Estrogen modulates the expression of myosin heavy chain in detrusor smooth muscle

The effect of low serum estrogen levels on urinary bladder function remains poorly understood. Using a rabbit model, we analyzed the effects of estrogen on the expression of the isoforms of myosin, the molecular motor for muscle contraction, in detrusor smooth muscle. Expression of myosin heavy chain (MHC) isoforms, which differ in the COOH-terminal (SM1 and SM2) and the NH(2)-terminal (SM-A and SM-B) regions as a result of alternative splicing of the mRNA at either the 3'- or 5'-ends, was analyzed in age-matched female rabbits that were sham operated, ovariectomized (Ovx), and given estrogen after ovariectomy (4 rabbits/group). Ovx rabbits showed a significant decrease in the overall MHC content per gram of wet detrusor smooth muscle compared with controls (P < 0.04), which was reversed by estrogen replacement (P < 0.02). MHC content, as a proportion of total milligram of protein in the bladder tissue extracted, was also increased in estrogen-treated Ovx rabbits. Quantitative competitive RT-PCR revealed 1.72-, 2.63-, and 5.82 x 10(6) copies of MHC mRNA/100 ng total mRNA in Ovx, control, and estrogen-treated rabbits, respectively (P < 0.01). RT-PCR analysis using oligonucleotides specific for the region containing the SM1/SM2 MHC alternative splice sites indicated a lower SM2-to-SM1 ratio in estrogen-treated compared with control and Ovx rabbits (P < 0.05). Similarly, SDS-PAGE analysis of extracted myosin from estrogen-treated rabbits revealed a significantly lower SM2-to-SM1 isoform ratio compared with control and Ovx rabbits (P < 0.05). Expression of the SM-A and SM-B isoforms was not affected. These results indicate that myosin content is increased upon estrogen replacement in Ovx rabbits and that the abundance of SM1 relative to SM2 is greater in estrogen-treated rabbits compared with normal and Ovx rabbits. These data suggest that estrogen affects alternative splicing at the 3'-end of the MHC pre-mRNA to increase the proportion of SM1 vs. SM2.     

Modeling and analysis of a virus that replicates selectively in tumor cells

Replication-competent viruses have shown considerable promise in overcoming the inefficient gene transduction experienced by traditional gene therapy approaches to cancer treatment. The viruses infect tumor cells and replicate inside them, eventually causing lysis. Virus particles released during lysis are then able to infect other tumor cells, and, in this way, continuous rounds of infection and lysis allow the virus to spread throughout the tumor. Motivated by this novel cancer treatment, we formulate and analyse a system of partial differential equations that is essentially a radially-symmetric epidemic model embedded in a Stefan problem. We compare three, alternative virus-injection strategies: a fixed fraction of cells pre-infected with the virus are introduced throughout the entire tumor volume, within the tumor core, or within the tumor rim. For all three injection methods, simple and accurate conditions that predict whether the virus will control the tumor are derived.     

Assessing screening policies for childhood obesity

To address growing concerns over childhood obesity, the United States Preventive Services Task Force (USPSTF) recently recommended that children undergo obesity screening beginning at age 6. An Expert Committee recommends starting at age 2. Analysis is needed to assess these recommendations and investigate whether there are better alternatives. We model the age- and sex-specific population-wide distribution of BMI through age 18 using National Longitudinal Survey of Youth (NLSY) data. The impact of treatment on BMI is estimated using the targeted systematic review performed to aid the USPSTF. The prevalence of hypertension and diabetes at age 40 are estimated from the Panel Study of Income Dynamics (PSID). We fix the screening interval at 2 years, and derive the age- and sex-dependent BMI thresholds that minimize adult disease prevalence, subject to referring a specified percentage of children for treatment yearly. We compare this optimal biennial policy to biennial versions of the USPSTF and Expert Committee recommendations. Compared to the USPSTF recommendation, the optimal policy reduces adult disease prevalence by 3% in relative terms (the absolute reductions are <1%) at the same treatment referral rate, or achieves the same disease prevalence at a 28% reduction in treatment referral rate. If compared to the Expert Committee recommendation, the reductions change to 6 and 40%, respectively. The optimal policy treats mostly 16-year olds and few children under age 14. Our results suggest that adult disease is minimized by focusing childhood obesity screening and treatment on older adolescents.     

Generation of a 3D model for human GABA transporter hGAT-1 using molecular modeling and investigation of the binding of GABA

A three-dimensional model of the human Na⁺/Cl^?-dependent γ-aminobutyric acid (GABA) transporter hGAT-1 was developed by homology modeling and refined by subsequent molecular modeling using the crystal structure of a bacterial homologue leucine transporter from Aquifex aeolicus (LeuT_Aa) as the template. Protein structure quality checks show that the resulting structure is particularly suited for the analysis of the substrate binding pocket and virtual screening experiments. Interactions of GABA and the substrate binding pocket were investigated using docking studies. The difference of 6 out of 13 substrate interacting side chains between hGAT-1 and LeuT_Aa lead to the different substrate preference which can be explained using our three-dimensional model of hGAT-1. In particular the replacement of serine 256 and isoleucine 359 in LeuT_Aa with glycine and threonine in hGAT-1 seems to facilitate the selection of GABA as the main substrate by changing the hydrogen bonding pattern in the active site to the amino group of the substrate. For a set of 12 compounds flexible docking experiments were performed using LigandFit in combination with the Jain scoring function. With few exceptions the obtained rank order of potency was in line with experimental data. Thus, the method can be assumed to give at least a rough estimate of the potency of the potential of GABA uptake inhibitors.     

Metabolic basis for contractile dysfunction following chronic partial bladder outlet obstruction in rabbits

Our study is designed to correlate nitrite concentration, an index of nitric oxide (NO) release with mast cell peroxidase (MPO), a marker of cardiac mast cell degranulation and cardioprotective effect of ischaemic preconditioning in isolated perfused rat heart subjected to 30 min of global ischaemia and 30 min of reperfusion. Ischaemic preconditioning, comprised of four episodes of 5 min global ischaemia and 5 min of reperfusion, markedly reduced the release of lactate dehydrogenase (LDH) and creatine kinase (CK) in coronary effluent and incidence of ventricular premature beats (VPBs) and ventricular tachycardia and fibrillation (VT/VF) during reperfusion phase. Ischaemia-reperfusion induced release of MPO was markedly reduced in ischaemic preconditioned hearts. Increased release of nitrite was noted during reperfusion phase after sustained ischaemia in preconditioned hearts as compared to control hearts. No alterations in the release of nitrite was observed immediately after ischaemic preconditioning. However, ischaemic preconditioning markedly increased the release of MPO prior to global ischaemia. It is proposed that cardioprotective and antiarrhythmic effect of ischaemic preconditioning may be ascribed to degranulation of cardiac mast cells. Depletion of cytotoxic mediators during ischaemic preconditioning and consequent decreased release of these mediators during sustained ischaemia-reperfusion may be associated with preservation of structures in isolated rat heart responsible for NO release.     

Metabolic studies on rabbit bladder smooth muscle and mucosa

Recent studies indicate that the mucosa of the urinary bladder may play a major role in the maintenance of normal bladder function. The mucosal surface of the urinary bladder serves as a protective layer against the irritative solutes found in the urine. The integrity of this barrier can be broken by overdistension, anoxia, detergents, alcohols, bacterial infection and by contact with agents to which the mucosa has been sensitized.In view that both anoxia and ischemia can mediate a breakdown in the role of the mucosal layer as a permeability barrier, it is reasonable to assume that this function is dependent on cellular metabolism. As an initial investigation we have compared a variety of biochemical and metabolic parameters between the mucosal layer (consisting of the lamina propria, urothelium, and any connective tissue and vascular tissue within this layer); and the muscularis layer.The results of these studies demonstrated that the rate of glucose metabolism to lactic acid (LA) of the mucosa was more than three-fold greater than that of the smooth muscle. The rate of CO₂ production of the mucosa was 60% greater than that of the unstimulated smooth muscle. The maximal activity of the mitochondrial enzyme citrate synthase was significantly greater in the mucosa than in the smooth muscle, however, the activity of malate dehydrogenase was similar for both tissues. The maximal activity of the cytosolic enzyme creatine kinase was more than two-fold greater in the bladder smooth muscle than in the mucosa; although the affinities of the creatine kinase isoforms of the mucosa were sigificantly greater than those of the muscle.Although the concentrations of ATP and ADP were similar in both muscle and mucosa, the level of creatine phosphate (CP) was over four-fold greater in the bladder muscle while the level of AMP in the muscle was only 58% of that in the mucosal epithelium.In summary, the rate of glucose metabolism was greater in the mucosa than in the smooth muscle although the concentrations of high energy phosphates (ATP+CP) was significantly greater in the smooth muscle. Future studies will be directed at identifying the specific cellular processes within the mucosal layer that relate to the function of the urothelium as a permeability barrier.     

模拟氮沉降对藏北高寒草甸温室气体排放的影响

目前,高寒草甸对全球温室效应的贡献仍具有不确定性,而随着N沉降的增加,该系统温室体气排放也必将发生变化。为揭示高寒草甸对N沉降的响应机制,探讨其对全球变化的反馈作用,利用人工添加氮素的方法,于2014年生长季(6-9月)在那曲地区那曲县设置不同水平N添加梯度(0、7、20kg hm~(-2)a~(-1)和40 kg hm~(-2)a~(-1)),模拟氮沉降增加对藏北高寒草甸温室气体排放的影响。经过1a的研究结果表明:1)施氮显著促进了CO_2排放但对CH_4的吸收和N_2O的排放无显著影响。总体而言,添加氮素明显增加了温室气体排放总量,其中N2O处理下高寒草甸温室气体排放总量最高。2)回归分析结果表明,CO_2与NPP(总生物量)和TOC(土壤有机碳)线性相关(P0.05),而与TN(总氮)、NH_4~+-N和NO_3~--N均无显著相关关系(P0.05),CH_4与TN/NPP/TOC/NH_4~+-N/NO_3~--N均不相关(P0.05),N_2O与NPP/TOC/NO_3~--N均显著线性相关(P0.05),而与TN/NH_4~+-N不相关。综合初步研究结果,未来氮沉降增加条件下,藏北高寒草甸温室气体排放通量将有可能明显增加,从而对气候变化产生重要的反馈作用。