Expression and characterization of HlyX hemolysin from Leptospira interrogans serovar Copenhageni: potentiation of hemolytic activity by LipL32

The HlyX, a putative hemolysin identified from the Leptospira genomes, was cloned, expressed in Escherichia coli, purified, and its hemolytic activity was confirmed. Mouse polyclonal antiserum against the recombinant HlyX recognized HlyX-related antigens in a panel of Leptospira species extracts and it was also able to abolish the hemolytic activity of HlyX. A mixture of HlyX and LipL32, a known hemolysin from Leptospira, induced hemolysis in a synergistic way that was fully inhibited by antiserum against either protein. Moreover, sera from patients with leptospirosis also recognized the recombinant HlyX, showing that it is presented to the host immune system during Leptospira infection.     

Physiological and Biochemical Changes Associated with Macroconidial Germination in Microsporum gypseum

A study was made of the metabolic processes associated with macroconidial germination in Microsporum gypseum. The optimum conditions for stimulation of endogenous respiration, changes in chemical composition as germination proceeds, and the uptake and synthetic fates of amino acids, glucose, and uracil were investigated. The assimilation and conversion of (14)C-glucose, (14)C-amino acids, and (14)C-uracil into the cell pool and into trichloroacetic acid-precipitable material were studied during the early stages of germination (i.e., prior to germ-tube emergence). The macroconidia were not metabolically inert for any significant period of time after exposure to germination conditions. Rather, the spores rapidly assimilated all metabolites and slowly converted them into macromolecules. Investigations of the effect of inhibitors of nucleic acid and protein synthesis prior to germ-tube emergence and during early germ-tube elongation suggested significant changes in metabolism and cell permeability may be correlated with the emergence of germ tubes. Radioactivity of incorporated glucose was found to be associated largely with the lipid fractions of the macroconidia early in germination.     

Superoxide Dismutase Activity and Lipid Peroxidation in the Liver of Guinea Pig Infected with Leptospira interrogans

Superoxide dismutase (SOD) activity and the degree of lipid peroxidation were studied over a two week period in guinea pigs infected with Leptospira interrogans derived from wild mice. The total SOD activity in infected host liver increased by four-fold two days after infection; this was followed by a 20% decrease resulting in levels comparable to normal, uninfected liver. During the period of decreasing SOD activity after day two, the levels of TBA-reactive material (TBARS) are increased by three-fold in infected guinea pig, liver, compared to uninfected liver. The results indicate that SOD attenuates intracellular superoxide-mediated toxic effects in guinea pigs infected with L. interrogans. In addition, electron microscopy structure demonstrates correlated pathogenic shrinkage of mitochondrial and Kupffer cell structures.     

A comparative analysis of the attachment of Leptospira interrogans and L. borgpetersenii to mammalian cells

Leptospirosis, the world's most ubiquitous zoonosis, is caused by pathogenic Leptospira. As microbe-host interactions are specific in pathogenesis, it is likely that there are several molecules mediating the attachment of the Leptospira to mammalian cells. In this study, we analysed the attachment of Leptospira interrogans serovar Portlandvere and Leptospira borgpetersenii serovar Jules to untreated HEp-2 cells or HEp-2 cells treated with the various enzymes, lectins or sugars and to integrins αVβ3 and α5β1, relative to control wells. We found that both serovars bound equally well to HEp-2 cells; however, serovar Jules showed a higher level of attachment to integrins. Both serovars showed an increase in attachment to HEp-2 cells coated with lectins peanut agglutinin, Ulex europaeus agglutinin, soybean agglutinin and Erythrina cristagalli agglutinin (p < 0.05); in the case of Concanavalin A, Jules showed an increase, while Portlandvere showed a significant decrease in attachment. Trypsinizing monolayers resulted in a decrease in attachment for both serovars, while when chondroitinase, neuraminidase and heparinase were used an increase in attachment was recorded. Leptospires coated with sugars showed a decrease in attachment. These results show that serovar Jules' general greater affinity for the mediators examined may suggest a greater potential for virulence over serovar Portlandvere.     

A Leptospira interrogans enzyme with similarity to yeast Ste14p that methylates the 1-phosphate group of lipid A

Distinct from other spirochetes, cells of Leptospira interrogans contain orthologues of all the Escherichia coli lpx genes required for lipid A biosynthesis, but they synthesize a modified form of lipopolysaccharide that supposedly activates toll-like receptor 2 (TLR2) instead of TLR4. The recent determination of the L. interrogans lipid A structure revealed an unprecedented O-methylation of its 1-phosphate group (Que-Gewirth, N. L. S., Ribeiro, A. A., Kalb, S. R., Cotter, R. J., Bulach, D. M., Adler, B., Saint Girons, I., Werts, C., and Raetz, C. R. H. (2004) J. Biol. Chem. 279, 25420-25429). The enzymatic activity responsible for selective 1-phosphate methylation has not been previously explored. A membrane enzyme that catalyzes the transfer of a methyl group from S-adenosylmethionine (SAM) to the 1-phosphate moiety of E. coli Kdo2-[4'-(32)P]lipid A has now been discovered. The gene encoding this enzyme was identified based on the hypothesis that methylation of a phosphate group is chemically analogous to methylation of a carboxylate moiety at a membrane-water interface. Database searching revealed a candidate gene (renamed lmtA) in L. interrogans showing distant homology to the yeast isoprenylcysteine carboxyl methyltransferase, encoded by sterile-14, which methylates the a-type mating factor. Orthologues of lmtA were not present in E. coli, the lipid A of which normally lacks the 1-phosphomethyl group, or in other spirochetes, which do not synthesize lipid A. Expression of the lmtA gene behind the lac promoter on a low copy plasmid resulted in the appearance of SAM-dependent methyltransferase activity in E. coli inner membranes and methylation of about 30% of the endogenous E. coli lipid A. Inactivation of the ABC transporter MsbA did not inhibit methylation of newly synthesized lipid A. Methylated E. coli lipid A was analyzed by mass spectrometry and NMR spectroscopy to confirm the location of the phosphomethyl group at the 1-position. In human cells, engineered to express the individual TLR subtypes, 1-phosphomethyl-lipid A purified from lmtA-expressing E. coli potently activated TLR4 but not TLR2.     

A Model for the Acrosome Reaction in Mammalian Sperm

The acrosome reaction is a complex, calcium-dependent reaction that results in an exocytotic event required for successful fertilization of the egg. It has long been thought that the acrosome reaction occurs upon sperm binding to the zona pellucida, a viscoelastic layer surrounding the oocyte. Recent studies have suggested that the reaction may even occur before the sperm encounters the zona, perhaps mediated by progesterone or some other agonist. It has been particularly difficult to understand differences between progesterone-induced and zona-induced reactions experimentally and whether one substance is the more biologically relevant trigger. Until this present work, there has been little effort to mathematically model the acrosome reaction in sperm as a whole. Instead, attention has been paid to modeling portions of the pathways involved in other cell types. Here we present a base model for the acrosome reaction which characterizes the known biochemical reactions and behaviors of the system. Our model allows us to analyze several pathways that may act as a stabilizing mechanism for avoiding sustained oscillatory calcium responses often observed in other cell types. Such an oscillatory regime might otherwise prevent acrosomal exocytosis and therefore inhibit fertilization. Results indicate that the acrosome reaction may rely upon multiple redundant mechanisms to avoid entering an oscillatory state and instead maintain a high resting level of calcium, known to be required for successful acrosomal exocytosis and, ultimately, fertilization of the oocyte.     

A Spatial Analysis of Physiological Changes Associated with Infection of Cotyledons of Marrow Plants with Cucumber Mosaic Virus

Changes in host primary metabolism associated with the compatible interaction between cucumber mosaic virus and cotyledons of the marrow plant (Cucurbita pepo L.) have been localized, first by measuring activities of key enzymes in infected and uninfected regions of the cotyledon, and second by histochemical techniques applied to tissue prints of the infected region. A series of progressive metabolic changes occurs within the expanding infected lesion. Virus replication and the synthesis of viral protein at the periphery creates a strong sink demand associated with increased activities of anaplerotic enzymes, increased photosynthesis, and starch accumulation. Inside the lesion, when the synthesis of virus has declined, photosynthesis is reduced, starch is mobilized, and the emphasis of metabolism is shifted toward glycolysis and mitochondrial respiration. These changes are associated spatially with the onset of chlorosis. A decrease in total protein synthesis in this inner zone could be instrumental in some or all of these changes, leading to symptoms of viral infection.     

Regulation by Delta-Sleep-Inducing Peptide of the Neurochemical Changes in the Brain Associated with Dopaminergic System Hyperactivity

The influence of a single injection of delta-sleep-inducing peptide (DSIP; 30 g/kg body weight) on neurochemical parameters of rats' brain was studied under the conditions of chronic administration of dopamine analogs inducing DA-system hyperactivity – 50 mg/kg body weight of L-DOPA for 30 days or 2,5 mg/kg body weight of amphetamine for 21 days. The parameters of serotonergic system (MAO A activity, 5-HT, and 5-HIAA contents) and of dopaminergic system (MAO B activity, DA, NA, and HVA contents) were investigated in the cortex and caudate nucleus of control, DA or amphetamine, and DSIP receiving rats. Changes caused by the two DA-system activating drugs had both similarities and differences, and the corrective action of DSIP also had certain peculiarities depending on the pharmacological preparation used for the induction of DA-system hyperactivity and on the investigated brain structure. It is supposed that DSIP action might be based on the activation of serotonergic system that ensures the adaptive behavior of the animals.     

哺乳动物体细胞克隆及在动物转基因中的应用

哺乳动物体细胞克隆及转基因技术,近些年发展迅速。尤其1997年‘多利’羊的诞生,对推动哺乳动物克隆及转基因技术的发展具有里程碑式的意义。本文介绍了哺乳动物体细胞克隆的方法,存在的问题和体细胞克隆的机理研究。对这些方法和研究理论进行了讨论,并探讨如何利用克隆技术进行转基因动物的制备。     

小型田鼠:研究单配制进化与调控的模型

哺乳动物的单配制通常被认为是社会性单配制,它不是单纯地由性行为来决定,而是由诸多因素,包括长期的pair bond、夫妻双方共同抚育后代、免近亲交配以及雌雄两性相似等来决定的。在这篇综述中,我们论述了如何以啮齿类田鼠属(Microtus)为模型,通过比较研究来帮助我们理解社会性单配制的进化以及其神经调控机制。对田鼠属的研究不仅证实了单配制起源于艰苦的生存条件的假说,而且还证实了雌性性选择可能有利于维持单配制。不仅如此,哺乳动物单配制的进化还需要雄性的prosocial行为的不断强化。例如,亲近行为可以促进pairbond的形成并强化雄性对后代的哺育行为,而这种强化则来源于神经多肽催产素(OT)和加压素(AVP)与类固醇类激素的相互作用。催产素和加压素调控pairbond和双亲哺育行为的表达,而单配制和多配制田鼠的催产素和加压素受体在脑内的分布有显的不同。比较研究揭示了小型田鼠单配制的调控机制,而种内差异和行为上的可塑性则有助于我们进一步理解这种机制。比如,在某些条件下,多配制的草原田鼠(Microtus pennsylvanicu)的雄性个体具有哺育后代的行为。尽管草原田鼠的加压素Vla受体在脑内的分布与其他多配制的田鼠相似,但是如果脑室注射加压素,仍可以诱发其哺育后代的行为。同样是单配制的橙腹田鼠(Microtus ochrogaster),生活在：Illnois的显示出高水平的prosocial行为,而生活在Kansas的则表现出较低水平的社会性行为。即使两个种群的催产素或加压素Vla受体在脑内的分布相同,它们的雌激素受体表达水平显不同,这在雄性个体表现尤其明显。与Kansas的雄性个体相比,在终纹床核(bed rucleus of the stria tenninalis)和杏仁核中区(medial amygdala)这两个调控亲近行为和攻击行为的脑区,Illinois的雄性个体的α雌激素受体的水平要低得多。这些研究表明对雌激素的低敏感程度有利于高水平地表达prosocial行为并降低特定类型的攻击行为。     

A Mouse Model for Studying Viscerotropic Disease Caused by Yellow Fever Virus Infection

Mosquito-borne yellow fever virus (YFV) causes highly lethal, viscerotropic disease in humans and non-human primates. Despite the availability of efficacious live-attenuated vaccine strains, 17D-204 and 17DD, derived by serial passage of pathogenic YFV strain Asibi, YFV continues to pose a significant threat to human health. Neither the disease caused by wild-type YFV, nor the molecular determinants of vaccine attenuation and immunogenicity, have been well characterized, in large part due to the lack of a small animal model for viscerotropic YFV infection. Here, we describe a small animal model for wild-type YFV that manifests clinical disease representative of that seen in primates without adaptation of the virus to the host, which was required for the current hamster YF model. Investigation of the role of type I interferon (IFN-α/β) in protection of mice from viscerotropic YFV infection revealed that mice deficient in the IFN-α/β receptor (A129) or the STAT1 signaling molecule (STAT129) were highly susceptible to infection and disease, succumbing within 6–7 days. Importantly, these animals developed viscerotropic disease reminiscent of human YF, instead of the encephalitic signs typically observed in mice. Rapid viremic dissemination and extensive replication in visceral organs, spleen and liver, was associated with severe pathologies in these tissues and dramatically elevated MCP-1 and IL-6 levels, suggestive of a cytokine storm. In striking contrast, infection of A129 and STAT129 mice with the 17D-204 vaccine virus was subclinical, similar to immunization in humans. Although, like wild-type YFV, 17D-204 virus amplified within regional lymph nodes and seeded a serum viremia in A129 mice, infection of visceral organs was rarely established and rapidly cleared, possibly by type II IFN-dependent mechanisms. The ability to establish systemic infection and cause viscerotropic disease in A129 mice correlated with infectivity for A129-derived, but not WT129-derived, macrophages and dendritic cells in vitro, suggesting a role for these cells in YFV pathogenesis. We conclude that the ability of wild-type YFV to evade and/or disable components of the IFN-α/β response may be primate-specific such that infection of mice with a functional IFN-α/β antiviral response is attenuated. Consequently, subcutaneous YFV infection of A129 mice represents a biologically relevant model for studying viscerotropic infection and disease development following wild-type virus inoculation, as well as mechanisms of 17D-204 vaccine attenuation, without a requirement for adaptation of the virus.     

A Novel Model to Combine Clinical and Pathway-Based Transcriptomic Information for the Prognosis Prediction of Breast Cancer

Breast cancer is the most common malignancy in women worldwide. With the increasing awareness of heterogeneity in breast cancers, better prediction of breast cancer prognosis is much needed for more personalized treatment and disease management. Towards this goal, we have developed a novel computational model for breast cancer prognosis by combining the Pathway Deregulation Score (PDS) based pathifier algorithm, Cox regression and L1-LASSO penalization method. We trained the model on a set of 236 patients with gene expression data and clinical information, and validated the performance on three diversified testing data sets of 606 patients. To evaluate the performance of the model, we conducted survival analysis of the dichotomized groups, and compared the areas under the curve based on the binary classification. The resulting prognosis genomic model is composed of fifteen pathways (e.g. P53 pathway) that had previously reported cancer relevance, and it successfully differentiated relapse in the training set (log rank p-value = 6.25e-12) and three testing data sets (log rank p-value<0.0005). Moreover, the pathway-based genomic models consistently performed better than gene-based models on all four data sets. We also find strong evidence that combining genomic information with clinical information improved the p-values of prognosis prediction by at least three orders of magnitude in comparison to using either genomic or clinical information alone. In summary, we propose a novel prognosis model that harnesses the pathway-based dysregulation as well as valuable clinical information. The selected pathways in our prognosis model are promising targets for therapeutic intervention.     

A Model for Fluid Drainage by the Lymphatic System

This study investigates the fluid flow through tissues where lymphatic drainage occurs. Lymphatic drainage requires the use of two valve systems, primary and secondary. Primary valves are located in the initial lymphatics. Overlapping endothelial cells around the circumferential lining of lymphatic capillaries are presumed to act as a unidirectional valve system. Secondary valves are located in the lumen of the collecting lymphatics and act as another unidirectional valve system; these are well studied in contrast to primary valves. We propose a model for the drainage of fluid by the lymphatic system that includes the primary valve system. The analysis in this work incorporates the mechanics of the primary lymphatic valves as well as the fluid flow through the interstitium and that through the walls of the blood capillaries. The model predicts a piecewise linear relation between the drainage flux and the pressure difference between the blood and lymphatic capillaries. The model describes a permeable membrane around a blood capillary, an elastic primary lymphatic valve and the interstitium lying between the two.     

Volvox as a Model System for Studying the Ontogeny and Phylogenyof Multicellularity and Cellular Differentiation

   apd: 1 March 2001