Prenatal SARS-CoV-2 Spike Protein Exposure Induces Autism-Like Neurobehavioral Changes in Male Neonatal Rats.

Recent research on placental, embryo, and brain organoids suggests that the COVID-19 virus may potentially affect embryonic organs, including the brain. Given the established link between SARS-CoV-2 spike protein and neuroinflammation, we sought to investigate the effects of exposure to this protein during pregnancy. We divided pregnant rats into three groups: Group 1 received a 1 ml/kg saline solution, Group 2 received 150 µg/kg adjuvant aluminum hydroxide (AAH), and Group 3 received 40 µg/kg spike protein + 150 µg/kg AAH at 10 and 14 days of gestation. On postnatal day 21 (P21), we randomly separated 60 littermates (10 male-female) into control, AAH-exposed, and spike protein-exposed groups. At P50, we conducted behavioral analyses on these mature animals and performed MR spectroscopy. Subsequently, all animals were sacrificed, and their brains were subject to biochemical and histological analysis. Our findings indicate that male rats exposed to the spike protein displayed a higher rate of impaired performance on behavioral studies, including the three-chamber social test, passive avoidance learning analysis, open field test, rotarod test, and novelty-induced cultivation behavior, indicative of autistic symptoms. Exposure to the spike protein (male) induced gliosis and neuronal cell death in the CA1-CA3 regions of the hippocampus and cerebellum. The spike protein-exposed male rats exhibited significantly greater levels of malondialdehyde (MDA), tumor necrosis factor alpha (TNF-α), interleukin-17 (IL-17), nuclear factor kappa B (NF-κB), and lactate and lower levels of brain-derived neurotrophic factor (BDNF) than the control group. Our study suggests a potential association between prenatal exposure to COVID-19 spike protein and neurodevelopmental problems, such as ASD. These findings highlight the importance of further research into the potential effects of the COVID-19 virus on embryonic and fetal development and the potential long-term consequences for neurodevelopment.

Evaluation of Atherosclerotic Risk by Oxidative Contributors in Alcohol Use Disorder.

Objective: Alcohol Use Disorder (AUD) is a condition described as the inability to control or stop alcohol consumption. The patients with AUD have an increased risk of developing atherosclerosis-related diseases. The present study aimed to evaluate oxidative contributors of atherosclerotic risk factors in patients with AUD. Methods: The male subjects diagnosed with AUD (n = 45) and the male subjects as control (n = 35) were enrolled in this study. All participants were undergone psychiatric evaluation and sociodemographic tests. Also, serum oxidative contributors of atherosclerosis including myeloperoxidase (MPO), ferroxidase, catalase (CAT), and lipid hydroperoxides (LOOH) were measured. Additionally, serum lipid profile tests and atherogenic indicators including atherogenic index of plasma (AIP) and non-high-density lipoprotein (HDL) cholesterol were also analyzed. Results: The AUD subject had significantly elevated MPO activity and LOOH levels with decreased antioxidant capacity. AIP and non-HDL cholesterol levels, the atherogenic indicators, were also higher in AUD group compared to the control group. We found the MPO activity and LOOH levels were positively correlated with AIP, non-HDL cholesterol levels, and amount of alcohol consumption. Additionally, CAT activity was negatively correlated with duration of alcohol consumption. Conclusion: Our results revealed that MPO and LOOH levels were elevated by severe alcohol intake and the atherogenic indicators, AIP and non-HDL cholesterol, were significantly correlated alcohol induced elevated oxidative risk factors. Therefore, it can be suggested that MPO activity and LOOH levels may be useful to determine jeopardy of atherosclerotic and the therapeutic interventions that reduce oxidative load could be taken into account to prevent atherosclerotic diseases before clinical manifestation.

An Investigation into the Protective Effects of Various Doses of Boric Acid on Liver, Kidney, and Brain Tissue Damage Caused by High Levels of Acute Alcohol Consumption.

Acute high-dose alcohol consumption can lead to oxidative stress, which can cause harm to organs. In this study we aim to determine whether administering boric acid (BA) can protect certain organs (liver, kidney, and brain) from the damaging effects of alcohol by reducing oxidative stress. We used 50 and 100 mg/kg of BA. Thirty-two Sprague Dawley (12-14-week-old) male rats in our study were separated into four groups (n=8); control, ethanol, ethanol+50 mg/kg BA, and ethanol+100 mg/kg BA groups. Acute ethanol was given to rats by gavage at 8 g/kg. BA doses were given by gavage 30 min before ethanol administration. Alanine transaminase (ALT) and aspartate transaminase (AST) measurements were made in blood samples. The total antioxidant status (TAS), total oxidant status (TOS), OSI (oxidative stress index) (TOS/TAS), malondialdehyde (MDA) levels and superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) activities were measured to determine the oxidative stress induced by high-dose acute ethanol in the liver, kidney, and brain tissue, and the antioxidant effects of BA doses. According to our biochemical results, acute high-dose ethanol increases oxidative stress in liver, kidney, and brain tissues, while BA reduces the damage in tissues with its antioxidant effect. For the histopathological examinations, hematoxylin-eosin staining was performed. As a result, we found that the effect of alcohol-induced oxidative stress on liver, kidney, and brain tissues was different, and that giving boric acid reduces the increased oxidative stress in tissues due to its antioxidant effect. It was found that 100mg/kg BA administration had a higher antioxidant effect than in the 50mg/kg group.

Modafinil Improves Autism-like Behavior in Rats by Reducing Neuroinflammation.

To evaluate the ameliorating effect of Modafinil on neuroinflammation, behavioral, and histopathological alterations in rats induced by propionic acid (PPA). Thirty male Wistar rats were used in the study, divided into 3 groups of ten subjects. One group served as a control, the subjects in the other two were given 250 mg/kg/day of PPA by intraperitoneal injection over the course of 5 days to induce autism. The experimental design was as follows: Group 1: Normal control (orally-fed control, n = 10); Group 2 (PPA + saline, n = 10): PPA and 1 ml/kg/day % 0.9 NaCl saline via oral gavage; Group 3 (PPA + Modafinil, n = 10) PPA and 30 mg/kg/day Modafinil (Modiodal tablets 100 mg, Cephalon) via oral gavage. All of the groups were investigated for behavioral, biochemical, and histological abnormality. Autism-like behaviors were reduced significantly in the rats treated with PPA. TNF-α, Nerve Growth Factor (NGF), IL-17, IL-2, and NF-KB levels as well as MDA levels and lactate were significantly higher in those treated with PPA compared to the control group. Using immunohistochemical methods, the number of neurons and GFAP immunoreactivity was significantly altered in PPA-treated rats compared to the control. Using Magnetic Resonance Spectroscopy (MRS), we found that lactate levels were significantly higher in the PPA-treated rats, while creatinine levels were significantly decreased. In the rats administered with Modafinil, behavior, neuroinflammation, and histopathological changes brought about by PPA were significantly reversed. Our results demonstrate the potential role of Modafinil in ameliorating PPA-induced neuroinflammation in rats.

Erythrocytic Reduced/Oxidized Glutathione and Serum Thiol/Disulfide Homeostasis in Patients with Opioid Use Disorder.

Background: This study aimed to evaluate oxidative damage by measuring erythrocytic reduced/oxidized glutathione as an intracellular thiol pool and serum thiol/disulfide homeostasis as an extracellular thiol pool in patients with opioid use disorder. Methods: In this prospective cross-sectional study, 33 male patients diagnosed with opioid use disorder and 30 healthy male controls were included. Sociodemographic characteristics and psychometric analyzes were performed and addiction characteristics (duration and amount of heroin use, usage methods) were recorded. For the evaluation of oxidative balance, intracellular reduced-oxidized glutathione (reduced glutathione and oxidized glutathione), and extracellular thiol-disulfide (native thiol and disulfide) levels were measured. Results: There was a decrease in reduced glutathione and native thiol levels and an increase in GSSG and SS levels. Similarly, while oxidized/reduced glutathione, oxidized/total glutathione%, and disulfide/native thiol % ratios increased, the ratio of reduced glutathione/total glutathione% and native thiol/total thiol% decreased. Moreover, a positive correlation was found between the level of both intracellular and extracellular oxidant molecules and the duration and amount of opioid use. Conclusion: Impaired intracellular reduced glutathione/oxidized glutathione and extracellular disulfide/native thiol homeostasis were found in patients with opioid use disorder. The intracellular and extracellular oxidative stress may cause complications related to chronic opioid use.

Vitamins C and E protect from sepsis-induced lung damage in rat and CT correlation.

BACKGROUND: Sepsis is one of the leading causes of death in intensive care units worldwide. Vitamins C and E are natural antioxidants and anti-inflammatory agents. Suppressing the inflammation is an important treatment target because it plays a role in the pathophysiology of sepsis. The purpose of this study was to investigate the effect of vitamins C and E treatment in rats with sepsis-induced lung damage. METHODS: In this animal study, fecal intraperitoneal injection procedure (FIP) was performed on 30 of 40 rats included for creating a sepsis model. Rats were randomly assigned into four groups: Group 1, control group (no procedure was applied, n = 10), Group 2, FIP (untreated septic group n = 10), Group 3, FIP+vitC (treated with 500 mg/kg/day ascorbic acid, n = 10), and Group 4, FIP+vitE (treated with 300 mg/kg/day alpha-tocopherol, n = 10). Chest CT was performed in all rats and density of the lungs was measured by using Hounsfield unit (HU). Histopathological examination of lung damage was performed, and blood samples were collected for biochemical analysis. RESULTS: TNF-α, CRP, IL 1-ß, IL-6, and MDA plasma levels in groups treated with vitamin C or vitamin E were lower than in the FIP group. Histological scores in groups treated either with vitamin C or vitamin E were significantly lower as compared to those in the FIP group. The HU value of lung in groups treated wither with vitamin C or vitamin E were lower than that in the FIP group (p < 0.05). CONCLUSION: The rats treated either with vitamin C or E showed improved results for sepsis. We think that they can be used as adjuvant therapy for septic patients because of their effectivity and low costs (Tab. 3, Fig. 2, Ref. 27).

Urethral monopolar cauterization: alternative infravesical obstruction model in male rats.

OBJECTIVE: We aimed to determine which method gives the most consistent results between urethral monopolar cauterization and standard urethral partial ligation methods for the urethral obstruction model. METHODS: Thirty male rats were randomly divided into control, partial ligation, and monopolar cauterization groups. Six weeks after experimental procedures, the experimental groups were evaluated cystometrically, biochemically, and histologically. RESULTS: According to the cystometric results, bladder capacity, baseline bladder pressure, and compliance data of the monopolar cauterization group were higher than those of the partial ligation and monopolar cauterization groups (p<0.05 and p<0.01, respectively). As a biochemical evaluation, malondialdehyde levels in bladder tissues of group control were higher than partial ligation and monopolar cauterization groups (p<0.05 and p<0.01, respectively). The collagen type I level of the control group was higher than the partial ligation and monopolar cauterization groups (p<0.01 and p<0.05, respectively). Collagen type III levels of the monopolar cauterization group were higher than those of the control group (p<0.01), but the Collagen type I/Collagen type III and transforming growth factor-ß levels of the monopolar cauterization group were significantly lower than those of the control group (p<0.001). As a histological evaluation (hematoxylin and eosin), fibrosis in the lamina propria was more prominent in the monopolar cauterization group than in the control group (p<0.05). In addition, the muscular thickness was higher in the monopolar cauterization group compared with control and partial ligation groups (p<0.001 and p<0.01, respectively). CONCLUSION: The needle-tipped monopolar cauterization of the posterior urethra may be the method of choice for creating a chronic infravesical obstruction model of infravesical obstruction in male rats.

Urethral monopolar cauterization: alternative infravesical obstruction model in male rats

SUMMARY OBJECTIVE: We aimed to determine which method gives the most consistent results between urethral monopolar cauterization and standard urethral partial ligation methods for the urethral obstruction model. METHODS: Thirty male rats were randomly divided into control, partial ligation, and monopolar cauterization groups. Six weeks after experimental procedures, the experimental groups were evaluated cystometrically, biochemically, and histologically. RESULTS: According to the cystometric results, bladder capacity, baseline bladder pressure, and compliance data of the monopolar cauterization group were higher than those of the partial ligation and monopolar cauterization groups (p<0.05 and p<0.01, respectively). As a biochemical evaluation, malondialdehyde levels in bladder tissues of group control were higher than partial ligation and monopolar cauterization groups (p<0.05 and p<0.01, respectively). The collagen type I level of the control group was higher than the partial ligation and monopolar cauterization groups (p<0.01 and p<0.05, respectively). Collagen type III levels of the monopolar cauterization group were higher than those of the control group (p<0.01), but the Collagen type I/Collagen type III and transforming growth factor-β levels of the monopolar cauterization group were significantly lower than those of the control group (p<0.001). As a histological evaluation (hematoxylin and eosin), fibrosis in the lamina propria was more prominent in the monopolar cauterization group than in the control group (p<0.05). In addition, the muscular thickness was higher in the monopolar cauterization group compared with control and partial ligation groups (p<0.001 and p<0.01, respectively). CONCLUSION: The needle-tipped monopolar cauterization of the posterior urethra may be the method of choice for creating a chronic infravesical obstruction model of infravesical obstruction in male rats.

Demonstration of ameliorating effect of vardenafil through its anti-inflammatory and neuroprotective properties in autism spectrum disorder induced by propionic acid on rat model.

INTRODUCTION: Autism spectrum disorder (ASD) is a neurodevelopmental disorder with complex etiology. In this study, we aimed to determine the ameliorating effects of vardenafil in the ASD rat model induced by propionic acid (PPA) in terms of neurobehavioral changes and also support these effects with histopathological changes, brain biochemical analysis and magnetic resonance spectroscopy (MRS) findings. MATERIALS AND METHODS: Twenty-one male rats were randomly assigned into three groups. Group 1 (control, 7 rats) did not receive treatment. Rats in groups 2 and 3 were given PPA at the dose of 250 mg/kg/day intraperitoneally for 5 days. After PPA administration, animals in group 2 (PPAS, 7 rats) were given saline and animals in group 3 (PPAV, 7 rats) were given vardenafil. Behavioral tests were performed between the 20th and 24th days of the study. The rats were taken for MRS on the 25th day. At the end of the study, brain levels of interleukin-2 (IL-2), IL-17, tumor necrosis factor-α, nerve growth factor, cGMP and lactate levels were measured. In the cerebellum and the CA1 and CA3 regions of the hippocampus, counts of neurons and Purkinje cells and glial fibrillary acidic protein (associated with gliosis) were evaluated histologically. RESULTS: Three chamber sociability and passive avoiding test, histopathological results, lactate levels derived from MRS, and biochemical biomarkers revealed significant differences among the PPAV and PPAS groups. CONCLUSION: We concluded that vardenafil improves memory and social behaviors and prevent loss of neuronal and Purkinje cell through its anti-inflammatory and neuroprotective effect.

Anti-Inflammatory Effects of Melatonin in Rats with Induced Type 2 Diabetes Mellitus.

INTRODUCTION: Insulin resistance is associated with a pro-inflammatory state increasing the risk for complications in patients with type 2 diabetes mellitus (T2DM). In addition to its chronobiotic effects, the pineal hormone melatonin is known to exert anti-inflammatory and antioxidant effects. Melatonin was also suggested to affect insulin secretion. The aim of this study was therefore to investigate the effect of melatonin on inflammation in diabetic rats and to study the possible involvement of the melatonin receptor, MT2. MATERIALS AND METHODS: Male Sprague Dawley rats were randomly divided into four experimental groups (n = 10 per group): (1) control, (2) streptozotocin/nicotinamide induced diabetes type 2 (T2DM), (3) T2DM treated with melatonin (500 µg/kg/day), and (4) T2DM treated with melatonin (500 µg/kg/day for 6 weeks) and the selective MT2 receptor antagonist luzindole (0.25 g/kg/day for 6 weeks). Blood samples were taken for biochemical parameters and various tissue samples (liver, adipose tissue, brain) were removed for immunohistochemistry (IHC), Western blot (WB), and Q-PCR analyses, respectively. RESULTS: Melatonin significantly reduced increased blood levels of liver transaminases (AST, ALT), blood urea nitrogen (BUN), triglyceride, very low-density lipoprotein (VLDL), and cholesterol in diabetic rats with luzindole treatment partly reversing this effect regarding the lipids. Furthermore, the liver and adipose tissues of T2DM rats treated with melatonin showed lower expression of the inflammatory markers IL-1ß, IL-6, TNF-α, and NF-κB as compared to the T2DM group without melatonin. The results also showed that the MT2 receptor is at least partly involved in the protective effects of melatonin. CONCLUSIONS: Our results suggest that melatonin exerts relevant anti-inflammatory effects on various tissues in type 2 diabetic rats.

The Comparative Effects of Inulin and Bacillus clausii on LPS-Induced Endotoxemic Rat Liver.

BACKGROUND: This paper sought to investigate the modifies of inulin and Bacillus clausii on the lipopolysaccharides (LPS) inducing oxidative stress signaling pathway in the endotoxemic rat model. METHODS: Wistar albino male rats (n = 36), divided into six groups, were formed randomly in the following stages: the control group; the prebiotic group (Inulin; 500 mg/kg); the probiotic group (Bacillus clausii; 1x109 CFU); the LPS group (1.5 mg/kg) as the endotoxemic model; the prebiotic group + LPS; and the probiotic group + LPS as treatment groups. RESULTS: The reactive oxygen species (ROS), advanced oxidation products of protein (AOPP), thiobarbituric acid reactive substances (TBARS), total oxidant status (TOS), oxidative stress index (OSI), and myeloperoxidase activity (MPO) levels increased in LPS-induced toxicity. Prebiotic treatment decreased LPS-induced hepatotoxicity on rat liver as observed in the decrease in the levels of oxidative stress parameters, such as ROS, TBARS, TOS, and OSI. The effect of the probiotic treatment on the ROS, AOPP, TOS, OSI levels was not statistically significant. However, it was determined that probiotic application was effective in the TBARS, TAS, and GSH levels. When the biochemical results of the prebiotic and probiotic treatment applications were compared, it was found that the prebiotic treatment was more effective on oxidative stress parameters (ROS, TBARS, TOS, and OSI). In addition, the histological damage score and MPO-staining results of the prebiotic treatment group were found to be more effective than the probiotic group. CONCLUSION: In this first study, where inulin and Bacillus clausii spores are used against liver damage caused by LPS, inulin provides much more effective protection than Bacillus clausii spores.

Effect of chronic alcohol consumption on myocardial apoptosis in the rat model of isoproterenol-induced myocardial injury and investigation on the cardioprotective role of calpain inhibitor 1.

We investigated the presence of myocardial apoptosis on isoproterenol (ISO)-induced myocardial injury (MI) after long-term high dose alcohol consumption and examined the antiapoptotic role of calpain inhibitor 1. Male Wistar Albino rats (n = 108) were divided into six groups: Control, alcohol (ethanol was given during 30 days for chronic alcohol consumption), MI (150 mg/kg ISO injection at last two days of alcohol consumption), alcohol + MI, alcohol + MI + calpain inhibitor 1 (10 mg/kg inhibitor was injected at 15 min before ISO injections) and Dimethyl Sulfoxide (DMSO) groups. Biochemical, histological, and morphometric methods determined apoptosis levels in the heart tissue of rats. Cytochrome c, caspase 3, and calpain levels were significantly high in alcohol, MI, and alcohol + MI groups. In contrast, mitochondrial cardiolipin content was found to be low in alcohol, MI, and alcohol + MI groups. These parameters were close to the control group in the therapy group. Histological and morphometric data have supported biochemical results. As a result of our biochemical data, myocardial apoptosis was seen in the alcohol, MI, and especially alcohol after MI groups. Calpain inhibitor 1 reduced apoptotic cell death and prevented myocardial tissue injury in these groups. The efficiency of calpain inhibitor was very marked in MI after long-term high dose alcohol consumption.

Effect of fulvic acid on gastric mucosa damage caused by chronic water avoidance stress.

We investigated the antioxidant and anti-ulcerogenic effects of fulvic acid (FA) on oxidative damage caused by water avoidance stress (WAS) in rat gastrointestinal mucosa. Three experimental groups were established: control (C), chronic stress (CS), and chronic stress + FA (CS + FA). After WAS, a single dose of FA was administered for 10 days to the CS + FA group. Samples of the pyloric region of the stomach were stained with hematoxylin and eosin (H & E) and periodic acid-Schiff (PAS). Immunohistochemical staining was performed for inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS). Total antioxidant status (TAS), total oxidant status (TOS), oxidative stress index (OSI), superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) levels were measured biochemically. By light microscopy, we observed loss of gastric epithelial cells and greater polymorphonuclear cell migration into the mucosa in the CS group compared to the C group. We found intact epithelial cell structure and a thick superficial mucus layer in the CS + FA group compared to the CS group. These findings in the CS + FA group were similar to those for group C. iNOS staining was stronger in the CS group compared to the C group. TOS and OSI levels in the CS + FA group were decreased compared to the CS group, but TAS, SOD, GPx and CAT levels were increased. We found that WAS caused damage to epithelium and connective tissue of the stomach mucosa and that this damage was prevented by FA. Therefore, administration of FA appears to prevent stress induced damage to rat stomach.

Dietary curcumin and capsaicin: Relationship with hepatic oxidative stress and apoptosis in rats fed a high fat diet.

BACKGROUND: Apoptosis plays a major role in fatty liver disease. High-fat diets are related to the onset of fatty liver disease and hepatic oxidant-antioxidant imbalance. Curcumin and capsaicin are somewhat beneficial in reducing hepatic triglycerides; this is most likely because they are known to downregulate reactive oxygen species (ROS) and apoptosis. OBJECTIVES: The aim of this study was to investigate the effects of curcumin and capsaicin on apoptosis through the oxidative effect in an animal model of fatty liver disease. MATERIAL AND METHODS: Male Sprague Dawley rats were fed a normal control diet, a high-fat diet (HFD; 60% of total calories from fat), a HFD+curcumin (1.5 g curcumin/kg HFD), a HFD+capsaicin (0.15 g capsaicin/kg HFD), or a HFD+curcumin+capsaicin (1.5 g curcumin and 0.15 g capsaicin/kg HFD). Liver lysate levels of BAX, Bcl-2 and caspase-3 were determined via immunoblotting. Caspase-3 activity was measured with a colorimetric caspase-3 measurement kit. Total antioxidant status (TAS) and total oxidant status (TOS) were assayed using commercial kits. The generation of hepatic ROS was measured with fluorimetry. Fragmentation of DNA was detected using the TUNEL method. RESULTS: High-fat diet caused increased expression of BAX and caspase-3, as well as increased TOS and caspase-3 activity, but decreased expression of Bcl-2. HFD+curcumin+capsaicin caused decreased BAX, caspase-3, TOS, and ROS levels as compared to HFD, but increased TAS and Bcl-2. A HFD +curcumin+capsaicin also decreased the number of TUNEL-positive cells. CONCLUSIONS: These results suggest that supplementation with curcumin and capsaicin balances the hepatic oxidant-antioxidant status and may have a protective role in the apoptotic process in an HFD-induced fatty liver model.

The antioxidant and antiapoptotic effect of boric acid on hepatoxicity in chronic alcohol-fed rats.

The harmful use of alcohol is a worldwide problem involving all ages. This study aims to investigate chronic alcohol exposure related hepatotoxicity on the rat liver and possible hepatoprotective effects of boric acid. Rats were separated into 4 different groups: control, ethanol, ethanol+boric acid, and boric acid. We measured (i) malondialdehyde (MDA), total sialic acid (TSA), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) levels, which are known to be the markers of alcohol damage; and also (ii) caspase-3, tumor necrosis factor-alpha (TNF-α), and the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) as the markers of apoptosis. In the ethanol group, MDA, TSA, and TNF-α levels increased whereas SOD and CAT levels decreased compared with the control group. Ethanol+boric acid group MDA, TSA, caspase-3, and TNF-α levels decreased whereas SOD and CAT levels increased compared with the ethanol group. Using histopathological evaluation of light microscope images, immunohistochemical caspase-3 and TNF-α activity in the ethanol+boric acid group were shown to be decreased compared with that in the ethanol group. Our results revealed that ethanol is capable of triggering oxidative stress and apoptosis in the rat liver. We propose that boric acid is an effective compound in protecting the rat liver against ethanol.

Signal integration at the PI3K-p85-XBP1 hub endows coagulation protease activated protein C with insulin-like function.

Coagulation proteases have increasingly recognized functions beyond hemostasis and thrombosis. Disruption of activated protein C (aPC) or insulin signaling impair function of podocytes and ultimately cause dysfunction of the glomerular filtration barrier and diabetic kidney disease (DKD). We here show that insulin and aPC converge on a common spliced-X-box binding protein-1 (sXBP1) signaling pathway to maintain endoplasmic reticulum (ER) homeostasis. Analogous to insulin, physiological levels of aPC maintain ER proteostasis in DKD. Accordingly, genetically impaired protein C activation exacerbates maladaptive ER response, whereas genetic or pharmacological restoration of aPC maintains ER proteostasis in DKD models. Importantly, in mice with podocyte-specific deficiency of insulin receptor (INSR), aPC selectively restores the activity of the cytoprotective ER-transcription factor sXBP1 by temporally targeting INSR downstream signaling intermediates, the regulatory subunits of PI3Kinase, p85α and p85ß. Genome-wide mapping of condition-specific XBP1-transcriptional regulatory patterns confirmed that concordant unfolded protein response target genes are involved in maintenance of ER proteostasis by both insulin and aPC. Thus, aPC efficiently employs disengaged insulin signaling components to reconfigure ER signaling and restore proteostasis. These results identify ER reprogramming as a novel hormonelike function of coagulation proteases and demonstrate that targeting insulin signaling intermediates may be a feasible therapeutic approach ameliorating defective insulin signaling.

Prenatal alcohol-induced neuroapoptosis in rat brain cerebral cortex: protective effect of folic acid and betaine.

PURPOSE: Alcohol consumption in pregnancy may cause fetal alcohol syndrome (FAS) in the infant. This study aims to investigate prenatal alcohol exposure related neuroapoptosis on the cerebral cortex tissues of newborn rats and possible neuroprotective effects of betaine, folic acid, and combined therapy. METHODS: Pregnant rats were divided into five experimental groups: control, ethanol, ethanol + betaine, ethanol + folic acid, and ethanol + betaine + folic acid combined therapy groups. We measured cytochrome c release, caspase-3, calpain and cathepsin B and L. enzyme activities. In order to observe apoptotic cells in the early stages, TUNEL method was chosen together with histologic methods such as assessing the diameters of the apoptotic cells, their distribution in unit volume and volume proportion of cortical intact neuron nuclei. RESULTS: Calpain, caspase-3 activities, and cytochrome c levels were significantly increased in alcohol group while cathepsin B and L. activities were also found to be elevated albeit not statistically significant. These increases were significantly reversed by folic acid and betaine + folic acid treatments. While ethanol increased the number of apoptotic cells, this increase was prevented in ethanol + betaine and ethanol + betaine + folic acid groups. Morphometric examination showed that the mean diameter of apoptotic cells was increased with ethanol administration while this increase was reduced by betaine and betaine + folic acid treatments. CONCLUSION: We observed that ethanol is capable of triggering apoptotic cell death in the newborn rat brains. Furthermore, folic acid, betaine, and combined therapy of these supplements may reduce neuroapoptosis related to prenatal alcohol consumption, and might be effective on preventing fetal alcohol syndrome in infants.

Effect of boric acid on oxidative stress in rats with fetal alcohol syndrome.

To the best of our knowledge, this is the first study concerning the effect of boric acid (BA) administration on fetal alcohol syndrome (FAS). In this study, the aim was to investigate prenatal alcohol-induced oxidative stress on the cerebral cortex of newborn rat pups and assess the protective and beneficial effects of BA supplementation on rats with FAS. Pregnant rats were divided into three groups, namely the control, alcohol and alcohol + boric acid groups. As markers of alcohol-induced oxidative stress in the cerebral cortex of the newborn pups, malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) levels were measured. Although the MDA levels in the alcohol group were significantly increased compared with those in the control group (P<0.05), the MDA level in the alcohol + boric acid group was shown to be significantly decreased compared with that in the alcohol group (P<0.01). The CAT activity of the alcohol + boric acid group was significantly higher than that in the alcohol group (P<0.05). The GPx activity in the alcohol group was decreased compared with that in the control group (P<0.05). These results demonstrate that alcohol is capable of triggering damage to membranes of the cerebral cortex of rat pups and BA could be influential in antioxidant mechanisms against oxidative stress resulting from prenatal alcohol exposure.

Biological assays on the effects of Acra3 peptide from Turkish scorpion Androctonus crassicauda venom on a mouse brain tumor cell line (BC3H1) and production of specific monoclonal antibodies.

Constitutes of the venom scorpion are a rich source of low molecular mass peptides which are toxic to various organisms, including man. Androctonus crassicauda is one of the scorpions from the Southeastern Anatolia of Turkey with public health importance. This work is focused on the investigation of biological effects of Acra3 peptide from Androctonus crassicauda. For this purpose, Acra3 isolated from crude venoms was tested for its cytotoxicity on BC3H1 mouse brain tumor cells using tetrazolium salt cleavage and lactate dehydrogenase activity assays. To determine whether the cytotoxic effects of Acra3 was related to the induction of apoptosis, the morphology of the cells and the nuclear fragmentation was examined by using Acridin Orange staining and DNA fragmentation assay, respectively. Caspase 3 and caspase 9 activities were measured spectrophotometrically and flow cytometric assay was performed using Annexin-V FITC and Propidium Iodide staining. Furthermore toxic peptide Acra3 was used as an antigen for immunological studies. Results showed that Acra3 exerted very strong cytotoxic effect on BC3H1 cells with an IC50 value of 5 µg/ml. Exposure of the cells to 0.1 and 0.5 µg/ml was resulted in very strong appearance of the apoptotic morphology in a dose dependent manner. On the other side, not any DNA fragmentation was observed after treatment of the cells. Caspase 3 and 9 activities were slightly decreased with Acra3. Results from flow cytometry and lactate dehydrogenase activity assays indicate that Acra3 exerts its effects by inducing a stronger necrosis than apoptosis in BC3H1 cells. To evaluate its immunogenicity, monoclonal antibody (MAb) specific for Acra3 antigen (5B9) was developed by hybridoma technology using spleen and lymph nodes of mice and immunoglobulin type of antibody was found to be IgM. We suggest that Acra3 may exert its effects by inducing both necrotic and apoptotic pathway in some way on mouse brain tumor cells. These findings will be useful for understanding the mechanism of cell death caused by venom in vitro. Anti-Acra3 monoclonal antibody can be further used as a bioactive tools for exploring the structure/function relationship and the pharmacological mechanism of scorpion peptide neurotoxins.

Monoclonal antibodies specific for hepatitis B e antigen and hepatitis B core antigen.

Despite effective vaccination programs in many countries, HBV infection is still a serious health problem throughout the world; more than 2 billion people have been infected with Hepatitis B virus (HBV). The serologic markers are crucial indicators in clinical diagnosis of HBV infection. The persistent presence of anti-HBc is associated with chronicity, and anti-HBe is an indicator for active viral replication. In the present study, two different hybridoma clones, 12E5 and 16F8, secreting anti-HBeAg and anti-HBc antibody were developed using hybridoma technology. BALB/c mice were immunized with HBe antigen (HBeAg), and monoclonal antibodies were generated from the spleen and lymph nodes of mice. Immunoglobulin types of antibodies were found to be IgG2a and IgG1, respectively. Monoclonal antibodies (MAbs) were produced in large scale, purified with affinity chromatography, and epitope analysis was performed. The results have shown that 12E5 and 16F8 monoclonal antibodies can be used for detection of HBcAg and HBeAg, indicating that they have the potential for use in clinical diagnosis.