N+注入和60Co-γ辐照对柠檬酸发酵菌黑曲霉的诱变效应

首次尝试了应用两种核技术手段(同时)对黑曲霉进行诱变,即将大剂量^60Co-γ辐照的黑曲霉孢于直接进行低能氮离子注入,使处于休眠状态下的黑曲霉抱子同时受至^60Co-γ辐照和N^ 注入的作用。通过溴甲酚绿指示性平板辅助筛选和摇瓶发酵。获得了1株产酸提高18．44％、转化率达1034．5％的M3代菌株CN05,为柠檬酸发酵菌黑曲霉的进一步育种工作提供了诱变参数和高产出发菌株。     

Electron tomography of fast frozen, stretched rigor fibers reveals elastic distortions in the myosin crossbridges

As a first step toward freeze-trapping and 3-D modeling of the very rapid load-induced structural responses of active myosin heads, we explored the conformational range of longer lasting force-dependent changes in rigor crossbridges of insect flight muscle (IFM). Rigor IFM fibers were slam-frozen after ramp stretch (1000 ms) of 1-2% and freeze-substituted. Tomograms were calculated from tilt series of 30 nm longitudinal sections of Araldite-embedded fibers. Modified procedures of alignment and correspondence analysis grouped self-similar crossbridge forms into 16 class averages with 4.5 nm resolution, revealing actin protomers and myosin S2 segments of some crossbridges for the first time in muscle thin sections. Acto-S1 atomic models manually fitted to crossbridge density required a range of lever arm adjustments to match variably distorted rigor crossbridges. Some lever arms were unchanged compared with low tension rigor, while others were bent and displaced M-ward by up to 4.5 nm. The average displacement was 1.6 +/- 1.0 nm. "Map back" images that replaced each unaveraged 39 nm crossbridge motif by its class average showed an ordered mix of distorted and unaltered crossbridges distributed along the 116 nm repeat that reflects differences in rigor myosin head loading even before stretch.     

Oxidative damage during chagasic cardiomyopathy development: role of mitochondrial oxidant release and inefficient antioxidant defense

In this study, we evaluated the oxidant status and antioxidant defense capabilities of the heart during the course of Trypanosoma cruzi infection and disease development in a murine model system. Our data show that the extent of protein carbonylation and lipid peroxidation is increased in the heart, but not the skeletal muscle, of infected mice. The level of oxidative injury biomarkers in the myocardium consistently increased with chronic disease severity. The antioxidant defense constituted by catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GSR), and reduced glutathione was increased in murine heart and skeletal tissue in response to the stress of T. cruzi infection. After the initial burst, CAT, GPx, and GSR remained unresponsive to the severity of chronic tissue damage in chagasic hearts. The cardiac level of Mn(2+) superoxide dismutase (MnSOD) was diminished in chagasic mice. Our data suggest that the host responds to acute injuries by activating antioxidant defenses that are of sufficient magnitude to scavenge the reactive oxidants in skeletal tissue. The myocardia of infected mice, however, sustain increased oxidative injuries with disease progression. We surmise that MnSOD deficiencies, resulting in the increased release of mitochondrial free radicals, lead to sustained oxidative stress that exceeds the cardiac antioxidant defense capacity and contribute to persistent oxidative damage in chagasic myocardium.     

Pituitary adenylate cyclase-activating polypeptide promotes differentiation of mouse neural stem cells into astrocytes

We have found that pituitary adenylate cyclase-activating polypeptide (PACAP) employed at the physiological concentrations induces the differentiation of mouse neural stem cells into astrocytes. The differentiation process was not affected by cAMP analogues such as dibutylic cAMP (db-cAMP) or 8Br-cAMP or by the specific competitive inhibitor of protein kinase A, Rp-adenosine-3',5'-cyclic monophosphothioate triethylamine salt (Rp-cAMP). Expression of the PACAP receptor (PAC1) in neural stem cells was detected by both RT-PCR and immunoblot using an affinity-purified antibody. The PACAP selective antagonist, PACAP(6-38), had an inhibitory effect on the PACAP-induced differentiation of neural stem cells into astrocytes. These results indicate that PACAP acts on the PAC1 receptor on the plasma membrane of mouse neural stem cells, with the signal then transmitted intracellularly via a PAC1-coupled G protein, does not involve Gs. This signaling mechanism may thus play a crucial role in the differentiation of neural stem cells into astrocytes.     

Purification and characterization of a vaterite-inducing peptide, pelovaterin, from the eggshells of Pelodiscus sinensis (Chinese soft-shelled turtle)

Proteins play a crucial role in the biomineralization of hard tissues such as eggshells. We report here the purification, characterization, and in vitro mineralization studies of a peptide, pelovaterin, extracted from eggshells of a soft-shelled turtle. It is a glycine-rich peptide with 42 amino acid residues and three disulfide bonds. When tested in vitro, the peptide induced the formation of a metastable vaterite phase. The floret-shaped morphology formed at a lower concentration ( approximately 1 microM) was transformed into spherical particles at higher concentrations (>500 microM). The solution properties of the peptide are investigated by circular dichroism (CD), fluorescence emission spectroscopy, and dynamic light scattering (DLS) experiments. The conformation of pelovaterin changed from an unordered state at a low concentration to a beta-sheet structure at high concentrations. Fluorescence emission studies indicated that the quantum yield is significantly decreased at higher concentrations, accompanied by a blue shift in the emission maximum. At higher concentrations a red-edge excitation shift was observed, indicating the restricted mobility of the peptide. On the basis of these observations, we discuss the presence of a peptide concentration-dependent monomer-multimer equilibrium in solution and its role in controlling the nucleation, growth, and morphology of CaCO(3) crystals. This is the first peptide known to induce the nucleation and stabilization of the vaterite phase in solution.     

遗传密码的新排列和起源探讨

根据DNA核苷酸组分的动态变化规律将遗传密码的传统排列按密码子对GC和嘌呤含量的敏感性进行了重排.新密码表可划分为2个半区（或1/2区）和4个四分区（或1/4区）.就原核生物基因组而言,当GC含量增加时,物种蛋白质组所含的氨基酸倾向于使用GC富集区和嘌呤不敏感半区所编码的氨基酸,它们均使用四重简并密码,对DNA序列的突变具有相对鲁棒性（Robustness）.当GC含量降低时,大多数密码子处于AU富集区和嘌呤敏感半区,这个区域编码的氨基酸具有物理化学性质的多样性.因为当密码子第三位核苷酸（CP3）在嘌呤和嘧啶之间发生转换时,密码子所编码的氨基酸也倾向于发生变化.关于遗传密码的进化存在多种假说,包括凝固事件假说、共进化假说和立体化学假说等,每种假说均试图解释遗传密码所表现出来的某些化学和生物学规律.基于遗传密码的物理化学性质、基因组变异的规律和相关的生物学假说,本研究提出了遗传密码分步进化假说（The Stepwise Evolution Hypothesis for the Genetic Code）.在人们推断的最原始的RNA世界里,原初（Primordial）遗传密码从只能识别嘌呤和嘧啶开始,编码一个或两个简单而功能明确的氨基酸.由于胞嘧啶C的化学不稳定性,最初形成的遗传密码应该仅仅由腺嘌呤A和尿嘧啶U来编码,却可得到一组7个多元化的氨基酸.随着生命复杂性的增加,鸟嘌呤G从主载操作信号的功能中释放出来,再伴随着C的引入,使遗传密码逐步扩展到12,15和20个氨基酸,最终完成全部进化步骤.遗传密码的进化过程同时也伴随以蛋白质为主体的分子机制和细胞过程的进化,包括氨酰tRNA合成酶（AARS）从初始翻译机器上的脱离、DNA作为信息载体而取代RNA以及AARS和tRNA共进化等基本过程.分子机制和细胞过程是生命的基本组成元件,它们不但自己不断地趋于完善,也促使生命体走?     

Thickness sensing of hMSCs on collagen gel directs stem cell fate

Mechanically compliant substrate provides crucial biomechanical cues for multipotent stem cells to regulate cellular fates such as differentiation, proliferation and maintenance of their phenotype. Effective modulus of which cells sense is not only determined by intrinsic mechanical properties of the substrate, but also the thickness of substrate. From our study, it was found that interference from underlying rigid support at hundreds of microns away could induce significant cellular response. Human mesenchymal stem cells (hMSCs) were cultured on compliant biological gel, collagen type I, of different thickness but identical ECM composition and local stiffness. The cells sensed the thin gel (130 μm) as having a higher effective modulus than the thick gel (1440 μm) and this was reflected in their changes in morphology, actin fibers structure, proliferation and tissue specific gene expression. Commitment into neuronal lineage was observed on the thin gel only. Conversely, the thick gel (1440 μm) was found to act like a substrate with lower effective modulus that inhibited actin fiber polymerization. Stem cells on the thick substrate did not express tissue specific genes and remained at their quiescent state. This study highlighted the need to consider not only the local modulus but also the thickness of biopolymer gel coating during modulation of cellular responses.     

Hepatitis B Virus (HBV) Surface Antigen Interacts with and Promotes Cyclophilin A Secretion: Possible Link to Pathogenesis of HBV Infection

Cyclophilin A (CypA), predominantly located intracellularly, is a multifunctional protein. We previously reported decreased CypA levels in hepatocytes of transgenic mice expressing hepatitis B virus (HBV) surface antigen (HBsAg). In this study, we found that expression of HBV small surface protein (SHBs) in human hepatoma cell lines specifically triggered CypA secretion, whereas SHBs added extracellularly to culture medium did not. Moreover, CypA secretion was not promoted by the expression of a secretion deficient SHBs mutant, suggesting a close association between secretion of CypA and SHBs. Interaction between CypA and SHBs was observed by using coimmunoprecipitation and glutathione S-transferase pull-down assays. Hydrodynamic injection of the SHBs expression construct into C57BL/6J mice resulted in increased serum CypA levels and ALT/AST levels, as well as the infiltration of inflammatory cells surrounding SHBs-positive hepatocytes. The inflammatory response and serum ALT/AST level were reduced when the chemotactic effect of CypA was inhibited by cyclosporine and anti-CD147 antibody. Furthermore, higher serum CypA levels were detected in chronic hepatitis B patients than in healthy individuals. In HBV patients who had received liver transplantation, serum CypA levels declined dramatically after the loss of HBsAg as a consequence of liver transplantation. Taken together, these results indicate that expression and secretion of SHBs can promote CypA secretion, which may contribute to the pathogenesis of HBV infection.Hepatitis B virus (HBV) infects more than 350 million people worldwide and is a major cause of chronic viral hepatitis and hepatocellular carcinoma (25). Three morphologically distinct forms of viral particles exist in the sera of HBV-infected patients, namely, the 22-nm-diameter spherical particles, tubular particles, and 42-nm-diameter virions (19). Strikingly, the subviral particles (spheres and tubules), containing only viral envelop glycoproteins and host-derived lipids, typically outnumber the virions by a factor of 1,000- to 10,000-fold (6, 11). There are three HBV envelop glycoproteins collectively known as HBV surface antigen (HBsAg), including the large (LHBs), middle (MHBs), and small (SHBs) surface proteins. Among them, SHBs is the most abundant viral envelop protein in virion and subviral particles. Although excess HBsAg subviral particles have been suggested to sequester the neutralizing antibody against HBV and contribute to a state of immune tolerance, thereby enabling the survival of infectious virions and leading to persistent infections (6, 27), the biological and pathological significance of the overproduction of HBsAg subviral particles still remains elusive.HBsAg has been proved extremely effective in inducing protective antibodies (anti-HBs) and thus has been used as the prophylactic vaccine. Thus far, most studies on HBsAg have focused on the development of hepatitis B vaccines (41), identification of HBsAg-interacting membrane proteins as potential host HBV receptors (9, 13), and characterization of the impact of naturally occurring HBsAg mutations on its antigenicity (12, 43). However, specific interactions between HBsAg and host intracellular factors have not been extensively studied.To address this issue, SHBs-secreting cell lines and lineages of HBV transgenic mice persistently expressing HBsAg were used in our previous studies (28, 34, 44). We found that the level of cyclophilin A (CypA) decreased markedly in the livers of HBsAg transgenic mice but increased significantly in their sera (44). CypA is a multifunctional cellular protein. It is the major binding protein for the immunosuppressive drug cyclosporine (Cs) (14) and exhibits peptidyl-prolyl cis-trans isomerase activity (35). Recently, it was found CypA played important roles in regulating inflammatory responses and viral infections. Regarding these newly recognized physiological functions, CypA was speculated to be involved in HBV infection. In the present study, the mechanism and clinical implications of elevated secretion of CypA induced by SHBs were explored in detail, including studies in cell cultures, hydrodynamic injected mouse models, and chronic hepatitis B patients. Our findings indicate that expression and secretion of SHBs can trigger the secretion of CypA, which may induce liver inflammation and contribute to the pathogenesis of HBV infection.     

Solution structure and dynamics of the I214V mutant of the rabbit prion protein

Background

The conformational conversion of the host-derived cellular prion protein (PrP^C) into the disease-associated scrapie isoform (PrP^Sc) is responsible for the pathogenesis of transmissible spongiform encephalopathies (TSEs). Various single-point mutations in PrP^Cs could cause structural changes and thereby distinctly influence the conformational conversion. Elucidation of the differences between the wild-type rabbit PrP^C (RaPrP^C) and various mutants would be of great help to understand the ability of RaPrP^C to be resistant to TSE agents.

Methodology/Principal Findings

We determined the solution structure of the I214V mutant of RaPrP^C(91–228) and detected the backbone dynamics of its structured C-terminal domain (121–228). The I214V mutant displays a visible shift of surface charge distribution that may have a potential effect on the binding specificity and affinity with other chaperones. The number of hydrogen bonds declines dramatically. Urea-induced transition experiments reveal an obvious decrease in the conformational stability. Furthermore, the NMR dynamics analysis discloses a significant increase in the backbone flexibility on the pico- to nanosecond time scale, indicative of lower energy barrier for structural rearrangement.

Conclusions/Significance

Our results suggest that both the surface charge distribution and the intrinsic backbone flexibility greatly contribute to species barriers for the transmission of TSEs, and thereby provide valuable hints for understanding the inability of the conformational conversion for RaPrP^C.