Search

We have previously described local aldosterone synthesis in mouse colon. In the renin-angiotensin-aldosterone system (RAAS), angiotensin II (Ang II) peptide is the physiological factor which stimulates aldosterone synthesis in the adrenal glands. We have recently demonstrated that Ang II stimulates aldosterone synthesis also in mouse colon. Here, we conducted a 75-min ex vivo incubation of murine colonic tissue and evaluated the effects of three other Ang peptides, Ang I (1 μM), Ang III (0.1 μM) and Ang (1-7) (0.1 μM) on aldosterone synthesis. As a possible mechanism, their effects on tissue levels of the rate-limiting enzyme, aldosterone synthase (CYP11B2) were measured by ELISA and Western blot. Ang III significantly elevated the amount of tissue CYP11B2 protein in colon. The values of released aldosterone in colon tissue incubation were increased over the control in the presence of Ang I, II or III, however, being statistically non-significant. In Western blot analysis, the values of tissue CYP11B2 protein content were elevated by Ang I and II. Ang (1-7) alone in colon did not influence CYP11B2 protein levels in the incubation experiment but showed higher aldosterone release without statistical significance. Ang (1-7) showed an antagonistic effect towards Ang II in release of aldosterone in adrenal gland. An overall estimation of a single peptide (three measured variables), the results were always in an increasing direction. The responses of aldosterone synthesis to high levels of glucose (44 mM) and potassium (18.8 mM) as physiological stimulators in vivo were investigated in the colon incubation. Glucose, equal to four times the concentration of the control buffer in the incubation, showed higher values of aldosterone release in colon than control without statistical significance similarly to the effect seen in adrenal glands. Increasing the concentration of potassium in the incubation buffer exerted no effect on colonic aldosterone production. Intriguingly, no correlation was found between aldosterone release and the tissue CYP11B2 protein content in colon. In summary, the response of colonic aldosterone synthesis to different Ang peptides resembles, but is not identical to, the situation in the adrenal glands.

Purpose: Diabetes and dyslipidemia are among the most common chronic diseases with increasing global disease burdens, and they frequently occur together. The study aimed to investigate differences in the heritability of glycemic traits and serum lipid indicators and differences in overlapping genetic and environmental influences between them across age groups., Methods: This study included 1189 twin pairs from the Chinese National Twin Registry and divided them into three groups: aged ≤ 40, 41-50, and > 50 years old. Univariate and bivariate structural equation models (SEMs) were conducted on glycemic indicators and serum lipid indicators, including blood glucose (GLU), glycated hemoglobin A1c (HbA 1c ), total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C), in the total sample and three age groups., Results: All phenotypes showed moderate to high heritability (0.37-0.64). The heritability of HbA 1c demonstrated a downward trend with age (HbA 1c : 0.50-0.79), while others remained relatively stable (GLU: 0.55-0.62, TC: 0.58-0.66, TG: 0.50-0.63, LDL-C: 0.24-0.58, HDL-C: 0.31-0.57). The bivariate SEMs demonstrated that GLU and HbA 1c were correlated with each serum lipid indicator (0.10-0.17), except HDL-C. Except for HbA 1c and LDL-C, as well as HbA 1c and HDL-C, differences in genetic correlations underlying glycemic traits and serum lipids between age groups were observed, with the youngest group showing a significantly higher genetic correlation than the oldest group., Conclusion: Across the whole adulthood, genetic influences were consistently important for GLU, TC, TG, LDL-C and HDL-C, and age may affect the shared genetic influences between glycemic traits and serum lipids. Further studies are needed to elucidate the role of age in the interactions of genes related to glycemic traits and serum lipids., (© 2023. The Author(s), under exclusive licence to Italian Society of Endocrinology (SIE).)

Objective: To investigate the role and related mechanism of the highly expressed circular RNA molecule 103124 (hsa&#95;circRNA&#95;103124) in macrophage differentiation, pyroptosis and inflammation in peripheral blood mononuclear cells (PBMC) of patients with active Crohn's disease (CD). Methods: Patients with active CD (CD group) admitted to the Affiliated Suzhou Hospital of Nanjing Medical University from April to September 2018 and healthy people (control group) from the physical examination center of the hospital from July to October 2018 were retrospectively selected. The levels of hsa&#95;circRNA&#95;103124 and Toll-like receptor 4 (TLR4) in PBMC of the two groups were detected by real-time quantitative polymerase chain reaction (RT-qPCR). Tohoku hospital pediatrics-1 (THP1) cell line was used as a model for the study of hsa&#95;circRNA&#95;103124 regulating macrophage differentiation. Lentivirus infection was used to construct hsa&#95;circRNA&#95;103124 overexpressed or down-regulated THP1 cells to induce macrophage-like differentiation. According to the expression level of hsa&#95;circRNA&#95;103124, THP1 cell lines were divided into the following four groups: pLC5-ciR was overexpression control group; hsa&#95;circRNA&#95;103124 OE was the overexpression group; ShRNActrl was down-regulated expression control group; hsa&#95;circRNA&#95;103124 ShRNA was the down-regulated expression group. Flow cytometry was used to detect levels cluster of differentiation (CD) 68, CD80, interleukin (IL)-6, tumor necrosis factor α (TNF-α) and reactive oxygen species (ROS). The expression levels of IL-6, TNF-α, IL-1β, TLR4 and myeloid differentiation factor 88 (MyD88) were detected by RT-qPCR. The levels of gasdermin D (GSDMD), IL-18 and NOD-like receptor thermal protein domain associated protein 3 (NLRP3) were determined by immunofluorescence and RT-qPCR. Pearson correlation analysis was used to analyze the correlation between the abundance of hsa&#95;circRNA&#95;103124 and TLR4 expression level or Crohn's disease activity index (CDAI). Results: A total of 50 patients were included in the CD group, including 36 males and 14 females, aged (35±10) (19-64) years. A total of 30 subjects were included in the control group, including 22 males and 8 females, aged (38±9) (24-64) years. hsa&#95;circRNA&#95;103124 [(0.009±0.016) vs (0.003±0.002), P =0.042] and TLR4 [(0.005±0.003) vs (0.001±0.001), P <0.001] were all upregulated in the PBMC of patients in the CD group, compared with the control group. And hsa&#95;circRNA&#95;103124 was positively correlated with TLR4 ( r =0.40, P =0.004). hsa&#95;circRNA&#95;103124 level was positively correlated with CDAI ( r =0.32, P =0.024). The expression of CD68 ( P =0.002) and CD80 ( P <0.001) were enhanced. hsa&#95;circRNA&#95;103124 promoted production of ROS and the expression of IL-6, TNF-α, IL-1β, TLR4, MyD88, GSDMD, IL-18 and NLRP3 in macrophage-like M1 differentiated THP1 cells (all P <0.05). Conclusion: High expresion of hsa&#95;circRNA&#95;103124 in PBMC of patients with active CD may promote macrophage M1 differentiation, pyroptosis and inflammation through enhancing the expression of TLR4, MyD88, NLRP3 and GSDMD.

Objective: To investigate the diagnostic and evaluation value of plasma interleukin 9 (IL9) in the mucosal healing (MH) in patients with inflammatory bowel disease (IBD) treated with biological agents. Methods: Cohort study. IBD patients (137 cases) treated in the Affiliated Suzhou Hospital to Nanjing Medical University (Suzhou Municipal Hospital) from September 2019 to January 2022 were prospective selected. Each patient was treated with biological agents [Infliximab (IFX, 56 cases), Adalimumab (ADA, 20 cases), Ustekinumab (UST, 18 cases), Vedolizumab (VDZ, 43 cases)]. According to different therapeutic drugs, the IFX, ADA, UST, and VDZ group were divided. Clinical symptoms, inflammatory indicators and imaging examinations etc. were evaluated every 8 weeks, and the degree of MH was evaluated by endoscopy at the 54th week. The expression of plasma IL9 was detected by ELISA after initial enrollment (W 0) and 8 weeks of biological treatment (W 8). Receiver operating characteristic curve (ROC) was used to evaluate the diagnostic efficacy of IL9 for MH. Select the cut off value for the optimal ROC threshold based on the highest value of the Youden index. Spearman's rank correlation was used to analyze the correlation between IL9 and Simple Endoscopic Score for CD (SES-CD) and Mayo Endoscopic Score (MES), so as to evaluate the predictive value of IL9 for MH in IBD patients treated with biologic agents. Results: Among the 137 patients, there were 97 Crohn's disease (CD) patients, 53 males and 44 females, aged (31.6±10.3) years (18-60 years). There were 40 ulcerative colitis (UC) patients, 22 males and 18 females, aged (37.5±14.7) years (18-67 years). Among the CD patients, 42 cases (43.3%) achieved MH on endoscopy at the 54th week, and 60 patients (61.9%) achieved clinical remission. Among the UC patients, 22 cases (55.0%) achieved MH and 30 cases (75.0%) achieved clinical remission. At W 0, the relative expression of IL9 in patients in IBD patients who achieved MH after 54 weeks of biological treatment was lower than that in the non-MH patients [x¯±s, (127.42±34.43) vs (146.82±45.64) ng/L, (113.01±44.88) vs (146.12±48.66) ng/L, respectively, both P <0.05]. At W 8, the relative expression of IL9 in the MH group was lower than that in the non-MH patients (both P <0.05). The relative expression of IL9 in the MH patients after IFX treatment was lower than that in the non-MH group ( P <0.05). There was no significant difference among the other groups between MH and non-MH patients (all P >0.05). IL9 at W 8 showed high value in predicting MH in IBD [CD patients: area under curve (AUC)=0.716(95% CI : 0.616-0.817, P <0.001), sensitivity and specificity were 80.77%(95% CI :67.64%-88.45%) and 48.89%(95% CI : 35.53%-64.47%), respectively; UC patients: AUC=0.821, sensitivity and specificity were 77.78% and 72.73%, respectively]. At W 8, the cut off values for CD and UC patients were IL9>80.77 ng/L and IL9>77.78 ng/L, respectively. IL9 was positively correlated with endoscopic MH score parameters [ M ( Q 1 , Q 3 ),SES-CD: 3.0(8.5, 18.5); MES: 2.0(1.0, 3.0)] ( r =0.55, 0.72, respectively, both P <0.001) at W8. Conclusion: The plasma IL-9 at the week 8 after biological agents treatment can be used to diagnose and evaluate the MH of patients with IBD.

Aldosterone is the most important mineralocorticoid hormone regulating water and electrolyte absorption in the distal convoluted tubule of the kidney. Recently, we detected the presence of the whole chain of aldosterone production from the precursor corticosterone, transcription factor liver receptor homologue-1 (LRH-1), the aldosterone synthase enzyme protein (CYP11B2) as well as the gene to the final product aldosterone in murine large intestine. Here, we decided to correlate the amount of this synthase protein with its enzymatic activity in different parts of gastrointestinal tract and also with the aldosterone concentration in the respective tissue. Considering the physiological behavior of the animals in light and dark environment, we measured these variables at four time points - two in the light, the others during darkness. In vitro activity of CYP11B2 was measured as the amount of aldosterone formed from the precursor deoxycorticosterone using enzyme preparations from homogenized intestinal sections. CYP11B2 enzyme activity was higher in the large than in the small intestine. In ileum and colon, the CYP11B2 activity increased in the dark time. The highest aldosterone concentration was detected in the dark in the large intestine. In summary, enzyme activity of CYP11B2 was present in all parts of intestine; the large intestine formed more aldosterone during the darkness. No difference was seen in any of the variables between the early and late light hours.

Aldosterone, the main physiological mineralocorticoid, regulates sodium and potassium balance in the distal convoluted tubule of the kidney. Aldosterone is synthesized from cholesterol in the adrenal cortex in a sequence of enzymatic steps. Recently however, several tissues or cells e.g. brain, heart, blood vessels, kidneys and adipocytes have been shown to possess capability to produce aldosterone locally, and there is some evidence that this occurs also in the intestine. Colon expresses mineralocorticoid receptors and is capable of synthesizing corticosterone, the second last intermediate on the route to aldosterone from cholesterol. Based on such reports and on our preliminary finding, we hypothesized that aldosterone could be synthesized locally in the intestine and therefore we measured the concentration of aldosterone as well as the protein and gene expression of aldosterone synthase (CYP11B2), an enzyme responsible on aldosterone synthesis, from the distal section of the gastrointestinal tract of 10-week-old Balb/c male mice. It is known that sodium deficiency regulates aldosterone synthesis in adrenal glands, therefore we fed the mice with low (0.01%), normal (0.2%) and high-sodium (1.6%) diets for 14 days. Here we report that, aldosterone was detected in colon and cecum samples. Measurable amounts of CYP11B2 protein were detected by Western blot and Elisa analysis from both intestinal tissues. We detected CYP11B2 gene expression from the large intestine along with immunohistochemical findings of CYP11B2 in colonic wall. Sodium depletion increased the aldosterone concentration in plasma compared to control and high-sodium groups as well as in the intestine compared to mice fed with the high-sodium diet. To summarize, this study further supports the presence of aldosterone and the enzyme needed to produce this mineralocorticoid in the murine large intestine.

Fat deposition is an important economic trait in farm animals. However, it is difficult to genetically improve intramuscular fat deposition via trait-based cattle breeding. The main objectives of this study were to analyze the factors about beef flavor, and to detect functional microRNA (miRNA, miR) associated with intramuscular fat deposition in Yanbian cattle. Longissimus dorsi samples from six steers were separated into high- and low-fat groups (n = 3 each) based on the marbling score, and transcriptomic analysis was performed using miRNA sequencing. A total of 33 miRNAs and 38 genes were found to be differentially expressed in the high- and low-fat groups. Quantitative real-time polymerase chain reaction was performed to validate the sequencing results. Integrated miRNA-mRNA analysis revealed that miRNA-associated target genes were primarily associated with skeletal muscle development. However, some of the miRNAs (miR-424 etc.) and genes (ATF3 etc.) were also associated with fat metabolism. A targeted relationship between miR-22-3p and the WFIKKN2 gene and its involvement in adipocyte differentiation were confirmed experimentally. The study findings may provide potential candidate molecular targets for the selection of cattle with improved meat quality., (© 2021 Stichting International Foundation for Animal Genetics.)

Aim: Research on prevention and cure of banana wilt is important to ensure the healthy development of the banana industry. In this study, antifungal mechanism of Streptomyces ma. FS-4 on fusarium wilt of banana was investigated., Methods and Results: The physiological strain of banana fusarium pathogen Fusarium oxysporum f. sp. cubense Race 4 (FOC.4) was used as the target fungus, and the antifungal mechanism of the crude extract of Streptomyces ma. FS-4 was investigated. Eighteen different compounds identified by gas chromatography-mass spectrometry were composed of aldehydes, methyl, hydrocarbons, amides, esters and acids. FS-4 significantly inhibited the spore germination of the target fungi, with an EC 50 of 22·78 μg ml -1 . After treatment with 100 μg ml -1 FS-4 crude extract, the N-acetylglucosamine content in the mycelium increased 1·95-fold. However, the extract had no significant effect on β-1,3-glucanase. At the FS-4 crude extract dose of 100 μg ml -1 , the total sugar and protein contents decreased by 28·6 and 29·1% respectively, and the fat content was 41·3%. FS-4 significantly inhibited the activity of the mitochondrial complex III of Foc4, which was reduced by 52·45%. Moreover FS-4 reduced the activity of succinate dehydrogenase, a key enzyme in the Krebs cycle, by 60·2%. However, FS-4 had no significant effect on malate dehydrogenase. The membrane potential on the mitochondrial inner membrane was significantly reduced at the test concentration of 100 μg ml -1 . ROS gradually accumulated in the Foc4 hypha, and the burst was 3·97 times higher than the control., Conclusions: This study demonstrated that the antifungal mechanism of Streptomyces ma. FS-4 against Foc4 includes the destruction of the plasma membrane and mitochondrial dysfunction and finally induction of cell apoptosis., Significance and Impact of the Study: These results may indicate the prevention and control of banana wilt, which is of great significance to the healthy development of banana industry system., (© 2020 The Society for Applied Microbiology.)

The COllaborative project of Development of Anthropometrical measures in Twins (CODATwins) project is a large international collaborative effort to analyze individual-level phenotype data from twins in multiple cohorts from different environments. The main objective is to study factors that modify genetic and environmental variation of height, body mass index (BMI, kg/m2) and size at birth, and additionally to address other research questions such as long-term consequences of birth size. The project started in 2013 and is open to all twin projects in the world having height and weight measures on twins with information on zygosity. Thus far, 54 twin projects from 24 countries have provided individual-level data. The CODATwins database includes 489,981 twin individuals (228,635 complete twin pairs). Since many twin cohorts have collected longitudinal data, there is a total of 1,049,785 height and weight observations. For many cohorts, we also have information on birth weight and length, own smoking behavior and own or parental education. We found that the heritability estimates of height and BMI systematically changed from infancy to old age. Remarkably, only minor differences in the heritability estimates were found across cultural-geographic regions, measurement time and birth cohort for height and BMI. In addition to genetic epidemiological studies, we looked at associations of height and BMI with education, birth weight and smoking status. Within-family analyses examined differences within same-sex and opposite-sex dizygotic twins in birth size and later development. The CODATwins project demonstrates the feasibility and value of international collaboration to address gene-by-exposure interactions that require large sample sizes and address the effects of different exposures across time, geographical regions and socioeconomic status.

The Chinese National Twin Registry (CNTR) currently includes data from 61 566 twin pair from 11 provinces or cities in China. Of these, 31 705, 15 060 and 13 531 pairs are monozygotic, same-sex dizygotic and opposite-sex dizygotic pairs, respectively, determined by opposite sex or intrapair similarity. Since its establishment in 2001, the CNTR has provided an important resource for analysing genetic and environmental influences on chronic diseases especially cardiovascular diseases. Recently, the CNTR has focused on collecting biologic specimens from disease-concordant or disease-discordant twin pairs or from twin pairs reared apart. More than 8000 pairs of these twins have been registered, and blood samples have been collected from more than 1500 pairs. In this review, we summarize the main findings from univariate and multivariate genetic effects analyses, gene-environment interaction studies, omics studies exploring DNA methylation and metabolomic markers associated with phenotypes. There remains further scope for CNTR research and data mining. The plan for future development of the CNTR is described. The CNTR welcomes worldwide collaboration., (© 2019 The Association for the Publication of the Journal of Internal Medicine.)

Objective: To explore the expression of microRNA-155 in colonic mucosa and peripheral blood in patients with inflammatory bowel disease(IBD), and to examine the clinical value and significance of microRNA-155 in the diagnosis of IBD. Methods: Quatitative reverse-transcription PCR was performed to detect the expression of microRNA-155 in 20 patients with Crohn disease(CD), 21 patients with ulcerative colitis (UC), 18 patients with IBD type unclassified(IBDU), 25 healthy people(control group), 12 patients with infection colitis and 19 patients with ischemia colitis.Receiver operating characteristic (ROC) curve was performed to analyze the clincal value of microRNA-155 in diagnosis of IBD. Results: The expression of microRNA-155 in colonic mucosa in CD, UC and IBDU group was significantly higher than that in control group( P <0.05). MicroRNA-155 expression was also significantly higher in UC group in comparison to CD group (35.4±3.0 vs 18.6±5.9, P <0.01), IBDU group in comparison to CD group (23.0±3.7 vs 18.6±5.9, P <0.05) and UC group in comparison to IBDU group (35.4±3.0 vs 23.0±3.7, P <0.01). The plasma level of microRNA-155 in UC group (55.6±2.5) and IBDU group (48.1±6.2) was significantly higher than that in control group( P <0.05), while no significant difference in CD group was observed when compared with control group( P >0.05). ROC curve shows an AUC of 0.83 and 95% CI of 0.679-0.986 of microRNA-155 expression in colonic mucosa.The sensitivity and specificity of microRNA-155 expression in colonic mucosa in diagnosis of IBD was 68.4% and 78.6%, respectively. Conclusions: MicroRNA-155 showed high expression in colonic mucosa and peripheral blood in patients with IBD.MicroRNA-155 shows promise as a biomarker in diagnosis of IBD.Furthermore, the aberrant expression indicates that microRNA-155 may be involved in pathogenesis and progression of IBD.

Objective: To investigate the application value of serum calprotectin in assessing the activity of IBD Methods: Eighty-five Crohn's disease(CD) and eighty-five Ulcerative colitis(UC) patients at Suzhou Municipal Hospital(North Branch) from August 2015 to January 2017 were enrolled in this study, and eighty-five healthy subjects were selected as controls. Serum level of calprotectin was determined by ELISA. Correlation of serum calprotectin with clinical features of IBD was analyzed. The diagnostic efficacy for CD and UC were assessed by ROC curve. Results: The level of serum calprotectin was significantly higher in IBD patients than in healthy subjects. Positive correlations were found between serum calprotectin level and CRP, ESR, fecal calprotectin level of IBD(ρ＝0.341, P <0.001；ρ＝0.438, P <0.001；ρ＝0.542, P <0.001，respectively). Besides, positive correlations were found between serum calprotectin level with disease activity index(CDAI or Mayo score) of IBD(ρ＝0.309, P ＝0.004；ρ＝0.227, P ＝0.036，respectively)．Nevertheless, no correlation was found between serum calprotectin level with location of IBD and disease subtypes( P >0.05)．The area under curve(AUC) of serum calprotectin for diagnosis of CD and UC were 0.946 and 0.906, respectively. Conclusions: Serum level of calprotectin is associated with the activity of IBD. As a result，serum level of calprotectin may has the potential to be served as a clinical index of IBD activity.

It is still controversial whether maternal anti-HBV antibodies (anti-HBVs) affect the infants' immune response to hepatitis B virus (HBV) vaccination. This multicentre study aims to address this question. First, we determined whether the transplacental transfer of maternal anti-HBVs occurs by measuring the titres of 90 anti-HBVs-positive pregnant women and their newborns. The anti-HBVs-positive rates of newborns ranged from 89.7% to 100.0%, depending on the maternal anti-HBVs titres. Secondly, we investigated the effects of maternal anti-HBVs on the immune response of infants to HBV vaccination. A total of 1063 mother-and-infant pairs were enrolled and divided into three groups with maternal anti-HBVs titres of <10 IU/L (negative - 37.9%), 10-499 and ≥500 IU/L. The infants' anti-HBVs-positive rate and titres were negatively correlated with maternal anti-HBVs titres: the anti-HBVs-positive rate of infants were 88.9% (360/405), 84.5% (381/451) and 77.3% (160/207) in mothers with low, intermediate and high antibody titres, respectively, P<.0001. Median titres of anti-HBVs (IU/L) among infants were 169.1, 141.0 and 79.4, respectively, P=.020. One hundred and sixty-two infants were negative for anti-HBVs after the standard vaccination, and 120 of 131 of these infants (91.6%) reached anti-HBVs positivity after the first "booster" dose. The maternal anti-HBVs titres did not significantly affect infant response to this booster. In summary, transplacental transfer of anti-HBVs occurs and high titres of maternal anti-HBVs may suppress the immune response of infants to the standard HBV vaccination. The current schedule of the 0, 1 and 6 month may not be the optimal choice of infants with anti-HBVs-positive mothers., (© 2016 John Wiley & Sons Ltd.)

Respiratory tract infection with Pseudomonas aeruginosa is a major cause of hospital&mdash;acquired pneumonia in immune&mdash;compromised individuals. Lung infection with P. aeruginosa is often associated with production of various inflammatory cytokines including IL&mdash;1&#946;. Production of IL&mdash;1&#946; requires proteolytic cleavage by a multiprotein complex termed inflammasome. AIM2 inflammasome recognizes foreign cytosolic double stranded DNA. A role of AIM2 in P. aeruginosa infection has not been reported previously. In this study, we found that P. aeruginosa infection induced degradation of AIM2 protein in macrophages and induction of AIM2 mRNA expression in macrophages and in the lung of mice. Interestingly, P. aeruginosa infection induced a similar level of IL&mdash;1&#946;, IL&mdash;6 and TNF production in wild&mdash;type and AIM2&mdash;deficient mice. Similarly, no significant differences in bacterial clearance, neutrophil infiltration and NF&mdash;&#954;B activation were observed between wild&mdash;type and AIM2&mdash;deficient mice following P. aeruginosa lung infection. Our data suggest that AIM2 inflammasome is dispensable for the host defense against P. aeruginosa infection.

Objective: The guizhi fuling (GZFL) capsule has been a traditional Chinese medicine for the treatment of gynecological inflammation for the past thousands of years. However, as a formula, its therapeutic mechanism has not been clearly elucidated. The aim of this study is to investigate the role of apoptosis during GZFL capsule therapy for the treatment of endometrial hyperplasia., Methods: The rat model of endometriosis was established, and the rats were given different doses of GZFL capsule. Uterine histomorphometric analysis, real-time quantitative PCR (qPCR) and Western blotting were performed. The terminal deoxynucleotidyl transferase (TdT)-mediated dUTP biotin nick end labeling (TUNEL) method was performed to analyze the apoptosis induced by the GZFL capsule., Results: The TUNEL assay showed that different doses of GZFL capsule were able to induce apoptosis in rat endometriotic cells. qPCR and Western blot analysis showed that the GZFL capsule can inhibit the mRNA levels of the survivin gene. In addition, the GZFL capsule can inhibit the mRNA level of the mitochondrial apoptotic pathway-related apoptosis-inhibiting factor Bcl-2 but increases the mRNA level of apoptosis- promoting factor Bax., Conclusions: These results indicate that the GZFL capsule can induce apoptosis of endometriotic cells and inhibit cell proliferation and metastasis of endometriotic cells through the mitochondrial apoptotic pathway.

Executive control of attention regulates our thoughts, emotion and behavior. Individual differences in executive control are associated with task-related differences in brain activity. But it is unknown whether attentional differences depend on endogenous (resting state) brain activity and to what extent regional fluctuations and functional connectivity contribute to individual variations in executive control processing. Here, we explored the potential contribution of intrinsic brain activity to executive control by using resting-state functional magnetic resonance imaging (fMRI). Using the amplitude of low-frequency fluctuations (ALFF) as an index of spontaneous brain activity, we found that ALFF in the right precuneus (PCUN) and the medial part of left superior frontal gyrus (msFC) was significantly correlated with the efficiency of executive control processing. Crucially, the strengths of functional connectivity between the right PCUN/left msFC and distributed brain regions, including the left fusiform gyrus, right inferior frontal gyrus, left superior frontal gyrus and right precentral gyrus, were correlated with individual differences in executive performance. Together, the ALFF and functional connectivity accounted for 67% of the variability in behavioral performance. Moreover, the strength of functional connectivity between specific regions could predict more individual variability in executive control performance than regionally specific fluctuations. In conclusion, our findings suggest that spontaneous brain activity may reflect or underpin executive control of attention. It will provide new insights into the origins of inter-individual variability in human executive control processing., (Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.)

Background/objectives: Epidemiological studies have suggested that dietary fiber intake may be associated with a decreased risk of stroke, but the findings have been inconsistent. We aimed to assess this association by conducting a meta-analysis of prospective cohort studies., Subjects/methods: We performed a literature search on PubMed database through July 2012 to identify prospective studies of dietary fiber intake in relation to risk of stroke. We also comprehensively reviewed the reference lists of the retrieved articles to identify additional studies. We used a random-effects model to compute the summary risk estimates., Results: Six prospective cohort studies containing a total of 314 864 subjects and 8920 stroke cases were included. The summary relative risk (RR) of stroke for the highest vs lowest category of dietary fiber intake was 0.87 (95% confidence interval (CI), 0.77-0.99). The corresponding RR in the subgroup analyses for men and women was 0.95 (95% CI, 0.83-1.08) and 0.80 (95% CI, 0.66-0.96), respectively; and for ischemic stroke and hemorrhagic stroke was 0.83(95% CI, 0.72-0.96) and 0.86 (95% CI, 0.70-1.06), respectively. Meta-regression indicated no significant difference between gender (P-interaction=0.18), or stroke subtypes (P-interaction =0.85). The dose-response analysis suggested a 12% (RR=0.88; 95% CI, 0.79-0.97) reduction in risk of stroke for each 10 g per day increment in dietary fiber intake. Moderate heterogeneity emerged in some of analyses, but disappeared after removing one study substantially contributing to the heterogeneity. Little evidence of publication bias was detected., Conclusion: Findings of this meta-analysis indicate a significant inverse dose-response relationship between dietary fiber intake and risk of stroke.

Background/objectives: Epidemiological evidence is suggestive, but inconclusive, for an association between consumption of red and processed meat and risk of stroke. We aimed to assess this association by conducting a meta-analysis of prospective cohort studies., Subjects/methods: We performed a literature search on PubMed database through June 2012 to identify prospective cohort studies of red and processed meat intake in relation to risk of stroke. Reference lists of the retrieved articles were also reviewed. Both fixed-effects and random-effects model were assumed to compute the summary risk estimates., Results: Five large independent prospective cohort studies were identified. These studies contained a total of 2 39 251 subjects and 9593 stroke events. Comparing the highest category of consumption with lowest category, the pooled relative risks (RRs) of total stroke were 1.15 (95% confidence interval (CI), 1.05-1.25) for total meat (red and processed meat combined) (n=4), 1.09 (95% CI, 1.01-1.18) for red meat (n=5) and 1.14 (95% CI, 1.05-1.25) for processed meat (n=5); the corresponding RRs of ischemic stroke (highest vs lowest quintile) were 1.15 (95% CI, 1.04-1.28), 1.13(95% CI, 1.01-1.25) and 1.19 (95% CI, 1.08-1.31). Consumption of red and/or processed meat was not associated with hemorrhagic stroke. In the dose-response analysis, the risk of stroke increased significantly by 10% and 13% for each 100 g per day increment in total and red meat consumption, respectively, and by 11% for each 50 g per day increment in processed meat consumption., Conclusion: Findings from this meta-analysis indicate that consumption of red and/or processed meat increase risk of stroke, in particular, ischemic stroke.

Epidemiologic studies have suggested that magnesium intake may be associated with a decreased risk of colorectal cancer (CRC), but the findings have been inconsistent. We aimed to assess this association by conducting a meta-analysis of prospective studies. We performed a literature search on PubMed database through July 2012 to identify prospective studies of magnesium intake in relation to CRC risk. Reference lists of the retrieved articles were also reviewed. A random-effects model was used to compute the summary risk estimates. Eight prospective studies containing 338,979 participants and 8000 CRC cases met the inclusion criteria. The summary relative risk (RR) for the highest vs lowest category of magnesium intake for CRC was 0.89 (95% CI, 0.79-1.00), with little evidence of heterogeneity. Restricting the analysis to six studies that have adjusted for calcium intake yielded a similar result. For colon and rectal cancer, the pooled RR was 0.81 (95% CI, 0.70-0.93) and 0.94 (95% CI, 0.72-1.24), respectively. In the dose-response analyses, the summary RRs for an increment of magnesium intake of 50 mg/day for colorectal, colon and rectal cancer were, respectively, 0.95 (95% CI, 0.89-1.00), 0.93 (95% CI, 0.88-0.99) and 0.93 (95% CI, 0.83-1.04), and there was some evidence of heterogeneity; omitting one study that substantially contributed to the heterogeneity yielded generally similar results, but with low heterogeneity. We detected no indication of publication bias. On the basis of the findings of this meta-analysis, a higher magnesium intake seems to be associated with a modest reduction in the risk of CRC, in particular, colon cancer.

The purpose of this study is to investigate how to scale pixel intensity acquired from one exposure time to another. This is required when comparing grayscale images acquired at different exposure times and other image processing such as autofluorescence removal. Pixel intensity is linear to exposure time as long as images are acquired at the linear range of a camera, but importantly there exists an intercept, which is set by the camera. We termed this intercept as dark pixel intensity, as it is the pixel intensity under conditions of no light and zero exposure time. Dark pixel intensity is determined by camera's readout noise (electron/pixel), gain, and DC offset. Knowing dark pixel intensity, image acquired from one exposure time can be linearly scaled to an image at a different exposure time. Dark pixel intensity can be directly measured by obtaining an image at no light and zero (or minimum) exposure time. It can be also indirectly calculated by capturing images at a series of exposure times. Finally, the prestained and poststained images were acquired at their optimal exposures and autofluorescence was completely removed by normalizing images with the exposure time ratio and dark pixel intensity followed by subtraction., (© 2011 General Electric Journal of Microscopy © 2011 Royal Microscopical Society.)

Aims/hypothesis: The common genetic and environmental effects on endophenotypes related to the metabolic syndrome have been investigated using bivariate and multivariate twin models. This paper extends the pairwise analysis approach by introducing independent and common pathway models to Chinese twin data. The aim was to explore the common genetic architecture in the development of these phenotypes in the Chinese population., Methods: Three multivariate models including the full saturated Cholesky decomposition model, the common factor independent pathway model and the common factor common pathway model were fitted to 695 pairs of Chinese twins representing six phenotypes including BMI, total cholesterol, total triacylglycerol, fasting glucose, HDL and LDL. Performances of the nested models were compared with that of the full Cholesky model., Results: Cross-phenotype correlation coefficients gave clear indication of common genetic or environmental backgrounds in the phenotypes. Decomposition of phenotypic correlation by the Cholesky model revealed that the observed phenotypic correlation among lipid phenotypes had genetic and unique environmental backgrounds. Both pathway models suggest a common genetic architecture for lipid phenotypes, which is distinct from that of the non-lipid phenotypes. The declining performance with model restriction indicates biological heterogeneity in development among some of these phenotypes., Conclusions/interpretation: Our multivariate analyses revealed common genetic and environmental backgrounds for the studied lipid phenotypes in Chinese twins. Model performance showed that physiologically distinct endophenotypes may follow different genetic regulations.

Background: Converging evidence has implicated endogenous neurotensin (NT) in the pathophysiology of brain processes relevant to schizophrenia. Prepulse inhibition of the startle reflex (PPI) is a measure of sensorimotor gating and considered to be of strong relevance to neuropsychiatric disorders associated with psychosis and cognitive dysfunction. Mice genetically engineered to not express NT display deficits in PPI that model the PPI deficits seen in schizophrenia patients. NT1 receptors have been most strongly implicated in mediating the psychosis relevant effects of NT such as attenuating PPI deficits. To investigate the role of NT1 receptors in the regulation of PPI, we measured baseline PPI in wildtype (WT) and NT1 knockout (KO) mice. We also tested the effects of amphetamine and dizocilpine, a dopamine agonist and NMDA antagonist, respectively, that reduce PPI as well as the NT1 selective receptor agonist PD149163, known to increase PPI in rats., Methods: Baseline PPI and acoustic startle response were measured in WT and NT1 KO mice. After baseline testing, mice were tested again after receiving intraperatoneal (IP) saline or one of three doses of amphetamine (1.0, 3.0 and 10.0 mg/kg), dizocilpine (0.3, 1.0 and 3.0 mg/kg) and PD149163 (0.5, 2.0 and 6.0 mg/kg) on separate test days., Results: Baseline PPI and acoustic startle response in NT1 KO mice were not significantly different from NT1 WT mice. WT and KO mice exhibited similar responses to the PPI-disrupting effects of dizocilpine and amphetamine. PD149163 significantly facilitated PPI (P < 0.004) and decreased the acoustic startle response (P < 0.001) in WT but not NT1 KO mice., Conclusions: The data does not support the regulation of baseline PPI or the PPI disruptive effects of amphetamine or dizocilpine by endogenous NT acting at the NT1 receptor, although they support the antipsychotic potential of pharmacological activation of NT1 receptors by NT1 agonists.

Electroencephalogram (EEG) is able to indicate states of mental activity ranging from concentrated cognitive efforts to sleepiness. Such mental activity can be reflected by EEG energy. In particular, intrusion of EEG theta wave activity into the beta activity of active wakefulness has been interpreted as ensuing sleepiness. Pupil behavior can also provide information regarding alertness. This paper develops an innovative signal classification method that is capable of differentiating subjects with sleep disorders which cause excessive daytime sleepiness (EDS) from normal control subjects who do not have a sleep disorder based on EEG and pupil size. Subjects with sleep disorders include persons with untreated obstructive sleep apnea (OSA) and narcolepsy. The Yoss pupil staging rule is used to scale levels of wakefulness and at the same time theta energy ratios are calculated from the same 2-s sliding windows by Fourier or wavelet transforms. Then, an artificial neural network (NN) of modified adaptive resonance theory (ART2) is utilized to identify the two groups within a combined group of subjects including those with OSA and healthy controls. This grouping from the NN is then compared with the actual diagnostic classification of subjects as OSA or controls and is found to be 91% accurate in differentiating between the two groups. The same algorithm results in 90% correct differentiation between narcoleptic and control subjects.

Objective: To investigate the effects of interlukin-2 (IL-2) on the in vitro invasiveness and the expression of several cell surface antigens related to invasive and metastatic potentials of human hepatocellular carcinoma (HCC) QGY-7701 cell line., Methods: QGY-7701 cells were incubated with high concentration of IL-2 or low concentration of IL-2 in different time. The expression of ICAM-1, CD(44) and HLA-I of the tumor cells was determined by fluorescence-activated cell sorter (FACS) analysis The tumor cell binding affinity to extracellular matrix components was measured by cell attachment assay. The degree of homotypic aggregation was quantified by cell aggregation assay., Results: IL-2 treatment exhibited enhanced expression of ICAM-1 (from 8.3% to 20.5% after high concentration of IL-2 treatment and 17.3% after low concentration of IL-2 treatment) and HLA-I (from 9.8% to 25.4% and 22.1%, respectively after high and low concentration of IL-2 treatment), suppression of CD(44) (from 26.4% to 12.5% and 11.6%, respectively) on HCC cell line and decreased binding affinity to type IV collagen (from 23.5% to 12.4%, 32.3% to 13.8%, 45.7% to 19.6% at 20 min, 40 min and 60 min, respectively after high concentration of IL-2 treatment, and 9.6%, 12.5% and 17.9%, respectively after low concentration of IL-2 treatment) and fibronectin (from 18.6% to 14.1%, 31.2% to 18.4%, 44.5% to 20.5% at 20 min, 40 min and 60 min, respectively after high concentration of IL-2 treatment, and 14.6%, 17.1% and 18.9%, respectively after low concentration of IL-2 treatment) and the degree of homotypic aggregation (from 58.3% to 26.5%, 85.4% to 37.6%, 88.6% to 42.3% at 20 min, 40 min and 60 min, respectively after high concentration of IL-2 treatment, and 25.0%, 36.4% and 42.6%, respectively after low concentration of IL-2 treatment)of HCC cells., Conclusions: IL-2 may directly alter tumor properties associated with invasive and metastatic phenotypes of HCC cells, and can inhibit the invasive and metastatic potentials of HCC cells.

The synaptic transmission and intrinsic membrane properties of spiny neurons in rat neostriatum were studied after unilateral dopamine depletion using in vivo intracellular recording and staining techniques. Two to four weeks after dopamine denervation, the spontaneous firing rate of spiny neurons increased and the spontaneous membrane potential fluctuation stayed at a more depolarized state for longer periods of time. The amplitude of cortically evoked initial excitatory postsynaptic potentials increased and a late excitatory postsynaptic potential that was occasionally found in control neurons was elicited from 23% of spiny neurons after dopamine denervation. No significant changes in intrinsic membrane properties of spiny neurons were observed after dopamine denervation. These results suggest that dopamine inhibits excitatory synaptic transmission of spiny neurons in naïve animals.

ANADPH-dependent 2-Oxoaldehyde reductase was isolated and purified from a marine bacteria Bacillus sp. The purification procedure involved ammonium sulfate fractionation and Q Sepharose FF, Hydroxyapatite, Sephadex G-100 column chromatographies. The specific activity of the purified enzyme was increased by 141.1 folds over crude extract and the recovery yield was 11.4%. 2-Oxoaldehyde compounds were found to be speciall good substrates. The optimum pH of the enzyme activity was 6.2-6.6. The Km coefficient for 3-deoxyglucosone was 2.5 mmol/L. The molecular weight of the enzyme was estimated to be 33 kD The enzyme activity is stable below 30 degrees C and pH 5.0-8.0. EDTA, beta-mercaptoethanol and dithiothreitol enhanced the enzyme activity. On the other hand, the enzyme activity was partially lost by idoacetic acid or N-ethylmaleimide.

Spiny neurons in neostriatum are vulnerable to cerebral ischemia. To reveal the mechanisms underlying the postischemic neuronal damage, the spontaneous activities, evoked postsynaptic potentials and membrane properties of spiny neurons in rat neostriatum were compared before and after transient forebrain ischemia using intracellular recording and staining techniques in vivo. In control animals the membrane properties of spiny neurons were about the same between the left and right neostriatum but the inhibitory synaptic transmission was stronger in the left striatum. After severe ischemia, the spontaneous firing and membrane potential fluctuation of spiny neurons dramatically reduced. The cortically evoked initial excitatory postsynaptic potentials were suppressed after ischemia indicated by the increase of stimulus threshold and the rise time of these components. The paired-pulse facilitation test indicated that such suppression might involve presynaptic mechanisms. The inhibitory postsynaptic potentials in spiny neurons were completely abolished after ischemia and never returned to the control levels. A late depolarizing postsynaptic potential that was elicited from approximately 5% of the control neurons by cortical stimulation could be evoked from approximately 30% of the neurons in the left striatum and approximately 50% in the right striatum after ischemia. The late depolarizing postsynaptic potential could not be induced after acute thalamic transection. The intrinsic excitability of spiny neurons was suppressed after ischemia evidenced by the significant increase of spike threshold and rheobase as well as the decrease of repetitive firing rate following ischemia. The membrane input resistance and time constant increased within 6 h following ischemia and the amplitude of fast afterhyperpolarization significantly increased after ischemia. These results indicate the depression of excitatory monosynaptic transmission, inhibitory synaptic transmission and excitability of spiny neurons after transient forebrain ischemia whereas the excitatory polysynaptic transmission in neostriatum was potentiated. The facilitation of excitatory polysynaptic transmission is stronger in the right neostriatum than in the left neostriatum after ischemia. The suppression of inhibitory component and the facilitation of excitatory polysynaptic transmission may contribute to the pathogenesis of neuronal injury in neostriatum after transient cerebral ischemia.

Precerebellin (Cbln1) is the precursor of the brain-specific hexadecapeptide cerebellin. Although cerebellin has properties of a conventional neuropeptide, its function is controversial because Cbln1 has structural features characteristic of circulating atypical collagens. Cbln1 is related to the three subunits of the complement C1q complex. Therefore, we hypothesized that Cbln1 participated in analogous heteromeric complexes with precerebellin-related proteins. Using LexA-Cbln1 as bait in a yeast two-hybrid screen, we isolated a cDNA encoding a novel Cbln1-related protein, designated Cbln3. The gene encoding cbln3 had the same intron-exon structure as cbln1 but mapped to a different mouse chromosome (14). The deduced amino acid sequence of Cbln3 was 55% identical to Cbln1 and also contained a C1q signature domain and signal sequence for secretion. In addition to binding avidly to Cbln3, Cbln1 also formed homomeric complexes. In contrast, Cbln3 homomeric association was weak. These interactions exhibited specificity because C1qB bound to neither Cbln1 nor Cbln3. Like cbln1, cbln3 was expressed in the cerebellum and dorsal cochlear nucleus in which it was detected in granule neurons. Because Cbln1 and Cbln3 are coexpressed in the brain and interact avidly, they may function as a secreted heteromeric complex in vivo.

The aim of this study was to evaluate the histologic characteristics of hemangioblastoma (HB) and investigate the histogenesis of its stromal cells. Sixty-eight cases of hemangioblastoma were studied by light microscopy and thirty-three cases were examined by immunohistochemical stainning. Forty-eight of the 68 patients were males and 20 were females. The average age was 37. Fifty-two cases (76.6%) had been present for two months to one year before the diagnosis was made. Sixty cases (88.2%) arose in the cerebellum. Fifty cases (73.5%) appeared as cystic nodules. Histologically, the 68 cases of HB were further categorized into three subtypes: typical (30 cases), cellular (21 cases) and reticular (17 cases). In 33 cases, the stromal cell were positive for NSE, but negative for F-VIII, UEA-1, GFAP and EMA. In summary, there are three essential histologic categories of hemangioblastoma, and its stromal cells may well have the characters of neuroendocrine differentiation.

To investigate the exact mechanism of epileptogenesis induced by coriaria lactone (CL), the effect of CL on NMDA receptor mediated current (IAsp) in rat hippocampal CA1 neurons was investigated by using nystatin perforated whole-cell patch clamp. 10(-6)-10(-4) mol/L Asp acted on NMDA receptors and elicited an inward current (IAsp) at a holding potential (VH) of -40 mV in presence of 10(-6) mol/L glycine and absence of Mg2+ extracellularly. CL enhanced NMDA receptor mediated current induced by Asp, but had no effect on threshold concentration, EC50, Hill coefficient as well as maximal-effect concentration and reversal potential of IAsp. The effect had no relationship with holding potential. These results showed that CL could enhance NMDA receptor mediated current to increase [Ca2+]i of neurons by acting on Gly site, thereby inducing epilepsy.

The mitochondrial inhibitors malonate and 3-nitropropionic (3NP) acid are potent neurotoxins in vivo. Administration of these compounds results in neuronal loss similar to that seen in Huntington's disease. Although the mechanism of cell death produced by these compounds likely involves activation of N-methyl-D-aspartate receptors, it remains unclear why the striatum demonstrates regional susceptibility to the toxicity of these and other mitochondrial poisons. We hypothesized that dopamine, a weak neurotoxin that occurs in high concentrations in the striatum, may contribute to the neuronal damage caused by mitochondrial inhibition. We investigated whether depletion of striatal dopamine using the catecholaminergic toxin 6-hydroxydopamine would attenuate lesions induced by mitochondrial inhibition. We found that dopamine depletion reduced significantly the extent of histological damage in the striatum elicited by both intraparenchymal injections of 0.8 micromol malonate and 20 mg/kg systemic administration of 3NP. These data suggest that dopamine or one of its metabolites may contribute to mitochondrial toxin-induced cell death., (Copyright 1998 Academic Press.)

Comparative study of oxidatively modified low-density lipoprotein (Ox-LDL) and malondialdehyde-modified low-density lipoprotein (MDA-LDL) is important for further understanding the biological properties of Ox-LDL, such as its toxic effects, immunogenicity and multiplicity of scavenger receptor binding. In this study, the characteristics of Ox-LDL and MDA-LDL binding and degradation were compared. The results show that when their degree of modification (as determined by relative electrophoretic mobility) was similar, the binding and degradation of Ox-LDL by the macrophage cell line P388D1 were greater than those of MDA-LDL. The binding and degradation of Ox-LDL by macrophages and human umbilical vein endothelial cells increased with the degree of modification. In addition, Ox-LDL or MDA-LDL could competitively inhibit binding of labelled Ox-LDL or labelled MDA-LDL to their respective macrophage receptors, and could partially inhibit each other.

Epidemiological and serological survey on five types of viral hepatities (hepatitis A-hepatitis E) was conducted from October 1994 to December 1996 among 119 labourers returned from abroad, including 55 female prostitutes, 86 medical workers, 88 STD patients and 88 paid blood donors. HAV infection rates for all of the 5 groups were above 83%. HBV infection rate showed significantly different (chi 2 = 3.86, P < 0.05) between medical workers (53.49%) and the control group (30.43%). A significant difference (chi 2 = 5.36, P < 0.05) between HBV infection among prostitutes (52.73%) and control group (26.67%) was also noticed. In all the five groups, none was found to be anti-HDV positive. The HEV infection rates for the medical workers, prostitutes, blood donors, returned labourers and STD patients were 1.16%, 3.64%, 2.27%, 5.88% and 6.82%, whereas the anti-HCV positive rates were 1.16%, 3.64%, 0.00%, 0.00% and 1.14%, among them respectively.

Low-density lipoprotein (LDL) was modified with copper ions (Cu2+) and malondialdehyde (MDA), and the differences were compared. The results show that both oxidative and MDA modification produce a decrease in free amino groups in LDL and enhance its electrophoretic mobility on agarose gel, and that these observations are linked. However, differences produced by the two forms of modification were observed. Oxidative modification of LDL involves free radical-mediated lipid peroxidation which produces large amounts of thiobarbituric acid-reactive substances (TBARS) and conjugated dienes. In addition, vitamin E was reduced considerably and cholesterol lowered, and apolipoprotein B (apo B) fragmentation and aggregation were seen. Similar changes were not seen with MDA modification which does not involve lipid peroxidation. Fluorescence emission spectra of both forms of modified LDL show that emission intensity increases gradually as the modification progresses, but maximum emission wavelength and spectrum patterns are not the same. These findings may be of some significance in the study of the antigenicity, scavenger receptor multiplicity and toxic effects of modified LDL.

Deposits of amyloid beta-peptide (A beta), reduced glucose uptake into brain cells, oxidative damage to cellular proteins and lipids, and excitotoxic mechanisms have all been suggested to play roles in the neurodegenerative process in Alzheimer's disease. Synapse loss is closely correlated with cognitive impairments in Alzheimer's disease, suggesting that the synapse may be the site at which degenerative mechanisms are initiated and propagated. We report that A beta causes oxyradical-mediated impairment of glucose transport, glutamate transport, and mitochondrial function in rat neocortical synaptosomes. A beta induced membrane lipid peroxidation in synaptosomes that occurred within 1 h of exposure; significant decreases in glucose transport occurred within 1 h of exposure to A beta and decreased further with time. The lipid peroxidation product 4-hydroxynonenal conjugated to synaptosomal proteins and impaired glucose transport; several antioxidants prevented A beta-induced impairment of glucose transport, indicating that lipid peroxidation was causally linked to this adverse action of A beta. FeSO4 (an initiator of lipid peroxidation), A beta, and 4-hydroxynonenal each induced accumulation of mitochondrial reactive oxygen species, caused concentration-dependent decreases in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction, and reduced cellular ATP levels significantly. A beta also impaired glutamate transport, an effect blocked by antioxidants. These data suggest that A beta induces membrane lipid peroxidation, which results in impairment of the function of membrane glucose and glutamate transporters, altered mitochondrial function, and a deficit in ATP levels; 4-hydroxynonenal appears to be a mediator of these actions of A beta. These data suggest that oxidative stress occurring at synapses may contribute to the reduced glucose uptake and synaptic degeneration that occurs in Alzheimer's disease patients. They further suggest a sequence of events whereby oxidative stress promotes excitotoxic synaptic degeneration and neuronal cell death in a variety of different neurodegenerative disorders.

Normal human diploid fibroblasts (HF) have a limited life span, undergo senescence, and rarely, if ever, spontaneously immortalize in culture. Introduction of the gene for T antigen encoded by the DNA virus SV40 extends the life span of HF and increases the frequency of immortalization; however, immortalization requires both T-dependent and T-independent functions. We previously generated independent SV40-transformed non-immortal (pre-immortal) HF cell lines from which we then obtained immortal sublines as part of a multifaceted approach to identify functions responsible for immortalization. In this study we undertook a search for cellular mRNAs which are differentially expressed upon immortalization. A lambda cDNA library was prepared from a pre-immortal SV40-transformed HF (HF-C). We screened the library with a subtracted probe enriched for sequences present in HF-C and reduced in immortal AR5 cells. A more limited screen was also employed for sequences overexpressed in AR5 using a different strategy. Alterations in the level of mRNAs in AR5 encoding functions relevant to signal transduction pathways were identified; however, most cDNAs encoded novel sequences. In an effort to clarify which of the altered mRNAs are most relevant to immortalization, we performed Northern analysis with RNA prepared from three paired sets of independent pre-immortal and immortal (4 cell lines) SV40-transformants using eight cloned cDNAs which show reduced expression in AR5. Three of these were reduced in additional immortal cell lines as well; one, J4-4 (unknown function) is reduced in all the immortal cell lines tested; a second, J4-3 (possible PP2C type phosphatase) is reduced in 2 of the 3 matched sets; and a third, J2-2 (unknown function) is reduced in 2 unrelated immortal cell lines. Although the roles of these genes are as yet unclear, their further analysis should extend our understanding of the molecular bases for immortalization. In particular, the patterns of expression of J4-4 and J4-3 strongly suggest that they are involved in the process of immortalization and/or can serve as target genes for assessing regulators of gene expression in this process.

Impaired energy metabolism may play an important role in neuronal cell death after brain ischemia and in late-onset neurodegenerative diseases. Both excitotoxic necrosis and apoptosis have been implicated in cell death induced by metabolic impairment. However, the factors that determine whether cells undergo apoptosis or necrosis are not known. In the present study, metabolic impairment was induced by 3-nitropropionic acid (3-NP), a suicide inhibitor of succinate dehydrogenase. Treatment of cultured rat hippocampal neurons with 3-NP resulted in two types of cell death with distinct morphological, pharmacological, and biochemical features. A rapid necrotic cell death, characterized by cell swelling and nuclear shrinkage, could be completely prevented by the NMDA receptor antagonist MK-801 (10 microM) and dose-dependently potentiated by low micromolar levels of extracellular glutamate. A slowly evolving apoptotic death, characterized by nuclear fragmentation, was not attenuated by MK-801 but was prevented by cycloheximide (1 microg/ml). The combination of MK-801 and cycloheximide resulted in an almost complete protection against 3-NP-induced cell death. DNA fragmentation, detected by the terminal deoxynucleotidyl transferase-mediated dUTP-X 3' nick end-labeling technique, was a late event in apoptosis and also occurred after necrotic cell death. ATP depletion was an early event in the 3-NP-induced neuronal degeneration, and the decline in ATP was exacerbated by glutamate. We conclude that 3-NP triggers two separate cell death pathways: an excitotoxic necrosis as a result of NMDA receptor activation and a delayed apoptosis that is NMDA receptor-independent. Mildly elevated levels of extracellular glutamate shift the cell death mechanism from apoptosis to necrosis.

A deficit in glucose uptake and a deposition of amyloid beta-peptide (A beta) each occur in vulnerable brain regions in Alzheimer's disease (AD). It is not known whether mechanistic links exist between A beta deposition and impaired glucose transport. We now report that A beta impairs glucose transport in cultured rat hippocampal and cortical neurons by a mechanism involving membrane lipid peroxidation. A beta impaired 3H-deoxy-glucose transport in a concentration-dependent manner and with a time course preceding neurodegeneration. The decrease in glucose transport was followed by a decrease in cellular ATP levels. Impairment of glucose transport, ATP depletion, and cell death were each prevented in cultures pretreated with antioxidants. Exposure to FeSO4, an established inducer of lipid peroxidation, also impaired glucose transport. Immunoprecipitation and Western blot analyses showed that exposure of cultures to A beta induced conjugation of 4-hydroxynonenal (HNE), an aldehydic product of lipid peroxidation, to the neuronal glucose transport protein GLUT3. HNE induced a concentration-dependent impairment of glucose transport and subsequent ATP depletion. Impaired glucose transport was not caused by a decreased energy demand in the neurons, because ouabain, which inhibits Na+/K(+)-ATPase activity and thereby reduces neuronal ATP hydrolysis rate, had little or no effect on glucose transport. Collectively, the data demonstrate that lipid peroxidation mediates A beta-induced impairment of glucose transport in neurons and suggest that this action of A beta may contribute to decreased glucose uptake and neuronal degeneration in AD.

In this paper, we describe the status of the exploration and use of the Ginkgo biloba leaves in China. We emphasize the need for careful studies of the intra-specific genetic diversity of Ginkgo biloba since genetic variation within this species may result in significant differences between the chemical and pharmacological properties of the different sub-species. It is therefore imperative that we catalog the intraspecific diversity of Ginkgo biloba L., and we stress that it is important to conserve this diversity since different subspecies may have different pharmacochemical properties resulting in differential medical usages.

To determine if impaired energy metabolism might contribute to some aspects of Alzheimer disease (AD), including the vulnerability of the CA1 region of the hippocampal formation and the altered cytoskeleton evident in neurofibrillary tangles, we examined the effects of metabolic poisons on neuronal damage and cytoskeletal disruption in the hippocampal formation. Intrahippocampal injection of 3-nitropropionic acid (3-NP) and malonic acid resulted in neuronal death, particularly in CA1. Cytoskeletal disruption included loss of dendritic MAP2, but sparing of axonal gamma. MK-801 (a noncompetitive NMDA receptor antagonist) did not atentuate the lesions produced by intrahippocampal injection of malonate. MK-801, however, was effective against intrastriatal malonate. Acute systemic 3-NP resulted in neuronal damage and cytoskeletal disruption in the CA1 region of the hippocampal formation, including an extensive loss of MAP2 immuno-reactivity, but sparing of gamma. The neuronal loss in CA1 was delayed as compared to striatum. Chronic intraventricular infusion of 3-NP produced a different pattern of neuronal damage. Loss of gamma-1 immuno-reactivity was observed in CA3 and CA1 s. orients, whereas MAP2 immunostaining was preserved. These results demonstrate that chronic and acute administration of metabolic inhibitors produce distinct patterns of neuronal damage and cytoskeletal disruption. The results further suggest a differential involvement of the NMDA receptor in malonate-induced neuronal damage in striatum as compared to the hippocampus. The pattern of neuronal damage and cytoskeletal disruption observed following acute metabolic impairment resembled some aspects of neurofibrillary pathology in AD, but did not result in gamma hyperphosphorylation.

Impaired energy metabolism may contribute to the pathogenesis of late-onset neurodegenerative disorders such as Alzheimer's disease by increasing neuronal vulnerability to excitotoxic damage through the NMDA receptor. The effects of metabolic impairment on the striatum have been extensively examined, but relatively little is known regarding the vulnerability of the hippocampus. To examine the effect of metabolic impairment on the hippocampal formation, malonate (0.25-2.5 mumol), a reversible inhibitor of succinate dehydrogenase, was administered by stereotaxic injection into the hippocampus of male Sprague-Dawley rats. Neuronal loss was assessed by Nissl stain, and immunocytochemistry was used to examine cytoskeletal disruption. Malonate produced a dose-dependent lesion in which CA1 pyramidal neurons were most vulnerable, followed by CA3 and dentate gyrus. Cytoskeletal alterations included the loss of microtubule-associated protein 2 (MAP2) and dendritic MAP1B immunoreactivity, whereas axonal MAP1B and tau proteins were relatively spared. Spatially and temporally correlated with the loss of MAP2 was an increase in the immunoreactivity of calpain-cleaved spectrin. A similar pattern of neuronal damage and cytoskeletal disruption was produced by intrahippocampal injection of quinolinate (0.1 mumol), an NMDA agonist. Although these results are consistent with the hypothesis that metabolic impairment results in excitotoxic death, MK-801 (dizocilipine maleate), a noncompetitive NMDA receptor antagonist, did not attenuate the lesions produced by malonate but was effective against quinolinate. The results suggest that NMDA receptor activation is not required for malonate-induced damage in the hippocampal formation.

We have identified a multistep mechanism by which the DNA virus SV40 overcomes cellular senescence. Expression of SV40 T antigen is required for both transient extension of life span and unlimited life span or immortalization. These effects are mediated through inactivation of function of growth suppressors pRB and p53 via complex formation with T antigen. However, immortalization additionally requires inactivation of a novel growth suppressor gene, which has recently been identified to be on the distal portion of the long arm of chromosome 6, designated SEN6. We propose that SEN6 is responsible for cellular senescence in fibroblasts and other cells.

Investigations of neurofilament alterations in neurodegenerative disorders utilize postmortem human tissues obtained at autopsy. To determine if alterations in the levels or distribution of neurofilament proteins might occur during the interval between death and autopsy, the postmortem cooling curve of the human brain was modeled in Sprague-Dawley rats and neurofilament proteins were examined by immunocytochemistry and immunoblots. One hour after death, enhanced perikaryal immunostaining of NF-M and both phosphorylated and nonphosphorylated NF-H epitopes was observed throughout the hippocampal formation. A greater number of neurons exhibited increased somatic immunostaining 4-h postmortem. In addition, loss of neurofilament protein immunostaining was observed in the neuropil, particularly in the molecular layer of the dentate gyrus. This corresponded with, but lagged behind, the pattern of calpain activation determined using an antibody against calpain-cleaved alpha-spectrin. Immunoblots confirmed the postmortem loss of neurofilament proteins in both triton-soluble and insoluble fractions. These results demonstrate that the levels and localization of neurofilament proteins observed in tissues obtained at autopsy even with short postmortem intervals may not accurately reflect the premortem condition.

The complete nucleotide sequence of the N protein gene of rice yellow stunt rhabdovirus (RYSV) was determined by sequencing of cDNA clones derived from the viral genomic RNA. The 3' end of the N gene (messenger sense) was defined by sequence analysis of cDNA clones generated from the N protein mRNA by 3'RACE. The 5' end sequence of the gene was putatively assigned as 5'-AACAC-3'; this sequence is found in the presumed 3' leader/N gene junction region. The mRNA encoding the RYSV N protein is 1714 nt comprising a 15-nt untranslated 5' leader sequence followed by an open reading frame (ORF) of 1563 nt and a 136-nt untranslated 3' region. The calculated molecular mass of the N protein encoded by the ORF is 58,400 Da, which is larger in size than N proteins of other rhabdoviruses. Amino acid composition analysis shows that the RYSV N protein is rather basic with a predicted isoelectric point of 10.04; indeed, a large highly basic region could be found at the carboxy terminal portion of the protein. Amino acid sequence comparison between N proteins of RYSV and sonchus yellow net virus, both of which belong to the same genus Nucleorhabdovirus, revealed an overall 30% identity, with three relatively conserved blocks of 14-20 amino acid residues. Moreover, the hydropathy profiles of the two proteins are generally similar. The structural similarities between the N protein of RYSV and that of lettuce necrotic yellows virus, the type member of the genus Cytorhabdovirus, and those of animal rhabdoviruses, are less significant. Nucleotide sequence determination of 5' and 3' regions flanking the RYSV N gene identified a 14-nt common sequence that is very similar to the consensus gene junction sequences of other plant and animal rhabdoviruses.

The gene for black-ear coat colour pattern, commonly found among cattle of tropical origin, was observed in 809 animals of four breeds of local cattle in southern China. Gene frequencies for Tf, Hb and Alb demonstrated that these groups of cattle were quite divergent from Bos taurus. These breeds of cattle are thought to be descended from ancient Chinese cattle. At the same time certain influences observed in coat colour may be derived from Bali cattle.