Genetic Ablation of Prorenin Receptor in the Rostral Ventrolateral Medulla Influences Blood Pressure and Hydromineral Balance in Deoxycorticosterone-Salt Hypertension.

Non-enzymatic activation of renin via its interaction with prorenin receptor (PRR) has been proposed as a key mechanism of local renin-angiotensin system (RAS) activation. The presence of renin and angiotensinogen has been reported in the rostral ventrolateral medulla (RVLM). Overactivation of bulbospinal neurons in the RVLM is linked to hypertension (HTN). Previous studies have shown that the brain RAS plays a role in the pathogenesis of the deoxycorticosterone (DOCA)-salt HTN model. Thus, we hypothesized that PRR in the RVLM is involved in the local activation of the RAS, facilitating the development of DOCA-salt HTN. Selective PRR ablation targeting the RVLM (PRRRVLM-Null mice) resulted in an unexpected sex-dependent and biphasic phenotype in DOCA-salt HTN. That is, PRRRVLM-Null females (but not males) exhibited a significant delay in achieving maximal pressor responses during the initial stage of DOCA-salt HTN. Female PRRRVLM-Null subsequently showed exacerbated DOCA-salt-induced pressor responses during the "maintenance" phase with a maximal peak at 13 d on DOCA-salt. This exacerbated response was associated with an increased sympathetic drive to the resistance arterioles and the kidney, exacerbated fluid and sodium intake and output in response to DOCA-salt, and induced mobilization of fluids from the intracellular to extracellular space concomitant with elevated vasopressin. Ablation of PRR suppressed genes involved in RAS activation and catecholamine synthesis in the RVLM but also induced expression of genes involved in inflammatory responses. This study illustrates complex and sex-dependent roles of PRR in the neural control of BP and hydromineral balance through autonomic and neuroendocrine systems. Graphical abstract.

Cardiometabolic Effects of DOCA-Salt in Mice Depend on Ambient Temperature.

BACKGROUND: Mice prefer warmer environments than humans. For this reason, behavioral and physiological thermoregulatory responses are engaged by mice in response to a standard room temperature of 22 to 24 °C. Autonomic mechanisms mediating thermoregulatory responses overlap with mechanisms activated in hypertension, and, therefore, we hypothesized that housing at thermoneutral temperatures (TNs; 30 °C) would modify the cardiometabolic effects of deoxycorticosterone acetate (DOCA)-salt in mice. METHODS: The effects of DOCA-salt treatment upon ingestive behaviors, energy expenditure, blood pressure, heart rate (HR), and core temperature were assessed in C57BL/6J mice housed at room temperature or TN. RESULTS: Housing at TN reduced food intake, energy expenditure, blood pressure, and HR and attenuated HR responses to acute autonomic blockade by chlorisondamine. At room temperature, DOCA-salt caused expected increases in fluid intake, sodium retention in osmotically inactive pools, blood pressure, core temperature, and also caused expected decreases in fat-free mass, total body water, and HR. At TN, the effects of DOCA-salt upon fluid intake, fat gains, hydration, and core temperature were exaggerated, but effects on energy expenditure and HR were blunted. Effects of DOCA-salt upon blood pressure were similar for 3 weeks and exaggerated by TN housing in the fourth week. CONCLUSIONS: Ambient temperature robustly influences behavioral and physiological functions in mice, including metabolic and cardiovascular phenotype development in response to DOCA-salt treatment. Studying cardiometabolic responses of mice at optimal ambient temperatures promises to improve the translational relevance of rodent models.

Transcriptional Profiling of Primate Central Nucleus of the Amygdala Neurons to Understand the Molecular Underpinnings of Early-Life Anxious Temperament.

BACKGROUND: Children exhibiting extreme anxious temperament (AT) are at an increased risk for developing anxiety and depression. Our previous mechanistic and neuroimaging work in young rhesus monkeys linked the central nucleus of the amygdala to AT and its underlying neural circuit. METHODS: Here, we used laser capture microscopy and RNA sequencing in 47 young rhesus monkeys to investigate AT's molecular underpinnings by focusing on neurons from the lateral division of the central nucleus of the amygdala (CeL). RNA sequencing identified numerous AT-related CeL transcripts, and we used immunofluorescence (n = 3) and tract-tracing (n = 2) methods in a different sample of monkeys to examine the expression, distribution, and projection pattern of neurons expressing one of these transcripts. RESULTS: We found 555 AT-related transcripts, 14 of which were confirmed with high statistical confidence (false discovery rate < .10), including protein kinase C delta (PKCδ), a CeL microcircuit cell marker implicated in rodent threat processing. We characterized PKCδ neurons in the rhesus CeL, compared its distribution with that of the mouse, and demonstrated that a subset of these neurons project to the laterodorsal bed nucleus of the stria terminalis. CONCLUSIONS: These findings demonstrate that CeL PKCδ is associated with primate anxiety, provides evidence of a CeL to laterodorsal bed nucleus of the stria terminalis circuit that may be relevant to understanding human anxiety, and points to specific molecules within this circuit that could serve as potential treatment targets for anxiety disorders.

Dorsal Amygdala Neurotrophin-3 Decreases Anxious Temperament in Primates.

BACKGROUND: An early-life anxious temperament (AT) is a risk factor for the development of anxiety, depression, and comorbid substance abuse. We validated a nonhuman primate model of early-life AT and identified the dorsal amygdala as a core component of AT's neural circuit. Here, we combine RNA sequencing, viral-vector gene manipulation, functional brain imaging, and behavioral phenotyping to uncover AT's molecular substrates. METHODS: In response to potential threat, AT and brain metabolism were assessed in 46 young rhesus monkeys. We identified AT-related transcripts using RNA-sequencing data from dorsal amygdala tissue (including central nucleus of the amygdala [Ce] and dorsal regions of the basal nucleus). Based on the results, we overexpressed the neurotrophin-3 gene, NTF3, in the dorsal amygdala using intraoperative magnetic resonance imaging-guided surgery (n = 5 per group). RESULTS: This discovery-based approach identified AT-related alterations in the expression of well-established and novel genes, including an inverse association between NTRK3 expression and AT. NTRK3 is an interesting target because it is a relatively unexplored neurotrophic factor that modulates intracellular neuroplasticity pathways. Overexpression of the transcript for NTRK3's endogenous ligand, NTF3, in the dorsal amygdala resulted in reduced AT and altered function in AT's neural circuit. CONCLUSIONS: Together, these data implicate neurotrophin-3/NTRK3 signaling in the dorsal amygdala in mediating primate anxiety. More generally, this approach provides an important step toward understanding the molecular underpinnings of early-life AT and will be useful in guiding the development of treatments to prevent the development of stress-related psychopathology.

Reduction of nitrosative stress by methane: Neuroprotection through xanthine oxidoreductase inhibition in a rat model of mesenteric ischemia-reperfusion.

Our aim was to characterize the main components of the nitrosative response with quantitative changes of the nitrergic myenteric neurons in adjacent intestinal segments after transient superior mesenteric artery occlusion. We also tested the hypothesis that exogenous methane may modulate the evolution of nitroxidation by influencing xanthine oxidoreductase (XOR) activity. The microcirculatory consequences of a 50 min ischemia or ischemia-reperfusion were investigated in anesthetized rats (n = 124) inhaling normoxic air with or without 2.2% methane. XOR activities, nitrogen monoxide (NO), nitrite/nitrate (NOx), and nitrotyrosine levels were measured, together with relative nitrergic neuron ratios from duodenum, ileum and colon samples. The effects of methane on XOR were also examined in vitro. The intramural flow stopped only in the ileum during ischemia. The highest baseline XOR activity was found in the duodenum, which increased further during ischemia. NO and nitrotyrosine levels rose, and the nNOS-immunopositive neuron ratio and NOx level both dropped. Reperfusion uniformly elevated XOR activity and nitrotyrosine formation, with the highest level attained in the duodenum, where the nitrergic neuron ratio remained depressed. These alterations were eliminated in methane-treated animals, XOR activity and nitrotyrosine formation decreased in all sites, and the duodenal nitrergic neuron ratio was re-established. The inhibitory effect of methane on XOR-linked nitrate reductase activity was also demonstrated in vitro. With segment-specific microcirculatory alterations, the risk for nitrosative stress is highest in transiently hypoxic tissues with high endogenous XOR activities. The XOR-inhibitory effect of methane can reduce nitroxidation and protects the nitrergic neuron population in such conditions.

Real-Time Intraoperative MRI Intracerebral Delivery of Induced Pluripotent Stem Cell-Derived Neurons.

Induced pluripotent stem cell (iPSC)-derived neurons represent an opportunity for cell replacement strategies for neurodegenerative disorders such as Parkinson's disease (PD). Improvement in cell graft targeting, distribution, and density can be key for disease modification. We have previously developed a trajectory guide system for real-time intraoperative magnetic resonance imaging (RT-IMRI) delivery of infusates, such as viral vector suspensions for gene therapy strategies. Intracerebral delivery of iPSC-derived neurons presents different challenges than viral vectors, including limited cell survival if cells are kept at room temperature for prolonged periods of time, precipitation and aggregation of cells in the cannula, and obstruction during injection, which must be solved for successful application of this delivery approach. To develop procedures suitable for RT-IMRI cell delivery, we first performed in vitro studies to tailor the delivery hardware (e.g., cannula) and defined a range of parameters to be applied (e.g., maximal time span allowable between cell loading in the system and intracerebral injection) to ensure cell survival. Then we performed an in vivo study to evaluate the feasibility of applying the system to nonhuman primates. Our results demonstrate that the RT-IMRI delivery system provides valuable guidance, monitoring, and visualization during intracerebral cell delivery that are compatible with cell survival.

Maternal Western diet increases adiposity even in male offspring of obesity-resistant rat dams: early endocrine risk markers.

Maternal overnutrition or associated complications putatively mediate the obesogenic effects of perinatal high-fat diet on developing offspring. Here, we tested the hypothesis that a Western diet developmental environment increases adiposity not only in male offspring from obesity-prone (DIO) mothers, but also in those from obesity-resistant (DR) dams, implicating a deleterious role for the Western diet per se. Selectively bred DIO and DR female rats were fed chow (17% kcal fat) or Western diet (32%) for 54 days before mating and, thereafter, through weaning. As intended, despite chow-like caloric intake, Western diet increased prepregnancy weight gain and circulating leptin levels in DIO, but not DR, dams. Yet, in both genotypes, maternal Western diet increased the weight and adiposity of preweanlings, as early as in DR offspring, and increased plasma leptin, insulin, and adiponectin of weanlings. Although body weight normalized with chow feeding during adolescence, young adult Western diet offspring subsequently showed decreased energy expenditure and, in DR offspring, decreased lipid utilization as a fuel substrate. By mid-adulthood, maternal Western diet DR offspring ate more chow, weighed more, and were fatter than controls. Thus, maternal Western diet covertly programmed increased adiposity in childhood and adulthood, disrupted relations of energy regulatory hormones with body fat, and decreased energy expenditure in offspring of lean, genetically obesity-resistant mothers. Maternal Western diet exposure alone, without maternal obesity or overnutrition, can promote offspring weight gain.

Structural and molecular features of intestinal strictures in rats with Crohn's-like disease.

AIM: To develop a new rat model we wanted to gain a better understanding of stricture formation in Crohn's disease (CD). METHODS: Chronic colitis was induced locally by the administration of 2,4,6-trinitrobenzenesulfonic acid (TNBS). The relapsing inflammation characteristic to CD was mimicked by repeated TNBS treatments. Animals were randomly divided into control, once, twice and three times TNBS-treated groups. Control animals received an enema of saline. Tissue samples were taken from the strictured colonic segments and also adjacent proximally and distally to its 60, 90 or 120 d after the last TNBS or saline administrations. The frequency and macroscopic extent of the strictures were measured on digital photographs. The structural features of strictured gut wall were studied by light- and electron microscopy. Inflammation related alterations in TGF-beta 2 and 3, matrix metalloproteinases 9 (MMP9) and TIMP1 mRNA and protein expression were determined by quantitative real-time PCR and western blot analysis. The quantitative distribution of caspase 9 was determined by post-embedding immunohistochemistry. RESULTS: Intestinal strictures first appeared 60 d after TNBS treatments and the frequency of them increased up to day 120. From day 90 an intact lamina epithelialis, reversible thickening of lamina muscularis mucosae and irreversible thickening of the muscularis externa were demonstrated in the strictured colonic segments. Nevertheless the morphological signs of apoptosis were frequently seen and excess extracellular matrix deposition was recorded between smooth muscle cells (SMCs). Enhanced caspase 9 expression on day 90 in the SMCs and on day 120 also in myenteric neurons indicated the induction of apoptosis. The mRNA expression profile of TGF-betas after repeated TNBS doses was characteristic to CD, TGF-beta 2, but not TGF-beta 3 was up-regulated. Overexpression of MMP9 and down-regulation of TIMP1 were demonstrated. The progressive increase in the amount of MMP9 protein in the strictures was also obvious between days 90 and 120 but TIMP1 protein was practically undetectable at this time. CONCLUSION: These findings indicate that aligned structural and molecular changes in the gut wall rather than neuronal cell death play the primary role in stricture formation.

Overexpressing Corticotropin-Releasing Factor in the Primate Amygdala Increases Anxious Temperament and Alters Its Neural Circuit.

BACKGROUND: Nonhuman primate models are critical for understanding mechanisms underlying human psychopathology. We established a nonhuman primate model of anxious temperament (AT) for studying the early-life risk to develop anxiety and depression. Studies have identified the central nucleus of the amygdala (Ce) as an essential component of AT's neural substrates. Corticotropin-releasing factor (CRF) is expressed in the Ce, has a role in stress, and is linked to psychopathology. Here, in young rhesus monkeys, we combined viral vector technology with assessments of anxiety and multimodal neuroimaging to understand the consequences of chronically increased CRF in the Ce region. METHODS: Using real-time intraoperative magnetic resonance imaging-guided convection-enhanced delivery, five monkeys received bilateral dorsal amygdala Ce-region infusions of adeno-associated virus serotype 2 containing the CRF construct. Their cagemates served as unoperated control subjects. AT, regional brain metabolism, resting functional magnetic resonance imaging, and diffusion tensor imaging were assessed before and 2 months after viral infusions. RESULTS: Dorsal amygdala CRF overexpression significantly increased AT and metabolism within the dorsal amygdala. Additionally, we observed changes in metabolism in other AT-related regions, as well as in measures of functional and structural connectivity. CONCLUSIONS: This study provides a translational roadmap that is important for understanding human psychopathology by combining molecular manipulations used in rodents with behavioral phenotyping and multimodal neuroimaging measures used in humans. The results indicate that chronic CRF overexpression in primates not only increases AT but also affects metabolism and connectivity within components of AT's neural circuitry.

Gut region-specific accumulation of reactive oxygen species leads to regionally distinct activation of antioxidant and apoptotic marker molecules in rats with STZ-induced diabetes.

UNLABELLED: The aim of this study was to seek possible links between the regionality along the digestive tract and the accumulation of reactive oxygen species, the effectiveness of the antioxidant defense system and the sensitivity to the types of demise in different gut regions of rats with streptozotocin-induced diabetes. Significant changes were observed in the oxidative status and in the activity of the antioxidant defense system in the diabetic colon; the peroxynitrite production was doubled, the level of hemoxygenase-2 protein was increased 11-fold and the expression of anti-apoptotic bcl-2 was also increased. The segment-specific vulnerability of the gastrointestinal tract induced by hyperglycemia was also confirmed by electron microscopy, demonstrating the presence of severe necrosis in the colon of the diabetic rats. No remarkable histopathological alterations were seen in the duodenum of the diabetic animals and there were likewise no significant changes in the production of peroxynitrite in their duodenum, whereas the level of the free radical scavenger metallothionein-2 was increased ∼300-fold. CONCLUSION: The spatially restricted vulnerability observed along the digestive tract could originate from a high level of oxidative stress via peroxynitrite production.

Alleviated mucosal and neuronal damage in a rat model of Crohn's disease.

AIM: To establish a rat model suitable to investigate the repetitive relapsing inflammations (RRI) characteristic to Crohn's disease. METHODS: Colitis was induced by 2,4,6-trinitrobenzenesulfonic acid (TNBS). RRI were mimicked by repeating administrations of TNBS. Tissue samples were taken from control, once, twice and three times treated rats from the inflamed and adjacent non-inflamed colonic segments at different timepoints during the acute intestinal inflammation. The means of the ulcerated area were measured to evaluate the macroscopic mucosal damage. The density of myenteric neurons was determined on whole mounts by HuC/HuD immunohistochemistry. Heme oxygenase-1 (HO-1) expression was evaluated by molecular biological techniques. RESULTS: TNBS-treated rats displayed severe colitis, but the mortality was negligible, and an increase of body weight was characteristic throughout the experimental period. The widespread loss of myenteric neurons, and marked but transient HO-1 up-regulation were demonstrated after the first TNBS administration. After repeated doses the length of the recovery time and extent of the ulcerous colonic segments were markedly decreased, and the neuronal loss was on a smaller scale and was limited to the inflamed area. HO-1 mRNA level was notably greater than after a single dose and overexpression was sustained throughout the timepoints examined. Nevertheless, the HO-1 protein up-regulation after the second TNBS treatment proved to be transient. Following the third treatment HO-1 protein expression could not be detected. CONCLUSION: Experimentally provoked RRI may exert a protective preconditioning effect against the mucosal and neuronal damage. The persistent up-regulation of HO-1 mRNA expression may correlate with this.

Regionally distinct alterations in the composition of the gut microbiota in rats with streptozotocin-induced diabetes.

The aim of this study was to map the microbiota distribution along the gut and establish whether colon/faecal samples from diabetic rats adequately reflect the diabetic alterations in the microbiome. Streptozotocin-treated rats were used to model type 1 diabetes mellitus (T1D). Segments of the duodenum, ileum and colon were dissected, and the microbiome of the lumen material was analysed by using next-generation DNA sequencing, from phylum to genus level. The intestinal luminal contents were compared between diabetic, insulin-treated diabetic and healthy control rats. No significant differences in bacterial composition were found in the luminal contents from the duodenum of the experimental animal groups, whereas distinct patterns were seen in the ileum and colon, depending on the history of the luminal samples. Ileal samples from diabetic rats exhibited particularly striking alterations, while the richness and diversity obscured some of the modifications in the colon. Characteristic rearrangements in microbiome composition and diversity were detected after insulin treatment, though the normal gut flora was not restored. The Proteobacteria displayed more pronounced shifts than those of the predominant phyla (Firmicutes and Bacteroidetes) in the rat model of T1D. Diabetes and insulin replacement affect the composition of the gut microbiota in different, gut region-specific manners. The luminal samples from the ileum appear more suitable for diagnostic purposes than the colon/faeces. The Proteobacteria should be at the focus of diagnosis and potential therapy. Klebsiella are recommended as biomarkers of T1D.

The transcriptome of lae1 mutants of Trichoderma reesei cultivated at constant growth rates reveals new targets of LAE1 function.

BACKGROUND: The putative methyltransferase LaeA is a global regulator that affects the expression of multiple secondary metabolite gene clusters in several fungi. In Trichoderma reesei, its ortholog LAE1 appears to predominantly regulate genes involved in increasing competitive fitness in its environment, including expression of cellulases and polysaccharide hydrolases. A drawback in all studies related to LaeA/LAE1 function so far, however, is that the respective loss-of-function and overexpressing mutants display different growth rates. Thus some of the properties attributed to LaeA/LAE1 could be simply due to changes of the growth rate. RESULTS: We cultivated T. reesei, a Δlae1 mutant and a lae1-overexpressing strain in chemostats on glucose at two different growth rates (0.075 and 0.020 h-1) which resemble growth rates at repressing and derepressing conditions, respectively. Under these conditions, the effect of modulating LAE1 expression was mainly visible in the Δlae1 mutant, whereas the overexpressing strain showed little differences to the parent strain. The effect on the expression of some gene categories identified earlier (polyketide synthases, heterokaryon incompatibility proteins, PTH11-receptors) was confirmed, but in addition GCN5-N-acetyltransferases, amino acid permeases and flavin monooxygenases were identified as so far unknown major targets of LAE1 action. LAE1 was also shown to interfere with the regulation of expression of several genes by the growth rate. About a tenth of the genes differentially expressed in the Δlae1 mutant under either growth condition were found to be clustered in the genome, but no specific gene group was associated with this phenomenon. CONCLUSIONS: Our data show that - using T. reesei LAE1 as a model - the investigation of transcriptome in regulatory mutants at constant growth rates leads to new insights into the physiological roles of the respective regulator.

Gut region-specific rearrangement of the cellular and subcellular compartments of nitric oxide synthase isoforms after chronic ethanol consumption in rats.

We recently provided evidence of cell-type-specific differences in the subcellular distributions of the three nitric oxide synthase (NOS) isoforms in the myenteric neurons, enteric smooth muscle cells and the capillary endothelium of the rat duodenum. We hypothesized that the presence of three NOS isoforms in the same type of cells with differences in subcellular compartmentalization might reflect a functional plasticity. Therefore, investigation of the possible rearrangement of cellular and subcellular NOS compartments in different gut segments following chronic ethanol treatment was the aim of this study. Rats were randomly divided into two groups and received water or 20% ethanol solution, preceded by short periods of adaptation with 10% and 15% ethanol. After 8 weeks, segments of duodenum, ileum and colon of the control and the alcohol-treated rats were processed for post-embedding immunohistochemistry and RT-PCR. The quantitative differences in the numbers of gold particles indicative of the different NOSs and their relative mRNA levels between the two experimental groups varied greatly, depending on the gut segment, and also on the cellular and subcellular compartments investigated. The chronic ethanol administration had the opposite effect on the quantitative distribution of the neuronal and endothelial NOS labelling gold particles in the different cellular compartments and resulted in subcellular rearrangement of NOS labels along the gastrointestinal tract. The intestinal region-specific rearrangement of the cellular and subcellular NOS compartments may possibly result in functional plasticity and help to maintain the optimum NO level under pathological conditions.

Spatiotemporal expression pattern of DsRedT3/CCK gene construct during postnatal development of myenteric plexus in transgenic mice.

Cholecystokinin (CCK) is an early marker of both neuronal and endocrine cell lineages in the developing gastrointestinal tract. To determine the quantitative properties and the spatial distribution of the CCK-expressing myenteric neurones in early postnatal life, a transgenic mouse strain with a CCK promoter-driven red fluorescent protein (DsRedT3/CCK) was established. The cell-specific expression of DsRedT3/CCK was validated by in situ hybridization with a CCK antisense riboprobe and by in situ hybridization coupled with immunohistochemistry involving a monoclonal antibody to CCK. A gradual increase in the DsRedT3/CCK-expressing enteric neurones with clear regional differences was documented from birth until the suckling to weaning transition, in parallel with the period of rapid intestinal growth and functional maturation. To evaluate the proportion of myenteric neurones in which DsRedT3/CCK transgene expression was colocalized with the enteric neuronal marker peripherin, immunofluorescence techniques were applied. All DsRedT3/CCK neurones were peripherin-immunoreactive and the proportion of DsRedT3/CCK-expressing myenteric neurones in the duodenum was the highest after the third week of life, when the number of peripherin-immunoreactive myenteric neurones in this region had decreased. Nearly all of the DsRedT3/CCK-expressing neurones also expressed 5-hydroxytryptophan (5-HT). Thus, by utilizing a new transgenic mouse strain, we have demonstrated a small number of CCK-expressing myenteric neurones with a developmentally regulated spatiotemporal distribution. The coexistence of CCK and 5-HT in the majority of these neurones suggests their possible regulatory role in feeding at the suckling to weaning transition.

Identification of a permease gene involved in lactose utilisation in Aspergillus nidulans.

Lactose is intracellularly hydrolysed by Aspergillus nidulans. Classical mutation mapping data and the physical characteristics of the previously purified glycosyl hydrolase facilitated identification of the clustered, divergently transcribed intracellular ß-galactosidase (bgaD) and lactose permease (lacpA) genes. At the transcript level, bgaD and lacpA were coordinately expressed in response to d-galactose, lactose or l-arabinose, while no transcription was detectable in the additional presence of glucose. In contrast, creA loss-of-function mutants derepressed for both genes to a considerable extent (even) under non-inducing or repressing growth conditions. Lactose- and d-galactose induction nevertheless occurred only in the absence of glucose, indicating a regulatory role for CreA-independent repression. Remarkably, bgaD deletion mutants grew normal on lactose. In contrast, lacpA deletants grew at a much slower rate in lactose liquid medium than wild-type while strains that carried more than one copy of lacpA grew faster, showing that transport is the limiting step in lactose catabolism. The effect of lacpA gene deletion on lactose uptake was exacerbated at lower substrate concentrations, evidence for the existence of a second transport system with a lower affinity for this disaccharide in A. nidulans.

Gut region-specific diabetic damage to the capillary endothelium adjacent to the myenteric plexus.

OBJECTIVE: Damage in the capillaries supplying the MP has been proposed as a critical factor in the development of diabetic enteric neuropathy. We therefore investigated connections between STZ-induced diabetes and the BM morphology, the size of caveolar compartments, the width of TJs, the transport of albumin, and the quantitative features of Cav-1 and eNOS expression in these microvessels. METHODS: Gut segments from diabetic rats were compared with those from insulin-treated diabetics and those from controls. The effects of diabetes on the BM, the caveolar compartments, and the TJs were evaluated morphometrically. The quantitative features of the albumin transport were investigated by postembedding immunohistochemistry. The diabetes-related changes in Cav-1 and eNOS expression were assessed by postembedding immunohistochemistry and molecular method. RESULTS: Thickening of the BM, enlargement of the caveolar compartments, opening of the junctions, enhanced transport of albumin, and overexpression of Cav-1 and eNOS were documented in diabetic animals. Insulin replacement in certain gut segments prevented the development of these alterations. CONCLUSIONS: These data provide morphological, functional, and molecular evidence that the endothelial cells in capillaries adjacent to the MP is a target of diabetic damage in a regional manner.

D-Galactose uptake is nonfunctional in the conidiospores of Aspergillus niger.

The majority of black Aspergilli (Aspergillus section Nigri), including Aspergillus niger, as well as many other Ascomycetes fail to germinate on d-galactose as a sole carbon source. Here, we provide evidence that the ability of A. niger to transport D-galactose is growth stage dependent, being absent in the conidiospores but present in the mycelia. Despite earlier claims, we could identify galactokinase activity in growing cells and all genes of the Leloir pathway (responsible for channelling D-galactose into the EMP pathway) are well induced on D-galactose (and also on lactose, D-xylose and L-arabinose) in the mycelial stage. Expression of all Leloir pathway genes was also detectable in conidiospores, although galE (encoding a galactokinase) and galD (encoding a galactose-1-phosphate uridylyl transferase) were expressed poorly. These results suggest that the D-galactose-negative phenotype of A. niger conidiospores may be due to the lack of inducer uptake.

Genetic diversity of a Botrytis cinerea cryptic species complex in Hungary.

Botrytis cinerea has been described as a species complex containing two cryptic species, referred to as groups I and II. The first B. cinerea group I strains outside of Western Europe were collected in Hungary in 2008 from strawberry and rape plants. Sympatric B. cinerea cryptic species were analyzed using a population genetic approach and phenotypic markers. Statistically significant, but moderate population differentiation was found between the two groups in Hungary. Group I was originally typified by the lack of the transposable elements Boty and Flipper. However, all the Hungarian group I isolates carried the Boty element and one isolate additionally contained Flipper, indicating a much wider genetic variation than previously believed. Vegetative compatibility analyses showed that twelve of the thirteen B. cinerea group I isolates studied belonged to a unique vegetative compatibility group (VCG), but VCGs overlapped between groups. Phenotypic markers such as fenhexamid resistance or asexual spore size were found unsuitable to differentiate between the cryptic species. The results did not confirm the complete separation of the two cryptic species, previously determined with genealogical concordance of the phylogenetic species recognition using multiple gene sequences, and suggest instead the possibility of information exchange between them.

Subcellular distribution of nitric oxide synthase isoforms in the rat duodenum.

AIM: To study the cell-type specific subcellular distribution of the three isoforms of nitric oxide synthase (NOS) in the rat duodenum. METHODS: Postembedding immunoelectronmicroscopy was performed, in which primary antibodies for neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS), were visualized with protein A-gold-conjugated secondary antibodies. Stained ultrathin sections were examined and photographed with a Philips CM10 electron microscope equipped with a MEGAVIEW II camera. The specificity of the immunoreaction in all cases was assessed by omitting the primary antibodies in the labeling protocol and incubating the sections only in the protein A-gold conjugated secondary antibodies. RESULTS: Postembedding immunoelectronmicroscopy revealed the presence of nNOS, eNOS, and iNOS immunoreactivity in the myenteric neurons, the enteric smooth muscle cells, and the endothelium of capillaries running in the vicinity of the myenteric plexus of the rat duodenum. The cell type-specific distributions of the immunogold particles labeling the three different NOS isozymes were revealed. In the control experiments, in which the primary antiserum was omitted, virtually no postembedding gold particles were observed. CONCLUSION: This postembedding immunoelectronmicroscopic study provided the first evidence of cell-type-specific differences in the subcellular distributions of NOS isoforms.