes. The number of macrophages on the small intestine increased significantly after feeding for 2 d, while the β-glucuronidase activity was only slightly higher that of the controls. After a 7 d feeding period, the number of IgA secreting cells increased, while the values for cells secreting IgM and IgG and for T lymphocytes remained similar to those of the controls. The effect of giving yogurt on lymphoid cells associated with the large intestine was mainly on the numbers of IgA secreting B cells and T lymphocytes, with a marked increase during the whole feeding period in the latter type of cell. Since giving yogurt mainly enhanced the IgA secreting B cells in both small and large intestines, this increase would strengthen the host's defence mechanisms in the intestinal mucosa. Although the number of macrophages was increased, there was no enhancement in their activity, which might have harmed the host by producing an inflammatory response.

An indirect immunofluorescence assay was carry out to determine the IgM and IgG antibody responses to yeast and mycelial forms of Candida albicans in mice injected with a 5 x 5(5) and 5 x 10(7) live cells suspensions. Prior adsorption of the serum samples with heat-killed blastospores enabled us to follow the specific antimycelial response which were detected considerably later than expected. Slow level of antibodies were obtained within an infection of 5 x 10(5) cell for both antibody classes and for yeast and mycelial forms. When a 5 x 10(7) cell dose was used for inoculation, maximum titers of antibodies to blastospores and mycelium in non-adsorbed sera appeared almost simultaneously (days 15 and 13, respectively). When serum samples from mice infected with the same dose were previously adsorbed with blastospores, the antimycelium antibodies for both types of Igs, were detected delayed during the infection course. In this case the higher titer for IgG appeared on day 33 and on day 23 for IgM. We suggest that the high titer obtained with the blastospore forms for the 5 x 10(7) cell dose may be due to a major immunogenicity of this forms, for to induce an immune response in the host, or that the delay in the antimycelium antibodies detection could be due to that a blastospore form is the predominant in the infection early stages. Implications of this fact for pathogenesis are discussed.

There is a controversy in respect to the immunological response (humoral or cellular) concerning the defense against Candida albicans. Candidosis would induce sub-populations of suppressor cells in the host cell-immune response. This report tries to show the effect of different doses of C. albicans (alive or heat-killed) on the expression of cell-mediated and humoral immunity. The effect upon cell immunity was determined by inoculating different lots of singeneic mice, doses of varied concentration of C. albicans and checking for delayed-type hipersensitivity (D.T.H.). D.T.H. was also controlled in syngeneic normal mice which had previously been injected with inoculated mice spleen cells. Humoral immunity was assayed by measuring the induced blastogenesis by Pokeweed Mitogen on spleen mononuclear cells with different doses of C. albicans. Results obtained show that the different doses gave origin to: Suppression of humoral and cell response (10(8) alive); Suppression of only humoral response (10(6) alive); Suppression of cell response and increase of humoral response (10(9) dead); Increase of both responses (10(8) dead).

Inoculation of 10(8) C. albicans intraperitoneally into Balb/c mice at given dosage was reported to induce suppression of antigen-specific delayed-type hypersensitivity. Adoptive transfer of spleen cells into normal syngeneic mice pre-treated with Cyclophosphamide confirmed the existence of suppressor cells in mice. Such cells were sensitive to treatment with anti-theta serum and complement, non-adherent to Sephadex G-10. A pretreatment of the mice with Cyclophosphamide eliminated DTH suppression. Treatment with antimacrophage agents via intraperitoneal abrogated suppression only if being effected before inoculation of alive 10(8) Candida albicans. It is concluded that the spleen suppressor cell is a T-lymphocyte whose precursor is Cyclophosphamide-sensitive, requiring the macrophage to be induced.

Candida albicans produces germ-tubes (GT) when it is incubated in animal or human serum. This dimorphism is responsible for its invasive ability. The purpose of the present paper is (1) to evaluate the ability of rat peritoneal macrophages to inhibit GT production of ingested Candida albicans, obtained from immunized rats and then activated in vitro with Candida-induced lymphokines; (2) to determinate any possible alteration of phagocytic and candidacidal activities. The phagocytes were obtained from rats immunized with viable C. albicans. Some of them were exposed to Candida-induced lymphokines in order to activate the macrophages in vitro. The monolayers of activated, immune and normal macrophages were infected with a C. albicans suspension during 4 hr. Activated macrophages presented not only the highest phagocytic and candidacidal activities but a noticeable inhibition of GT formation and incremented candidacidal activity.

This study evaluated the ability of the probiotic organism Lactobacillus plantarum to inhibit the pathogenic activity of Pseudomonas aeruginosa, both in vitro and in vivo, and investigated the mechanisms involved in such protection. L. plantarum whole cultures, culture filtrates (acid filtrate and neutralised acid filtrate) and isolated, washed cells were tested in vitro for their effects on the production of the P. aeruginosa quorum-sensing signal molecules, acyl-homoserine-lactones (AHLs), and two virulence factors controlled by these signal molecules, elastase and biofilm. All were inhibited by L. plantarum cultures and filtrates, but not by isolated, washed cells. The acid L. plantarum growth medium itself had some inhibitory activity, but the greatest activity was exerted by the whole culture. To test the in-vivo activity of L. plantarum, a burned-mouse model was used in which burns infected with P. aeruginosa were treated with L. plantarum at 3, 4, 5, 7 and 9 days post-infection. Samples from skin, liver and spleen taken after 5, 10 and 15 days demonstrated inhibition of P. aeruginosa colonisation by L. plantarum. There was also an improvement in tissue repair, enhanced phagocytosis of P. aeruginosa by tissue phagocyctes, and a decrease in apoptosis at 10 days. These results indicate that L. plantarum and/or its by-products are potential therapeutic agents for the local treatment of P. aeruginosa burn infections.