链霉菌降解角蛋白的生化机制研究

对弗氏链霉菌S-221变种降解角蛋白的生化机制进行了初步研究。该菌在角蛋白底物作用下诱导产生角蛋白酶。它是一种复合蛋白酶,含有二硫键还原酶和多肽水解酶等多种酶活性组分。硫酸钠、亚硫酸钠和巯基乙醇对角蛋白酶具有强烈的激活作用,其主要表现作用于角蛋白酶中的二硫键还原酶。亚硫酸钠在0．01mol／L浓度下不仅作用于二硫键还原酶,而且还作用于多肽水解酶。硫代硫酸钠对二硫键还原酶有强烈的抑制作用。角蛋白酶降解羽毛角蛋白首先是角蛋白酶中的二硫键还原酶使角蛋白中二硫键裂解产生变性角蛋白,然后变性角蛋白在多肽水解酶的共同作用下逐步水解成多肽、寡肽和游离氨基酸,使角蛋白彻底降解。在角蛋白降解过程中,角蛋白中的硫也随之转化成巯基化合物,H2S和硫酸盐3种含硫化合物存在于降解产物中。     

F0F1-ATPase as biosensor to detect single virus

F(0)F(1)-ATPase within chromatophore was constructed as a biosensor (immuno-rotary biosensor) for the purpose of capturing single virus. Capture of virus was based on antibody-antigen reaction. The detection of virus based on proton flux change driven by ATP-synthesis of F(0)F(1)-ATPase, which was indicated by F1300, was directly observed by a fluorescence microscope. The results demonstrate that the biosensor loading of virus particles has remarkable signal-to-noise ratio (3.8:1) compared to its control at single molecular level, and will be convenient, quick, and even super-sensitive for detecting virus particles.     

Recognition and incision of oxidative intrastrand cross-link lesions by UvrABC nuclease

Nucleotide excision repair (NER) is a repair pathway that removes a variety of bulky DNA lesions in both prokaryotic and eukaryotic cells. The perturbation of DNA helix structure caused by the oxidative intrastrand lesions could render them good substrates for the NER pathway. Here we employed Escherichia coli NER enzymes, i.e., UvrA, UvrB, and UvrC, to examine the incision efficiency of duplex DNA carrying three different oxidative intrastrand cross-link lesions, that is, G[8-5]C, G[8-5m]mC, and G[8-5m]T, and two dithymine photoproducts, namely, the cis,syn-cyclobutane pyrimidine dimer (T[c,s]T) and the pyrimidine(6-4)pyrimidone product (T[6-4]T). Our results showed that T[6-4]T was the best substrate for UvrA binding, followed by G[8-5]C, G[8-5m]mC, and G[8-5m]T, and then by T[c,s]T. The efficiencies of the UvrABC incisions of these lesions were consistent with their UvrA binding affinities: the stronger the binding to UvrA, the higher the rate of incision. In addition, flanking DNA sequences appeared to have little effect on the binding affinity of UvrA for G[8-5]C as AG[8-5]CA was only slightly preferred over CG[8-5]CG. Consistently, these two sequences exhibited almost no difference in incision rates. Furthermore, we investigated the thermal stability of dodecameric duplexes containing G[8-5m]mC or G[8-5m]T, and our results revealed that these two lesions destabilized the duplex, due to an increase in the free energy for duplex formation at 37 degrees C, by approximately 5.4 and 3.6 kcal/mol, respectively. The destabilizations to the DNA helix caused by those lesions, for the most part, are correlated with the binding affinities of UvrA and incision rates of UvrABC. Taken together, the results from this study suggest that oxidative intrastrand lesions might be substrates for NER enzymes in vivo.     

Cell death induced by MPPa-PDT in prostate carcinoma in vitro and in vivo

Lack of effective photosensitizers has become a major limit for extensive application of photodynamic therapy. In this study, the photocytotoxicity and mode of death induced by a newly developed photosensitizer MPPa, a derivative of chlorophyll a, were investigated in PC-3M cell line, a highly metastatic variant of poorly differentiated androgen-independent proctanec adenocarcinoma PC-3. MTT reduction assay was used to measure cytotoxicity in both PC-3M and HUVEC, after which a flow cytometer was used to measure apoptotic rate and cell cycle, and then Caspase-3, -8, -9 were investigated. Finally, an animal model was set up to embody the curative effect and for histopathological examinations. The photocytotoxicity of MPPa showed both light- and drug-dose dependent characteristics and no significant dark cytotoxicity was observed in PC-3M cells. In HUVEC, MPPa exhibited an obviously low cytotoxicity. By other in vitro studies, we found MPPa-PDT induced apoptotic mainly via the mitochondrial/Caspase-9/Caspase-3 pathway and could restrain the cell cycle progression from the more sensitive G0/G1-phases. In vivo, the tumour growth was significantly inhibited after PDT, and many apoptotic cells could be seen by histopathological examinations. These results indicate the death way of cells induced by MPPa is mainly via mild apoptotic and the cure effect is obvious, suggesting that MPPa is a potential photosensitizer of photodynamic therapy for prostate cancer.     

Volume-sensitive outwardly rectifying chloride channels are involved in oxidative stress-induced apoptosis of mesangial cells

Volume-sensitive outwardly rectifying (VSOR) Cl- channels have been electrophysiologically identified in human and mouse mesangial cells, but the functional role of VSOR Cl- channels in mesangial cell apoptosis is not clear. The aim of the present study was to demonstrate the role of VSOR Cl- channels in oxidative stress-induced mesangial cell apoptosis. H2O2-induced Cl- currents showed phenotypic properties of VSOR Cl- channels, including outward rectification, voltage-dependent inactivation at more positive potentials, sensitivity to hyperosmolarity, and inhibition by VSOR Cl- channel blockers. Moreover, blockage of VSOR Cl- channels by DIDS (100 microM), NPPB (10 microM) or niflumic acid (10 microM) rescued mesangial cell apoptosis induced by H2O2. Treatment with 150 microM H2O2 for 2h resulted in significant reduction of cell volume, in contrast, nuclear condensation and/or fragmentation were not observed and the caspase-3 activity was also not increased. The early-phase alterations in cell volume were markedly abolished by pretreatment with VSOR Cl- channel blockers. We conclude that VSOR Cl- channels are involved in H2O2-induced apoptosis in cultured mesangial cells and its mechanism is associated with apoptotic volume decrease processes.     

EMG and MMG of agonist and antagonist muscles as a function of age and joint angle.

The aim of this study was to determine the effect of elbow joint position on electromyographic (EMG) and mechanomyographic (MMG) activities of agonist and antagonist muscles in young and old women. Surface EMG and MMG were recorded from the triceps and biceps brachii, and brachioradialis muscles during isometric elbow extensions in young and old women. The measurements were carried out at an optimal joint angle (A(o)), as well as at smaller (A(s) = A(o) - 30 degrees ) and larger (A(l) = A(o) + 30 degrees ) angles. The normalized to force EMG amplitude (RMS-EMG/F) was smaller in old women compared to young in all muscles. The RMS-EMG/F of the triceps brachii muscle was not affected by muscle length while that of the biceps brachii and brachioradialis muscles increased at shortest muscle length in both groups. The normalized to force MMG amplitude (RMS-MMG/F) was smaller in old than in young in the triceps brachii muscle only. There was an increase in RMS-MMG/F with triceps brachii and biceps brachii muscle shortening in both groups, and in the brachioradialis muscle -- in young only. Compared to young, older women exhibited a bigger force fluctuation during maximum voluntary contraction, but these did not contribute significantly to the RMS-MMG. Skinfold thickness accounted for the RMS-EMG/F and RMS-MMG/F differences seen between old and young women in the biceps brachii muscle only. It is concluded that, the EMG and MMG response to muscles length change in agonist and antagonist muscles is generally similar in old and young women but the optimal angle shifts toward a bigger value in older women.     

Nonhuman primate models of intrauterine cytomegalovirus infection

Congenital human cytomegalovirus (HCMV) infection has long been recognized as a threat to the developing fetus, even though studies have shown that only a subset of congenital infections results in clinical signs of disease. Among the estimated 8000 children who develop sequelae from congenital CMV infection each year in the United States alone, most suffer permanent developmental defects within the central nervous system. Because there is currently no approved vaccine for HCMV, and anti-HCMV drugs are not administered to gravid women with congenital infection because of potential toxicity to the fetus, there is a clear clinical need for effective strategies that minimize infection in the mother, transplacental transmission of the virus, and/or fetal disease. Animal models provide a method to understand the mechanisms of HCMV persistence and pathogenesis, and allow for testing of novel strategies that limit prenatal infection and disease. The rhesus macaque model is especially well suited for these tasks because monkeys and humans share strong developmental, immunological, anatomical, and biochemical similarities due to their close phylogenetic relationship. This nonhuman primate model provides an invaluable system to accelerate the clinical development of promising new therapies for the treatment of human disease. This review addresses salient findings with the macaque model as they relate to HCMV infection and potential avenues of discovery, including studies of intrauterine CMV infection. The complexity of the natural history of HCMV is discussed, along with the ethical and logistical issues associated with studies during pregnancy, the recent contributions of animal research in this field of study, and future prospects for increasing our understanding of immunity against HCMV disease.