Expression of nerve growth factor (NGF), and its receptors TrkA and p75 in the reproductive organs of the adult male rats

Immunolocalization of nerve growth factor (NGF) and its receptors, TrkA and p75 in the reproductive organs of adult male rats was investigated. Sections of the testis, efferent duct, epididymis, deferent duct, seminal vesicle, coagulating gland and prostate of adult male rats were immunostained by the avidin-biotin-peroxidase complex methods (ABC). NGF was expressed in Leydig cells, primary spermatocytes and pachytene spermatocytes in the testis. TrkA only immunoreacted to elongate spermatids and p75 showed positive immunostaining in the Sertoli cells, Leydig cells, the pachytene spermatocytes and elongate spermatids. Immunoreactions for NGF and its two receptors were detected in epithelial cells of efferent duct, deferent duct and epididymis. In addition, immunoreactions for NGF and its two receptors were also observed in columnar secretory epithelium lines of the seminal vesicles, prostate and coagulating gland. These results suggest that NGF is an important growth factor in gonadal function of adult male rats.     

Biosynthesisin vitro of neolactotetraosylceramide by a galactosyltransferase from mouse T-lymphoma: purification and kinetic studies; synthesis of neolacto and polylactosamine core

The galactosyltransferase, GalT-4, which catalyses the biosynthesisin vitro of neolactotetraosylceramide, nLcOse4Cer (Gal1-4GleNAc1-3Gal1-4Glc-Cer) from lactotriaosylceramide, LcOse3Cer (GlcNAc1-3Gal1-4Glc-Cer), and UDP-galactose has been purified 107 500-fold from a mineral oil induced mouse T-lyphoma P-1798, using affinity columns. The purified enzyme is partially stabilized in the presence of phospholipid liposomes. Two closely migrating protein bands of apparent molecular weights 56 kDa and 63 kDa were observed after sodium dodecyl sulfate polyacrylamide gel electrophoresis of highly purified mouse GalT-4. These two protein bands, when subjected to limited proteolysis, resulted in three peptides with identical mobilities indicating amino acid sequence identity between the proteins. Both protein bands from P-1798 gave a positive immunostain when tested with polyclonal antibody against bovine lactose synthetase (UDP-Gal:Glc 4-galactosyltransferase) following Western blot analysis on nitrocellulose paper. The enzyme has a pH optimum between 6.5 and 7.0 and like all other galactosyltransferases, GalT-4 has absolute requirements for divalent cation (Mn²⁺). TheK _m values for the substrate LcOse3Cer and donor UDP-galactose are 110 and 250 µm, respectively. Substrate competition studies with LcOse3Cer and either asialo-agalacto-1-acid glycoprotein orN-acetylglucosamine revealed that these reactions might be catalysed by the same protein. The only other glycolipid which showed acceptor activity toward the purified GalT-4 was iLcOse5Cer (GlcNAc1-1-3Gal1-4Lc3), the precursor for polylactosamine antigens. However, competition studies with these two active substrates using the most purified enzyme fraction, revealed that these two reactions might be catalysed by two different proteins since the experimental values were closer to the theoretical values calculated for two enzymes. Interestingly however, it seems that the GalT-4 from P-1798 has an absolute requirement for anN-acetylglucosamine residue in the substrate since the lyso-derivative (GlcNH₂1-3Gal1-4Glc-sphingosine) of the acceptor glycolipid LcOse3Cer is completely inactive as substrate while theK _m andV _max of the reacetylated substrate (GlcNac1-3Gal1-4Glc-acetylsphingosine) was comparable with LcOse3Cer. Autoradiography of the radioactive product formed by purified P-1798 GalT-4 confirmed the presence of nLcOse4Cer, as the product cochromatographed with authentic glycolipid. The monoclonal antibody IB-2, specific for nLcOse4Cer, also produced a positive immunostained band on TLC as well as giving a positive ELISA when tested with radioactive product obtained using a highly purified enzyme from mouse P-1798 T-lymphoma.Abbreviations EDTA ethylenediamine tetraacetate - ME -mercaptoethanol - PEG polyethylene glycol - PBS phosphate buffered saline - Suc sucrose - Mn²⁺ manganese - Gal galactose - GlcNAc N-acetylglucosamine - UDP-Gal Uridine diphosphate galactose - Ab antibody - SDS sodium dodecyl sulphate - PAGE polyacrylamide gel electrophoresis - ECB embryonic chicken brain - Cer ceramide - nLc4 or NlcOse4Cer Gal1-4GleNac1-3Gal1-4Glc-Cer, neoLactotetraosylceramide - Lc3 or LcOse3Cer GlcNac1-3Gal1-4Glc-Cer, lactotriaosylceramide - iLc5 iLcOse5Cer, GlcNAc1-3nLcOse4Cer - nLc6 nLcOse6Cer, Gal1-4iLcOse5Cer - SA^–Gal^–₁AGP asialo-agalacto1-acid glycoprotein - TLC thin layer chromatography     

Impact of the Economic Recession on Companion Animal Relinquishment,Adoption, and Euthanasia: A Chicago Animal Shelter's Experience

This study investigated how the current economic recession (since December 2007) has affected dog and cat relinquishment, adoption, and euthanasia at the Anti-Cruelty Society animal shelter in Chicago, Illinois. The study compared temporal patterns of the investigated statistics before (2000–2007) the start of the current recession with the patterns after the start of the recession (2008–2010). The results showed that once the guardianship (ownership) of a nonhuman animal had been established, the recession did not greatly affect the owner's decision on relinquishment—except for the relinquishment of senior dogs, which may be associated with increased costs of care. However, an unfavorable economic environment may have reduced adoption of animals. The consequences of a decline in adoptions might be reflected in an increase in the proportion or number of sheltered animals euthanized. This study demonstrated how monitoring changes in temporal patterns in these shelter statistics can help guide animal shelters to better prepare for the current recession.     

Heat shock protein 27 is increased in cyanotic tetralogy of Fallot myocardium and is associated with improved cardiac output and contraction

Tetralogy of Fallot (TOF) is a congenital heart condition in which the right ventricle is exposed to cyanosis and pressure overload. Patients have an increased risk of right ventricle dysfunction following corrective surgery. Whether the cyanotic myocardium is less tolerant of injury compared to non-cyanotic is unclear. Heat shock proteins (HSPs) protect against cellular stresses. The aim of this study was to examine HSP 27 expression in the right ventricle resected from TOF patients and determine its relationship with right ventricle function and clinical outcome. Ten cyanotic and ten non-cyanotic patients were studied. Western blotting was used to quantify HSP 27 in resected myocardium at (1) baseline (first 15 min of aortic cross clamp and closest representation of pre-operative status) and (2) after 15 min during ischemia until surgery was complete. The cyanotic group had significantly increased haematocrit, lower O₂ saturation, thicker interventricular septal wall thickness and released more troponin-I on post-operative day 1 (p?<?0.05). HSP 27 expression was significantly increased in the <15 min cyanotic compared to the <15 min non-cyanotic group (p?=?0.03). In the cyanotic group, baseline HSP 27 expression also significantly correlated with oxygen extraction ratio (p?=?0.028), post-operative basal septal velocity (p?=?0.036) and mixed venous oxygen saturation (p?=?0.02), markers of improved cardiac output/contraction. Increased HSP 27 expression and associated improved right ventricle function and systemic perfusion supports a cardio-protective effect of HSP 27 in cyanotic TOF.     

六盘水酸模属植物资源的初步调查研究

通过野外调查、标本采集、分类学研究及资料考证,对六盘水酸模属植物的资源进行了调查.结果表明,六盘水有酸模属植物7种,其中钝叶酸模(Rumex obtusifoliusL.)为贵州省的分布新纪录,并对酸模属植物药用价值、食用价值及生态价值等进行了分析.     

Glucocorticoid receptor and sequential P53 activation by dexamethasone mediates apoptosis and cell cycle arrest of osteoblastic MC3T3-E1 cells

Glucocorticoids play a pivotal role in the proliferation of osteoblasts, but the underlying mechanism has not been successfully elucidated. In this report, we have investigated the molecular mechanism which elucidates the inhibitory effects of dexamethasone on murine osteoblastic MC3T3-E1 cells. It was found that the inhibitory effects were largely attributed to apoptosis and G1 phase arrest. Both the cell cycle arrest and apoptosis were dependent on glucocorticoid receptor (GR), as they were abolished by GR blocker RU486 pre-treatment and GR interference. G1 phase arrest and apoptosis were accompanied with a p53-dependent up-regulation of p21 and pro-apoptotic genes NOXA and PUMA. We also proved that dexamethasone can't induce apoptosis and cell cycle arrest when p53 was inhibited by p53 RNA interference. These data demonstrate that proliferation of MC3T3-E1 cell was significantly and directly inhibited by dexamethasone treatment via aberrant GR activation and subsequently P53 activation.     

Validation and application of an LC–MS/MS method for quantitation of three fatty acid ethanolamides as biomarkers for fatty acid hydrolase inhibition in human plasma

Endogenous ethanolamides (fatty acid amides), including arachidonyl ethanolamide (anandamide, AEA), oleoyl ethanolamide (OEA), and palmitoyl ethanolamide (PEA), are substrates of fatty acid amide hydrolase (FAAH). FAAH may play an important role for pain, anxiety/depression, and metabolic disorders. Ethanolamides are considered to be potential pharmacodynamic biomarkers to determine target engagement for FAAH inhibition by novel pharmaceutical agents. A highly selective, sensitive, and high-throughput liquid chromatography tandem mass spectrometry (LC–MS/MS) method was developed and validated for simultaneous quantitation of AEA, OEA, and PEA in human plasma. The method employed D₄-AEA, D₄-OEA, and ¹³C₂-PEA as “surrogate analytes” to establish the concentration–mass response relationship, i.e. a regression equation. The concentrations of AEA, OEA, and PEA were calculated based on the regression equations derived from the surrogate analytes. This approach made it possible to prepare calibration standard and quality control (QC) samples in plasma devoid of interferences from the endogenous analytes. The analytical methodology required 150 μL of human plasma that was processed via liquid–liquid extraction (LLE) using a 96-well plate format. Chromatographic separation was achieved with a reversed-phase high performance liquid chromatography (HPLC) column using gradient elution, and the run time was 3 min. The method was fully validated and it demonstrated acceptable accuracy, precision, linearity, and specificity. The lower limit of quantitation (LLOQ) was 0.1/0.5/0.5 ng/mL for AEA/OEA/PEA, which was sensitive enough to capture the basal plasma levels in healthy subjects. Bench-top stability in plasma, freeze–thaw stability in plasma, frozen long-term stability in plasma, autosampler stability, and stock solution stability all met acceptance criteria (%Bias within ±12.0%). Characterization of stability in purchased/aged blood indicated that ethanolamides are subject to degradation mediated by intracellular membrane-bound FAAH, which has been shown to be inhibited by phenylmethylsulfonyl fluoride (PMSF). In the presence of PMSF, ethanolamide levels increased slightly over time, suggesting that blood cells release ethanolamides into plasma. Whole blood stability conducted in fresh blood immediately following collection revealed that there was significant elevation of ethanolamide concentrations (∼1.3–2.0-fold on ice and ∼1.5–3.0-fold at room temperature by 2 h), indicating that de novo synthesis and release from blood cells were the predominant factors affecting ethanolamide concentrations ex vivo. Accordingly, conditions that ensured rapid separation of plasma from blood cells and consistency in the blood harvesting procedures were established and implemented for clinical studies to minimize the ex vivo elevation of plasma ethanolamide concentrations. The variability (intra-subject and inter-subject) of plasma ethanolamide levels was evaluated in healthy subjects during a Phase 0 study (no drug administration) that simulated the design of single-ascending dose and multiple-ascending dose clinical trials in terms of sample collection time points, population, food, and activity. The data indicated there was relatively large inter- and intra-subject variation in plasma ethanolamide concentrations. In addition, apparent variations due to time of day and/or food effects were also revealed. Understanding the variability of ethanolamide levels in humans is very important for study design and data interpretation when changes in ethanolamide levels are used as target engagement biomarkers in clinical trials.     

Health Related Quality of Life and Influencing Factors among Welders

Background

Occupational exposure to welding fumes is a serious occupational health problem all over the world. Welders are exposed to many occupational hazards; these hazards might cause some occupational diseases. The aim of the study was to assess the health related quality of life (HRQL) of electric welders in Shanghai China and explore influencing factors to HRQL of welders.

Methods

301 male welders (without pneumoconiosis) and 305 non-dust male workers in Shanghai were enrolled in this study. Short Form-36 (SF-36) health survey questionnaires were applied in this cross-sectional study. Socio-demographic, working and health factors were also collected. Multiple stepwise regress analysis was used to identify significant factors related to the eight dimension scores.

Results

Six dimensions including role-physical (RP), bodily pain (BP), general health (GH), validity (VT), social function (SF), and mental health (MH) were significantly worse in welders compared to non-dust workers. Multiple stepwise regress analysis results show that native place, monthly income, quantity of children, drinking, sleep time, welding type, use of personal protective equipment (PPE), great events in life, and some symptoms including dizziness, discomfort of cervical vertebra, low back pain, cough and insomnia may be influencing factors for HRQL of welders. Among these factors, only sleep time and the use of PPE were salutary.

Conclusions

Some dimensions of HRQL of these welders have been affected. Enterprises which employ welders should take measures to protect the health of these people and improve their HRQL.     

Downregulation of Sirt1 as aging change in advanced heart failure

Background

In congestive heart failure the balance between cell death and cell survival in cardiomyocytes is compromised. Sirtuin 1 (Sirt1) activates cell survival machinery and has been shown to be protective against ischemia/reperfusion injury in murine heart. The role of Sirt1 in heart failure, especially in human hearts is not clear.

Results

The expression of Sirt1 and other (associated) downstream molecules in human cardiomyocytes from patients with advanced heart failure was examined. Sirt1 was down-regulated (54.92% ± 7.80% in advanced heart failure samples compared with healthy control cardiomyocytes). The modulation of molecules involved in cardiomyocyte survival and death in advanced heart failure were also examined. The expression of Mn-superoxide dismutase and thioredoxin1, as well as an antiapoptotic molecule, Bcl-xL, were all significantly reduced in advanced heart failure cardiomyoctes (0.71 ± 0.02-fold, 0.61 ± 0.05-fold, and 0.53 ± 0.08-fold vs. control, respectively); whereas the expression of proapoptotic molecule Bax was significantly increased (1.62 ± 0.18-fold vs. control). Increased TUNEL-positive number of cardiomyocytes and oxidative stress, confirmed by 8-hydorxydeoxyguanosine staining, were associated with advanced heart failure. The AMPK-Nampt-Sirt1 axis also showed inhibition in advanced heart failure in addition to severely impaired AMPK activation. Increased p53 (acetyl form) and decreased FoxO1 translocation in the nucleus may be the mechanism of down-regulation of antioxidants and up-regulation of proapoptotic molecules due to low expression of Sirt1.

Conclusion

In advanced heart failure, low Sirt1 expression, like aging change may be a significant contributing factor in the downregulation of antioxidants and upregulation of proapoptotic molecules through the p53, FoxO1, and oxidative stress pathways.     

糖基化终产物对原代培养乳鼠肾脏细胞损伤的影响

糖基化终产物(AGEs)在糖尿病肾病的发生发展过程中起着重要的作用.但目前其作用机制还不太清楚.通过体外乳鼠肾脏细胞的原代培养,探讨AGEs对肾细胞的损伤作用及可能的作用机制.取出生3天的SD大鼠的乳鼠肾脏进行体外原代细胞培养,并取传代到4-6代的细胞进行实验研究.分别用不同浓度的AGEs(0、1.2、2.5、5、10、20 mg/ml),不同的作用时间(6、12、18、24 h)作用于体外培养的肾细胞,用MTT法检测AGEs对肾细胞的增殖情况,用酶试剂盒法检测AGEs对肾细胞培养液中乳酸脱氢酶(LDH)、β-N-乙酰氨基葡萄糖苷酶(NAG)的含量,以及肾细胞内还原型谷胱甘肽(GSH)和超氧化物歧化酶(SOD)的含量.实验结果表明随着AGEs作用肾细胞时间的延长和浓度的增加,细胞存活率、细胞内GSH含量和SOD活性均逐渐下降,而细胞培养液中LDH和NAG的含量则逐渐升高,与正常培养的对照组细胞相比差异非常显著(P＜0.001),并且AGEs对细胞的作用与其浓度和作用时间呈显著的量效关系.实验结果说明AGEs对原代培养的肾细胞有明显的损伤作用,并随着AGEs作用浓度的增加和作用时间的延长对肾细胞的损伤越来越严重,实验结果也表明.肾细胞对AGEs的作用很敏感,其损伤细胞的途径和作用机制可能是由于改变了肾细胞膜的通透性和降低肾细胞抗氧化能力,该实验研究也进一步提示了AGEs是导致糖尿病肾脏并发症发生的重要原因之一.