Isolation of hypoxanthine phosphoribosyltransferase-defective mutants in chinese hamster V79 cells by tritium suicide

Tritium suicide was shown to be highly efficient method for isolating mutants defective in hypoxanthine incorporation in the Chinese hamster lung cell line V79. The tritium suicide procedure consisted of 3 kill cycles. Survivors of one kill cycle were used for the next kill cycle. The kill cycles involved incorporation of [³H]hypoxanthine for 5 or 10 min, followed by storage of ³H-labelled cells at ?70°C for 4–10 days. 12 clones that survived the 3rd kill cycle were tested for incorporation of [³H]hypoxanthine and all were found to be defective. At least 6 of the clones have defective hypoxanthine phosphoribosyltransferase (HPRT) activity. One mutant, H19, chosen for further characterization, had HPRT with a 13-fold elevation in apparent K_m for phosphoribosylpyrophosphate (PRPP). Thin-layer chromatography of cell extracts showed that this mutant was incapable of converting intracellular hypoxanthine to IMP or to other purine metabolites. In addition, H19 was resistant to 6-thioguanine.     

Sensitivity of human embryonic stem cells to different conditions during cryopreservation

Low cell recovery rate of human embryonic stem cells (hESCs) resulting from cryopreservation damages leads to the difficulty in their successful commercialization of clinical applications. Hence in this study, sensitivity of human embryonic stem cells (hESCs) to different cooling rates, ice seeding and cryoprotective agent (CPA) types was compared and cell viability and recovery after cryopreservation under different cooling conditions were assessed. Both extracellular and intracellular ice formation were observed. Reactive oxidative species (ROS) accumulation of hESCs was determined. Cryopreservation of hESCs at 1 °C/min with the ice seeding and at the theoretically predicted optimal cooling rate (TPOCR) led to lower level of intracellular ROS, and prevented irregular and big ice clump formation compared with cryopreservation at 1 °C/min. This strategy further resulted in a significant increase in the hESC recovery when glycerol and 1,2-propanediol were used as the CPAs, but no increase for Me₂SO. hESCs after cryopreservation under all the tested conditions still maintained their pluripotency. Our results provide guidance for improving the hESC cryopreservation recovery through the combination of CPA type, cooling rate and ice seeding.     

Calcium oscillations in human mesenteric vascular smooth muscle

Phenylephrine (PE)-induced oscillatory fluctuations in intracellular Ca²⁺ concentration ([Ca²⁺]_i) of vascular smooth muscle have been observed in many blood vessels isolated from a wide variety of mammals. Paradoxically, until recently similar observations in humans have proven elusive. In this study, we report for the first time observations of adrenergically-stimulated [Ca²⁺]_i oscillations in human mesenteric artery smooth muscle. In arterial segments preloaded with Fluo-4 AM and mounted on a myograph on the stage of a confocal microscope, we observed PE-induced oscillations in [Ca²⁺]_i, which initiated and maintained vasoconstriction. These oscillations present some variability, possibly due to compromised health of the tissue. This view is corroborated by our ultrastructural analysis of the cells, in which we found only (5 ± 2)% plasma membrane-sarcoplasmic reticulum apposition, markedly less than measured in healthy tissue from laboratory animals. We also partially characterized the oscillations by using the inhibitory drugs 2-aminoethoxydiphenyl borate (2-APB), cyclopiazonic acid (CPA) and nifedipine. After PE contraction, all drugs provoked relaxation of the vessel segments, sometimes only partial, and reduced or inhibited oscillations, except CPA, which rarely caused relaxation. These preliminary results point to a potential involvement of the sarcoplasmic reticulum Ca²⁺ and inositol 1,4,5-trisphosphate receptor (IP3R) in the maintenance of the Ca²⁺ oscillations observed in human blood vessels.     

Crystal Structure of the LasA Virulence Factor from Pseudomonas aeruginosa: Substrate Specificity and Mechanism of M23 Metallopeptidases

Pseudomonas aeruginosa is an opportunist Gram-negative bacterial pathogen responsible for a wide range of infections in immunocompromized individuals and is a leading cause of mortality in cystic fibrosis patients. A number of secreted virulence factors, including various proteolytic enzymes, contribute to the establishment and maintenance of Pseudomonas infection. One such is LasA, an M23 metallopeptidase related to autolytic glycylglycine endopeptidases such as Staphylococcus aureus lysostaphin and LytM, and to DD-endopeptidases involved in entry of bacteriophage to host bacteria. LasA is implicated in a range of processes related to Pseudomonas virulence, including stimulating ectodomain shedding of the cell surface heparan sulphate proteoglycan syndecan-1 and elastin degradation in connective tissue. Here we present crystal structures of active LasA as a complex with tartrate and in the uncomplexed form. While the overall fold resembles that of the other M23 family members, the LasA active site is less constricted and utilizes a different set of metal ligands. The active site of uncomplexed LasA contains a five-coordinate zinc ion with trigonal bipyramidal geometry and two metal-bound water molecules. Using these structures as a starting point, we propose a model for substrate binding by LasA that explains its activity against a wider range of substrates than those used by related lytic enzymes, and offer a catalytic mechanism for M23 metallopeptidases consistent with available structural and mutagenesis data. Our results highlight how LasA is a structurally distinct member of this endopeptidase family, consistent with its activity against a wider range of substrates and with its multiple roles in Pseudomonas virulence.     

乙状窦后锁孔入路的内镜和显微镜下解剖学研究及临床应用

目的:通过乙状窦后锁孔入路对该区周围进行内镜和显微解剖学观察,为临床该手术入路定位提供解剖学及形态学依据。方法:应用10%甲醛溶液充分固定的成人尸头标本8例(16侧),模拟乙状窦后锁孔入路进行内镜和显微解剖学观察,并测量尸头表面标志点位置关系,确定枕下乙状窦后锁孔位置。结果:1:锁孔位置为取耳后旁4cm以上项线为上点∫形切口3.0cm～4.0cm。暴露星点后在其下方骨孔直径2.0～2.5cm,可以充分暴露桥小脑角区。2:该锁孔入路虽然通道窄小,但利用内镜和显微镜暴露范围较广,可达中上斜坡。结论:熟悉桥小脑角区神经血管解剖结构的毗邻关系,准确到达解剖目标,有助于在显露血管和神经的同时保护脑组织的重要结构,减小创伤,内镜的应用能把手术中的副损伤降低到最低点。     

Adaptive response to oxidative stress: Bacteria, fungi, plants and animals

Reactive oxygen species (ROS) are continuously produced and eliminated by living organisms normally maintaining ROS at certain steady-state levels. Under some circumstances, the balance between ROS generation and elimination is disturbed leading to enhanced ROS level called "oxidative stress". The primary goal of this review is to characterize two principal mechanisms of protection against oxidative stress - regulation of membrane permeability and antioxidant potential. The ancillary goals of this work are to describe up to date knowledge on the regulation of the previously mentioned mechanisms and to identify areas of prospective research and emerging directions in investigation of adaptation to oxidative stress. The ubiquity for challenges leading to oxidative stress development calls for identification of common mechanisms. They are cysteine residues and [Fe,S]-clusters of specific regulatory proteins. The latter mechanism is realized via SoxR bacterial protein, whereas the former mechanism is involved in operation of bacterial OxyR regulon, yeast H(2)O(2)-stimulon, plant NPR1/TGA and Rap2.4a systems, and animal Keap1/Nrf2, NF-κB and AP-1, and others. Although hundreds of studies have been carried out in the field with different taxa, the comparative analysis of adaptive response is quite incomplete and therefore, this work aims to cover a plethora of phylogenetic groups to delineate common mechanisms. In addition, this article raises some questions to be elucidated and points out future directions of this research. The comparative approach is used to shed light on fundamental principles and mechanisms of regulation of antioxidant systems. The idea is to provide starting points from which we can develop novel tools and hypothesis to facilitate meaningful investigations in the physiology and biochemistry of organismic response to oxidative stress.     

Cryo-responses of two types of large unilamellar vesicles in the presence of non-permeable or permeable cryoprotecting agents

Cryo-responses of two types of large unilamellar vesicles (LUV) that were made from either egg yolk l-α-phosphatidylcholine (EPC) or 1,2-dipalmitoyl-rac-glycero-3-phosphocholine (DPPC), in the presence of non-permeable or permeable cryoprotective agents (CPA) was investigated. Partial ternary phase diagrams of CPA–salt–water with specific CPA to salt ratio (R), were constructed to estimate the phase volume of ice and unfrozen matrix of the LUV dispersion, which could aid in understanding the mechanistic actions of CPA. Leakage of both EPC and DPPC LUV was reduced if the sugar concentrations are above 10% (w/w) for disaccharides and 5% (w/w) for monosaccharides. Above these sugar concentrations, non-permeable CPA were more effective in preventing leakage of DPPC LUV than in EPC LUV. Below these sugar concentrations, EPC and DPPC LUV with limited mobility in the remaining unfrozen matrix were more likely to approach and interact with one and another, which were not anticipated when the LUV were completely embedded in the ice matrix. In the presence of Me₂SO or EG, EPC LUV that had been subjected to freezing and thawing processes were protected from leakage. At room temperature, Me₂SO and EG were detrimental to the DPPC LUV. This study suggests that the choice of CPA for cell cryopreservation depends on the type of phospholipids in plasma membranes, which vary in their acyl chain length and gel–liquid crystal phase transition temperature.     

Multidimensional approaches in dealing with prostate cancer

Prostate cancer is one of the most prevalent malignancies worldwide affecting the human male population. Different case-control, cohort or twin studies and segregation analyses point towards the presence of prostate cancer-susceptibility genes in the population. The studies have shown linkage of prostate susceptibility genes to multiple loci on chromosome 1 and single locus each on chromosomes 4, 8, 16, 17, 19, 20 and X chromosome. However, differences right from the mode of inheritance (autosomal dominant or X-linked recessive) to the target genes exist. There have been reports supporting no or weak linkage to these loci as well. Also, region (environmental factors), age and dietary habits have implications in different aspects of the disease. The important targets for treating prostate cancer are androgens and estrogen (synthesized from androgens by the action of enzyme aromatase) owing to their involvement in development and progression of prostate cancer. Further, prostate gland needs androgens (male hormones) for its normal maintenance and functioning. Besides, radiation therapy and surgical methods have also been used. The emerging areas include identifying and preparing successful vaccines from candidate peptides and gene therapy in several forms. This review deals with the paradox of linkage analyses and the various approaches in practice for treatment and management of prostate cancer.