Plant and Animal-Type Feedstuff Shape the Gut Microbiota and Metabolic Processes of the Chinese Mitten Crab Eriocheir sinensis.

In animals, growth and development are strongly correlated with the gut microbiota and metabolic profiles. In this study, gut microbiome communities, metabolic profiles, and growth performance of Eriocheir sinensis under three dietary feed types based on waterweed plants only, freshwater snails only, and waterweed plants combined with freshwater snails were studied by using 16S rRNA gene sequencing and liquid chromatography-mass spectrometry. Results indicated that different feed types dramatically affected the growth performances of E. sinensis by altering the gut microbiota and metabolic profiles. Aquatic plants, such as waterweeds, played essential roles in shaping gut microbiome communities, and the optimal Bacteroides-to-Firmicutes ratio might strongly promote growth performance. Waterweed plants also helped decrease maleficent Proteobacteria caused by excess animal-type feedstuff, such as freshwater snails, and might have positive roles in antibacterial functions in gut. A diet based on waterweeds only resulted in lipid metabolism disorders, which significantly retarded the growth of E. sinensis. In summary, E. sinensis cultured with a diet of waterweeds and freshwater snails showed superior growth performance due to their healthy gut microbiota and metabolic homeostasis. Our findings unveiled the roles of aquatic plants and animal-type food such as freshwater snail in shaping the gut microbiota and metabolic processes and provided guidance for the aquaculture of E. sinensis in future.

Differential Expression of Genes Related to Innate Immune Responses in Ex Vivo Spinal Cord and Cerebellar Slice Cultures Infected with West Nile Virus.

West Nile virus (WNV) infection results in a spectrum of neurological symptoms, ranging from a benign fever to severe WNV neuroinvasive disease with high mortality. Many who recover from WNV neuroinvasive infection present with long-term deficits, including weakness, fatigue, and cognitive problems. While neurons are a main target of WNV, other cell types, especially astrocytes, play an important role in promoting WNV-mediated central nervous system (CNS) damage. Conversely, it has been shown that cultured primary astrocytes secrete high levels of interferons (IFNs) immediately after WNV exposure to protect neighboring astrocytes, as well as neurons. However, how intrinsic responses to WNV in specific cell types and different regions of the brain modify immune protection is not fully understood. Here, we used a mouse ex vivo spinal cord slice culture (SCSC) and cerebellar slice culture (CSC) models to determine the innate immune responses specific to the CNS during WNV infection. Slices were prepared from the spinal cord and cerebellar tissue of 7⁻9-day-old mouse pups. Four-day-old SCSC or CSC were infected with 1 × 10³ or 1 × 105 PFU of WNV, respectively. After 12 h exposure to WNV and 3 days post-infection in normal growth media, the pooled slice cultures were processed for total RNA extraction and for gene expression patterns using mouse Affymetrix arrays. The expression patterns of a number of genes were significantly altered between the mock- and WNV-treated groups, both in the CSCs and SCSCs. However, distinct differences were observed when CSC data were compared with SCSC. CSCs showed robust induction of interferons (IFNs), IFN-stimulated genes (ISGs), and regulatory factors. Some of the antiviral genes related to IFN were upregulated more than 25-fold in CSCs as compared to mock or SCSC. Though SCSCs had twice the number of dysregulated genes, as compared CSCs, they exhibited a much subdued IFN response. In addition, SCSCs showed astrogliosis and upregulation of astrocytic marker genes. In sum, our results suggest that early anti-inflammatory response to WNV infection in CSCs may be due to large population of distinct astrocytic cell types, and lack of those specialized astrocytes in SCSC may make spinal cord cells more susceptible to WNV damage. Further, the understanding of early intrinsic immune response events in WNV-infected ex vivo culture models could help develop potential therapies against WNV.

Testosterone enhances tubuloglomerular feedback by increasing superoxide production in the macula densa.

Males have higher prevalence of hypertension and renal injury than females, which may be attributed in part to androgen-mediated effects on renal hemodynamics. Tubuloglomerular feedback (TGF) is an important mechanism in control of renal microcirculation. The present study examines the role of testosterone in the regulation of TGF responses. TGF was measured by micropuncture (change of stop-flow pressure, ΔPsf) in castrated Sprague-Dawley rats. The addition of testosterone (10(-7) mol/l) into the lumen increased the ΔPsf from 10.1 ± 1.2 to 12.2 ± 1.2 mmHg. To determine whether androgen receptors (AR) are involved, mRNA of AR was measured in the macula dense cells isolated by laser capture microdissection from kidneys, and a macula densa-like cell line (MMDD1). AR mRNA was expressed in the macula densa of rats and in MMDD1 cells. We next examined the effects of the AR blocker, flutamide (10(-5) mol/l) on the TGF response. The addition of flutamide blocked the effects of testosterone on TGF. The addition of Tempol (10(-4) mol/l) or polyethylene glycol-superoxide dismutase (100 U/ml) to scavenge superoxide blocked the effect of testosterone to augment TGF. We then applied apocynin to inhibit NAD(P)H oxidase and oxypurinol to inhibit xanthine oxidase and found the testosterone-induced augmentation of TGF was blocked. In additional experiments in MMDD1 cells, we found that testosterone increased O2(-) generation. Apocynin or oxypurinol blocked the testosterone-induced increases of O2(-), while blockade of COX-2 with NS-398 had no effect. These findings suggest that testosterone enhances TGF response by stimulating O2(-) production in macula densa via an AR-dependent pathway.

Mechanistic role for a novel glucocorticoid-KLF11 (TIEG2) protein pathway in stress-induced monoamine oxidase A expression.

Chronic stress is a risk factor for psychiatric illnesses, including depressive disorders, and is characterized by increased blood glucocorticoids and brain monoamine oxidase A (MAO A, which degrades monoamine neurotransmitters). This study elucidates the relationship between stress-induced MAO A and the transcription factor Kruppel-like factor 11 (KLF11, also called TIEG2, a member of the Sp/KLF- family), which inhibits cell growth. We report that 1) a glucocorticoid (dexamethasone) increases KLF11 mRNA and protein levels in cultured neuronal cells; 2) overexpressing KLF11 increases levels of MAO A mRNA and enzymatic activity, which is further enhanced by glucocorticoids; in contrast, siRNA-mediated KLF11 knockdown reduces glucocorticoid-induced MAO A expression in cultured neurons; 3) induction of KLF11 and translocation of KLF11 from the cytoplasm to the nucleus are key regulatory mechanisms leading to increased MAO A catalytic activity and mRNA levels because of direct activation of the MAO A promoter via Sp/KLF-binding sites; 4) KLF11 knockout mice show reduced MAO A mRNA and catalytic activity in the brain cortex compared with wild-type mice; and 5) exposure to chronic social defeat stress induces blood glucocorticoids and activates the KLF11 pathway in the rat brain, which results in increased MAO A mRNA and enzymatic activity. Thus, this study reveals for the first time that KLF11 is an MAO A regulator and is produced in response to neuronal stress, which transcriptionally activates MAO A. The novel glucocorticoid-KLF11-MAO A pathway may play a crucial role in modulating distinct pathophysiological steps in stress-related disorders.

Aldosterone blunts tubuloglomerular feedback by activating macula densa mineralocorticoid receptors.

Chronic aldosterone administration increases glomerular filtration rate, whereas inhibition of mineralocorticoid receptors (MRs) markedly attenuates glomerular hyperfiltration and hypertension associated with primary aldosteronism or obesity. However, the mechanisms by which aldosterone alters glomerular filtration rate regulation are poorly understood. In the present study, we hypothesized that aldosterone suppresses tubuloglomerular feedback (TGF) via activation of macula densa MR. First, we observed the expression of MR in macula densa cells isolated by laser capture microdissection and by immunofluorescence in rat kidneys. Second, to investigate the effects of aldosterone on TGF in vitro, we microdissected the juxtaglomerular apparatus from rabbit kidneys and perfused the afferent arteriole and distal tubule simultaneously. Under control conditions, TGF was 2.8±0.2 µm. In the presence of aldosterone (10(-8) mol/L), TGF was reduced by 50%. The effect of aldosterone to attenuate TGF was blocked by the MR antagonist eplerenone (10(-5) mol/L). Third, to investigate the effect of aldosterone on TGF in vivo, we performed micropuncture, and TGF was determined by maximal changes in stop-flow pressure P(sf) when tubular perfusion rate was increased from 0 to 40 nL/min. Aldosterone (10(-7) mol/L) decreased ΔP(sf) from 10.1±1.4 to 7.7±1.2 mm Hg. In the presence of l-NG-monomethyl arginine citrate (10(-3) mol/L), this effect was blocked. We conclude that MRs are expressed in macula densa cells and can be activated by aldosterone, which increases nitric oxide production in the macula densa and blunts the TGF response.

Aldosterone stimulates superoxide production in macula densa cells.

Two major factors which regulate tubuloglomerular feedback (TGF)-mediated constriction of the afferent arteriole are release of superoxide (O(2)(-)) and nitric oxide (NO) by macula densa (MD) cells. MD O(2)(-) inactivates NO; however, among the factors that increase MD O(2)(-) release, the role of aldosterone is unclear. We hypothesize that aldosterone activates the mineralocorticoid receptor (MR) on MD cells, resulting in increased O(2)(-) production due to upregulation of cyclooxygenase-1 (COX-2) and NOX-2, and NOX-4, isoforms of NAD(P)H oxidase. Studies were performed on MMDD1 cells, a renal epithelial cell line with properties of MD cells. RT-PCR and Western blotting confirmed the expression of MR. Aldosterone (10(-8) mol/l for 30 min) doubled MMDD1 cell O(2)(-) production, and this was completely blocked by MR inhibition with 10(-5) mol/l eplerenone. RT-PCR, real-time PCR, and Western blotting demonstrated aldosterone-induced increases in COX-2, NOX-2, and NOX-4 expression. Inhibition of COX-2 (NS398), NADPH oxidase (apocynin), or a combination blocked aldosterone-induced O(2)(-) production to the same degree. These data suggest that aldosterone-stimulated MD O(2)(-) production is mediated by COX-2 and NADPH oxidase. Next, COX-2 small-interfering RNA (siRNA) specifically decreased COX-2 mRNA without affecting NOX-2 or NOX-4 mRNAs. In the presence of the COX-2 siRNA, the aldosterone-induced increases in COX-2, NOX-2, and NOX-4 mRNAs and O(2)(-) production were completely blocked, suggesting that COX-2 causes increased expression of NOX-2 and NOX-4. In conclusion 1) MD cells express MR; 2) aldosterone increases O(2)(-) production by activating MR; and 3) aldosterone stimulates COX-2, which further activates NOX-2 and NOX-4 and generates O(2)(-). The resulting balance between O(2)(-) and NO in the MD is important in modulating TGF.

Fem1b antigen in the stool of ApcMin mice as a biomarker of early Wnt signaling activation in intestinal neoplasia.

BACKGROUND: Colorectal cancer is preventable by early detection and removal of precursor lesions. Central to early stages of colorectal neoplasia is activation of Wnt signaling, usually due to inactivation of the Apc tumor suppressor gene for which there is an established animal model, the Apc(Min) mouse. Immunodetection in stool of proteins up-regulated by aberrant Wnt signaling, within intestinal epithelial cells shed into the lumen, could be a rational approach to identify biomarkers of early intestinal neoplasia. Fem1b gene expression is up-regulated, following inactivation of Apc, in mouse intestinal epithelium. METHODS: We initially screened pooled random stool samples by immunoblotting and found that we could detect, in Apc(Min) mice but not wild-type mice, a fragment of Fem1b protein with an antibody (Li-50) directed against an epitope near the middle of the protein, but not with antibodies directed against N-terminus or C-terminus epitopes. We then evaluated freshly voided individual stool samples collected on four consecutive days from four each of male and female Apc(Min) mice and their wild-type littermates. RESULTS: The Fem1b antigen was detected with the Li-50 antibody in 15/16 samples from male Apc(Min) mice compared to 0/16 samples from male wild-type mice, and in 5/16 samples from female Apc(Min) mice compared to 0/16 samples from female wild-type mice. CONCLUSIONS: This study provides proof-of-principle that fragments of proteins, whose expression is increased by aberrant Wnt signaling early in intestinal neoplasia, can be immunodetected in stool. Excreted Fem1b protein fragments may be a useful biomarker for epithelial Wnt signaling and early intestinal neoplasia.

Ethanol increases TIEG2-MAO B cell death cascade in the prefrontal cortex of ethanol-preferring rats.

Brain cell loss has been reported in subjects with alcoholism. However, the molecular mechanisms are unclear. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and monoamine oxidase B (MAO B) reportedly play a role in cellular dysfunction with regards to ethanol exposure. We have recently reported that GAPDH protein expression was increased in the brains of rats fed with ethanol. Furthermore, GAPDH interacts with the transcriptional activator, transforming growth factor-beta-inducible early gene 2 (TIEG2), to augment TIEG2-mediated MAO B activation, resulting in neuronal cell damage due to ethanol exposure. The current study investigates whether the TIEG2-MAO B cascade is also active in the brains of rats fed with ethanol. Ten ethanol-preferring rats were fed with a liquid diet containing ethanol, with increasing amounts of ethanol up to a final concentration of 6.4% representing a final diet containing 36% of calories for 28 days. Ten control rats were fed the liquid diet without ethanol. The expression of TIEG2 protein, MAO B mRNA levels, MAO B catalytic activity, and the levels of anti-apoptotic protein Bcl 2 and apoptotic protein caspase 3 were determined in the prefrontal cortex of the rats. Ethanol significantly increased protein levels of TIEG2, active caspase 3, MAO B mRNA and enzyme activity, but significantly decreased Bcl 2 protein expression compared to control rats. In summary, ethanol increases the TIEG2-MAO B brain cell death cascade in rat brains, suggesting that the TIEG2-MAO B pathway is a novel pathway for brain cell damage resulting from ethanol exposure, and may contribute to chronic alcohol-induced brain damage.

The New Inhibitor of Monoamine Oxidase, M30, has a Neuroprotective Effect Against Dexamethasone-Induced Brain Cell Apoptosis.

Stress detrimentally affects the brain and body and can lead to or be accompanied by depression. Although stress and depression may contribute to each other, the exact molecular mechanism underlying the effects is unclear. However, there is a correlation between stress and an increase in glucocorticoid secretion which causes a subsequent increase in monoamine oxidase (MAO) activity during stress. Consequently, MAO inhibitors have been used as traditional antidepressant drugs. Cellular treatment with the synthetic glucocorticoid, dexamethasone (a cellular stressor), has been reported to markedly increase both MAO A and MAO B catalytic activities, as well as apoptosis. This study compares the neuroprotective abilities of M30 (a new generation inhibitor of both MAO A and MAO B) with rasagiline (Azilect(®), another new MAO B inhibitor) and selegiline (Deprenyl(®), a traditional MAO B inhibitor) in the prevention of dexamethasone-induced brain cell death and MAO activity in human neuroblastoma cells, SH-SY5Y. M30 demonstrated the highest inhibitory effect on MAO A; however, M30 showed the lowest inhibitory effect on MAO B enzymatic activity in comparison to rasagiline and selegiline. Although, M30 exhibited the greatest neuroprotective effect by decreasing cell death rates and apoptotic DNA damage compared to rasagiline and selegiline, these neuroprotective effects of M30 were, overall, similar to rasagiline. Summarily, M30 has a generally greater impact on neuroprotection than the MAO B inhibitors, selegiline and rasagiline. Our results suggest that M30 may have great potential in alleviating disorders involving increases in both MAO A and MAO B, such as stress-induced disorders.

Salt-sensitive splice variant of nNOS expressed in the macula densa cells.

Neuronal nitric oxide synthase (nNOS), which is abundantly expressed in the macula densa cells, attenuates tubuloglomerular feedback (TGF). We hypothesize that splice variants of nNOS are expressed in the macula densa, and nNOS-beta is a salt-sensitive isoform that modulates TGF. Sprague-Dawley rats received a low-, normal-, or high-salt diet for 10 days and levels of the nNOS-alpha, nNOS-beta, and nNOS-gamma were measured in the macula densa cells isolated with laser capture microdissection. Three splice variants of nNOS, alpha-, beta-, and gamma-mRNAs, were detected in the macula densa cells. After 10 days of high-salt intake, nNOS-alpha decreased markedly, whereas nNOS-beta increased two- to threefold in the macula densa measured with real-time PCR and in the renal cortex measured with Western blot. NO production in the macula densa was measured in the perfused thick ascending limb with an intact macula densa plaque with a fluorescent dye DAF-FM. When the tubular perfusate was switched from 10 to 80 mM NaCl, a maneuver to induce TGF, NO production by the macula densa was increased by 38 +/- 3% in normal-salt rats and 52 +/- 6% (P < 0.05) in the high-salt group. We found 1) macula densa cells express nNOS-alpha, nNOS-beta, and nNOS-gamma, 2) a high-salt diet enhances nNOS-beta, and 3) TGF-induced NO generation from macula densa is enhanced in high-salt diet possibly from nNOS-beta. In conclusion, we found that the splice variants of nNOS expressed in macula densa cells were alpha-, beta-, and gamma-isoforms and propose that enhanced level of nNOS-beta during high-salt intake may contribute to macula densa NO production and help attenuate TGF.

NOX2 is the primary source of angiotensin II-induced superoxide in the macula densa.

Macula densa (MD)-mediated regulation of renal hemodynamics via tubuloglomerular feedback is regulated by interactions between factors such as superoxide (O(2)(-)) and angiotensin II (ANG II). We have reported that NaCl-induced O(2)(-) in the MD is produced by the NOX2 isoform of NADPH oxidase (NOX); however, the source of ANG II-induced O(2)(-) in MD is unknown. Thus we determined the pathways by which ANG II increased O(2)(-) in the MD by measuring O(2)(-) in ANG II-treated MMDD1 cells, a MD-like cell line. ANG II caused MMDD1 O(2)(-) levels to increase by more than twofold (P < 0.01). This increase was blocked by losartan (AT(1) receptor blocker) but not PD-123319 (AT(2) receptor antagonist). Apocynin (a NOX inhibitor) decreased O(2)(-) by 86% (P < 0.01), whereas oxypurinol (a xanthine oxidase inhibitor) and NS-398 (a cyclooxygenase-2 inhibitor) had no significant effect. The NOX-dependent increase in O(2)(-) was due to the NOX2 isoform; a short interfering (si)RNA against NOX2 blunted ANG II-induced increases in O(2)(-), whereas the NOX4/siRNA did not. Finally, we found that inhibiting the Rac1 subunit of NOX blunted ANG II-induced O(2)(-) production in NOX4/siRNA-treated cells but did not further decrease it in NOX2/siRNA-treated cells. Our results indicate that ANG II stimulates O(2)(-) production in the MD primarily via AT(1)-dependent activation of NOX2. Rac1 is required for the full activation of NOX2. This pathway may be an important component of ANG II enhancement of tubuloglomerular feedback.

A novel role for glyceraldehyde-3-phosphate dehydrogenase and monoamine oxidase B cascade in ethanol-induced cellular damage.

BACKGROUND: Alcoholism is a major psychiatric condition at least partly associated with ethanol (EtOH)-induced cell damage. Although brain cell loss has been reported in subjects with alcoholism, the molecular mechanism is unclear. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and monoamine oxidase B (MAO B) reportedly play a role in cellular dysfunction under stressful conditions and might contribute to EtOH-induced cell damage. METHODS: Expression of GAPDH and MAO B protein was studied in human glioblastoma and neuroblastoma cell lines exposed to physiological concentrations of EtOH. Expression of these proteins was also examined in the prefrontal cortex from human subjects with alcohol dependence and in rats fed with an EtOH diet. Coimmunoprecipitation, subcellular fractionation, and luciferase assay were used to address nuclear GAPDH-mediated MAO B activation. To test the effects of inactivation, RNA interference and pharmacological intervention were used, and cell damage was assessed by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP Nick End Labeling (TUNEL) and hydrogen peroxide measurements. RESULTS: Ethanol significantly increases levels of GAPDH, especially nuclear GAPDH, and MAO B in neuronal cells as well as in human and rat brains. Nuclear GAPDH interacts with the transcriptional activator, transforming growth factor-beta-inducible early gene 2 (TIEG2), and augments TIEG2-mediated MAO B transactivation, which results in cell damage in neuronal cells exposed to EtOH. Knockdown expression of GAPDH or treatment with MAO B inhibitors selegiline (deprenyl) and rasagiline (Azilect) can block this cascade. CONCLUSIONS: Ethanol-elicited nuclear GAPDH augments TIEG2-mediated MAO B, which might play a role in brain damage in subjects with alcoholism. Compounds that block this cascade are potential candidates for therapeutic strategies.

Glyceraldehyde-3-phosphate dehydrogenase-monoamine oxidase B-mediated cell death-induced by ethanol is prevented by rasagiline and 1-R-aminoindan.

The inhibitors of monoamine oxidase B (MAO B) are effectively used as therapeutic drugs for neuropsychiatric and neurodegenerative diseases. However, their mechanism of action is not clear, since the neuroprotective effect of MAO B inhibitors is associated with the blockage of glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-death cascade, rather than the inhibition of MAO B. Here, we provide evidence that GAPDH potentiates the ethanol-induced activity of MAO B and brain cell toxicity. The levels of nuclear GAPDH and MAO B activity are significantly increased in brain-derived cell lines upon 75 mM ethanol-induced cell death. Over-expression of GAPDH in cells enhances ethanol-induced cell death, and also increases the ethanol-induced activation of MAO B. In contrast, the MAO B inhibitors rasagiline and selegiline (0.25 nM) and the rasagiline metabolite, 1-R-aminoindan (1 muM) decreases the ethanol-induced MAO B, prevents nuclear translocation of GAPDH and reduces cell death. In addition, GAPDH interacts with transforming growth factor-beta-inducible early gene (TIEG2), a transcriptional activator for MAO B, and this interaction is increased in the nucleus by ethanol but reduced by MAO B inhibitors and 1-R-aminoindan. Furthermore, silencing TIEG2 using RNAi significantly reduces GAPDH-induced MAO B upregulation and neurotoxicity. In summary, ethanol-induced cell death, attenuated by MAO B inhibitors, may result from disrupting the movement of GAPDH with the transcriptional activator into the nucleus and secondly inhibit MAO B gene expression. Thus, the neuroprotective effects of rasagiline or 1-R-aminoindan on ethanol-induced cell death mediated by a novel GAPDH-MAO B pathway may provide a new insight in the treatment of neurobiological diseases including alcohol-use disorders.

Comparative neuroprotective effects of rasagiline and aminoindan with selegiline on dexamethasone-induced brain cell apoptosis.

Stress can affect the brain and lead to depression; however, the molecular pathogenesis is unclear. An association between stress and stress-induced hypersecretion of glucocorticoids occurs during stress. Dexamethasone (a synthetic glucocorticoid steroid) has been reported to induce apoptosis and increase the activity of monoamine oxidase (MAO) (Youdim et al. 1989). MAO is an enzyme for the degradation of aminergic neurotransmitters; dopamine, noradrenaline and serotonin and dietary amines and MAO inhibitors are classical antidepressant drugs. In this study, we have compared the ability of rasagiline (Azilect) and its main metabolite, R-aminoindan with selegiline (Deprenyl) in prevention of dexamethasone-induced brain cell death employing human neuroblastoma SH-SY5Y cells and glioblastoma 1242-MG cells. Dexamethasone reduced cell viability as measured by MTT test, but rasagiline, selegiline, and 1-R-aminoindan could significantly prevent dexamethasone-induced brain cell death. Among three drugs, rasagiline had the highest neuroprotective effect. Furthermore, the inhibitory effects of these drugs on MAO B catalytic activity and on apoptotic DNA damage (TUNEL staining) were examined. Rasagiline exhibited highest inhibition on MAO B enzymatic activity and prevention on DNA damage as compared to selegiline and 1-R-aminoindan. In summary, the greater neuroprotective effect of rasagiline may be associated with the combination of the parent drug and its metabolite 1-R-aminoindan.

The Neuroprotective Effect of Antidepressant Drug via Inhibition of TIEG2-MAO B Mediated Cell Death.

Alcohol use disorders are common in the world. However, the development of novel drugs to prevent alcohol-induced brain damage is based upon an improved neurobiological understanding on the cellular changes that take place in the brain. We previously reported that ethanol exposure lowered cell proliferation and increased cell apoptosis in all cell types, but affects brain cell lines the most, while ethanol and the anti-depressant drug deprenyl, an monoamine oxidase B (MAO B) inhibitor, exposure in unison increases cell viability. Here we investigated the molecular mechanism of the neuroprotective effect of deprenyl (0.25 nM) on ethanol (75 mM)-induced harmful effect. Transforming growth factor-beta-inducible early gene 2 (TIEG2) is an activator for MAO B. MAO B levels increase has been shown to contribute to neuronal cell death. This study uses the neuronal cell line to address whether ethanol induced cell death is through the activation of TIEG2-MAO B apoptotic pathway, and whether deprenyl protects cells from the effects of alcohol through the inhibition of this pathway. We have found that ethanol exposure increases the levels of mRNA and protein/catalytic activity for both TIEG2 and MAO B, while ethanol and deprenyl exposure in unison reduce the expression of both TIEG2 and MAO B, however it increases cell viability. Additionally, TIEG2-overexpressed cells display more cellular death-induced by ethanol than control cells. In summary, this study demonstrates the role of TIEG2 in ethanol induced cell death. The inhibition of the TIEG2-MAO B pathway may be one of the mechanisms for the neuroprotective effect of deprenyl.

Abnormal glucose homeostasis and pancreatic islet function in mice with inactivation of the Fem1b gene.

Type 2 diabetes mellitus is a disorder of glucose homeostasis involving complex gene and environmental interactions that are incompletely understood. Mammalian homologs of nematode sex determination genes have recently been implicated in glucose homeostasis and type 2 diabetes mellitus. These are the Hedgehog receptor Patched and Calpain-10, which have homology to the nematode tra-2 and tra-3 sex determination genes, respectively. Here, we have developed Fem1b knockout (Fem1b-KO) mice, with targeted inactivation of Fem1b, a homolog of the nematode fem-1 sex determination gene. We show that the Fem1b-KO mice display abnormal glucose tolerance and that this is due predominantly to defective glucose-stimulated insulin secretion. Arginine-stimulated insulin secretion is also affected. The Fem1b gene is expressed in pancreatic islets, within both beta cells and non-beta cells, and is highly expressed in INS-1E cells, a pancreatic beta-cell line. In conclusion, these data implicate Fem1b in pancreatic islet function and insulin secretion, strengthening evidence that a genetic pathway homologous to nematode sex determination may be involved in glucose homeostasis and suggesting novel genes and processes as potential candidates in the pathogenesis of diabetes mellitus.

FEM1A is a candidate gene for polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is the most common endocrine disorder among women of reproductive age, and is characterized by infertility, hyperandrogenism and insulin resistance in skeletal muscle. There is evidence for a PCOS gene localized to chromosome 19p13.3. The FEMIA gene maps to chromosome 19p13.3 and is highly expressed in skeletal muscle. FEMIA is a homolog of fem-1, a sex-determination gene of Caenorhabditis elegans that controls masculinization. In a pilot study of Caucasian PCOS patients from our local clinic, we found that one of these five patients exhibited a heterozygous germline missense mutation in FEM1A, designated FEM1A*H500Y. This mutation alters an amino acid conserved from human to C. elegans, and was not found in any of 198 control chromosomes. This missense allele was not found in any of a separate group of 30 PCOS patients from a different regional/ethnic background. Immunostaining of mouse ovary demonstrated that the mouse homolog of FEM1A is expressed in androgen-producing secondary interstitial cells, with a marked increase in expression after puberty, consistent with a key feature of PCOS -- ovarian hyperandrogenism. In conclusion, FEM1A should be considered a candidate gene for PCOS, and more extensive analysis of FEM1A, both coding and regulatory sequences, is warranted in patients and families with PCOS.

The Fem1c genes: conserved members of the Fem1 gene family in vertebrates.

The fem-1 gene of Caenorhabditis elegans functions in a signaling pathway that controls sex determination. Homologs of fem-1 in mammals have been characterized, consisting of two family members, Fem1a and Fem1b. We report here on Fem1c, a third member of the Fem1 gene family, in three vertebrate species: human, mouse, and zebrafish. The proteins encoded by these Fem1c genes share >99% amino acid identity between human and mouse, 79% amino acid identity between mouse and zebrafish, and end with a C-terminal Arginine residue, which distinguishes them from other FEM-1 proteins reported thus far. The human and mouse Fem1c coding regions show conservation of intron-exon structure and expression pattern in adult tissues. Human FEM1C maps to 5q22, mouse Fem1c maps to chromosome 18, and zebrafish fem1c maps to Linkage Group 8. The Fem1c genes in vertebrates may play a conserved role in the development and/or physiologic function of these organisms.

The relation between HLA-DQA1 genes and genetic susceptibility to duodenal ulcer in Wuhan Hans.

AIM:To study the genetic susceptibility of HLA-DQA1 alleles to duodenal ulcer in Wuhan Hans.METHODS:Seventy patients with duodenal ulcer and fifty healthy controls were examined for HLA-DQA1 genotypes.HLA-DQA1 typing was carried out by digesting the locus specific polymerase chain reaction amplified products with alleles specific restriction enzymes (PCR-RFLP), i.e.,Apal I, Bsaj- I, Hph I, Fok I, Mbo II and Mnl I.RESULTS:The allele frequencies of DQA10301 and DQA1-0102 in patients with duodenal ulcer were significantly higher and lower respectively than those in healthy controls (0.40 vs 0.20, P = 0.003, P(corret) = 0.024) and (0.05 vs 0.14, P = 0.012, but P(corret) > 0.05), respectively.CONCLUSION: DQA1(*)0301 is a susceptible gene for duodenal ulcer in Wuhan Hans, and there are immunogenetic differences in HLA-DQA1 locus between duodenal ulcer patients and healthy controls.