Bacterial fimbriae stimulate proinflammatory activation in the endothelium through distinct TLRs

The major and minor fimbriae proteins produced by the human pathogen Porphyromonas gingivalis are required for invasion of human aortic endothelial cells and for the stimulation of potent inflammatory responses. In this study, we report that native forms of both the major and minor fimbriae proteins bind to and signal through TLR2 for this response. Major and minor fimbriae bound to a human TLR2:Fc chimeric protein with an observed K(d) of 28.9 nM and 61.7 nM, respectively. Direct binding of the major and minor fimbriae to a human chimeric CD14-Fc protein also established specific binding of the major and minor fimbriae to CD14 with classic saturation kinetics. Using a P. gingivalis major and minor fimbriae mutant, we confirmed that TLR2 binding in whole cells is dependent on the expression of the major and minor fimbriae. Although we did not observe binding with the major or minor fimbriae to the TLR4-Fc chimeric protein, signaling through TLR4 for both proteins was demonstrated in human embryonic kidney 293 cells transfected with TLR4 and only in the presence MD-2. Transient transfection of dominant-negative forms of TLR2 or TLR4 reduced IL-8 production by human aortic endothelial cells following stimulation with major or minor fimbriae. The ability of two well-defined microbe-associated molecular patterns to select for innate immune recognition receptors based on accessory proteins may provide a novel way for a pathogen to sense and signal in appropriate host environments.     

人类主要Cyclins在MOLT-4细胞阻断动力学下的表达规律

细胞周期素与相应的细胞周期素依赖性蛋白激酶相结合,驱动着细胞通过细胞周期各时相,而细胞周期素时相性规律大多来自酵母研究或同步化细胞的分析。本研究着重于人类非同步化细胞的细胞周期素时相性规律的揭示。采用人类白血病细胞株MOLT-4,使其处于对数生长期,加以有丝分裂中期阻滞法,应用多参数流式细胞术分析人类主要细胞周期素B1、A和E。分析发现,细胞周期素B1峰值在M期,降解于M期后,细胞周期素A峰值在G2期,降解于M期,细胞周期素E峰值在G1晚期,降解于S期。以上结果,使我们首次在人类非同步化培养细胞展现了主要细胞周期素的时相性表达规律。     

Isolation of the cDNA clone for mouse glycophorin, erythroid-specific membrane protein

The cDNA clone for a major mouse glycophorin, transmembrane glycoprotein of erythrocytes has been isolated from a mouse spleen erythroblast cDNA library. The primary structure of a major glycophorin indicates that the protein is a single polypeptide chain of 168 amino acids (aa) clearly organized in three domains distinct in the glycophorin of other species. A strong homology of the mouse major glycophorin with human glycophorin A or B, but not with human glycophorin C is observed only in the hydrophobic stretch of 23 nonpolar aa, indicating that the major mouse glycophorin species cloned is similar to human glycophorin A. The glycophorin mRNA is absent in all non-erythroid organs or cell lines examined. The glycophorin mRNA is induced during the differentiation of murine erythroleukemia cells with dimethyl sulfoxide.     

Lipid compositions of cells isolated from pig, human, and rat epidermis.

Epidermal slices from pig, human, and rat skin were treated with dilute buffered trypsin solution (0.005%, w/v), and suspensions of mixed basal and spinous cells were obtained in good yield. Total lipids accounted for approximately 8% of the pig, 10% of the human, and 20% of the rat epidermal cell (dry weight). Phospholipids in pig, human, and rat cells accounted for, respectively, 62%, 53%, and 35% of the total lipids. Phosphatidylcholine (34-38%), phosphatidylethanolamine (18-23%), and sphingomyelin (17-21%) were major compounds in all species. The major neutral lipids were sterols (mostly cholesterol) and triglycerides. Free fatty acids were a major lipid class in pig and human cells, whereas wax esters were a major component in rat epidermal cells. Nearly half (45%) of the sterols in rat cells but less than 10% of those in pig and human cells were esterified. Cholest-7-ene-3beta-ol accounted for 20% of the total sterols in rat cells. Cholesteryl sulfate and ceramide were minor lipids in the three species. The predominant glycosphingolipid (greater than 99%) was glucosylceramide, which accounted for 7% and 9%, respectively, of the total lipids in pig and human cells. A significant proportion (pig, 17%; human, 11%) of the fatty acids in the glucosylceramides were C26:0 and C28:0.     

Implications of hedgehog signaling antagonists for cancer therapy

The hedgehog (Hh) pathway, initially discovered in Drosophila by two Nobel laureates, Dr. Eric Wieschaus and Dr. Christiane Nusslein-Volhard, is a major regulator for cell differentiation, tissue polarity and cell proliferation. Studies from many laboratories, including ours, reveal activation of this pathway in most basal cell carcinomas and in approximately 30% of extracutaneous human cancers, including medulloblastomas, gastrointestinal, lung, breast and prostate cancers. Thus, it is believed that targeted inhibition of Hh signaling may be effective in treating and preventing many types of human cancers. Even more exciting is the discovery and synthesis of specific signaling antagonists for the Hh pathway, which have significant clinical implications in novel cancer therapeutics. This review discusses the major advances in the current understanding of Hh signaling activation in different types of human cancers, the molecular basis of Hh signaling activation, the major antagonists for Hh signaling inhibition and their potential clinical application in human cancer therapy.     

The major human rhinovirus receptor is ICAM-1

The major human rhinovirus receptor has been identified with monoclonal antibodies that inhibit rhinovirus infection. These monoclonal antibodies recognize a 95 kd cell surface glycoprotein on human cells and on mouse transfectants expressing a rhinovirus binding phenotype. Purified 95 kd protein binds to rhinovirus in vitro. Protein sequence from the 95 kd protein showed an identity with that of intercellular adhesion molecule-1 (ICAM-1); a cDNA clone obtained from mouse transfectants expressing the rhinovirus receptor had essentially the same sequence as ICAM-1. Thus, the major human rhinovirus receptor is ICAM-1. The gene for this receptor maps to human chromosome 19, which also contains the genes for a number of other picornavirus receptors.     

Human AP endonuclease possesses a significant activity as major 3'-5' exonuclease in human leukemia cells

Apurinic/apyrimidinic (AP) endonuclease (Ape1) is the major cellular enzyme responsible for repairing AP-sites in DNA. It can cleave the DNA phosphodiester backbone immediately 5(') to an AP-site. Ape1 also shows 3(')-phosphodiesterase activity, a 3(')-phosphatase activity, and an RNaseH activity. However, regarding its exonuclease activity, it remains controversial whether human Ape1 may possess a 3(')-5(') exonuclease activity. During the course of study to search for the major nuclease activity to double-stranded DNA in human leukemia cells, we purified a 37 kDa Mg(2+)-dependent exonuclease from cytosolic fraction of human leukemia U937 cells. Surprisingly, this exonuclease is Ape1. We demonstrated for the first time that Ape1 possesses a significant activity as major 3(')-5(') exonuclease in human leukemia cells. In addition, we also observed that translocation of cytoplasmic Ape1 into nucleus occurs during DNA damage.     

Biosynthetic labelling of the excretory and secretory antigens of Toxocara canis larvae

Toxocara canis larvae were cultured in vitro in medium containing [35S-]methionine for six days. The medium and the larval tissues were analysed for biosynthetically labelled polypeptides by sodium dodecyl sulphate polyacrylamide gel electrophoresis and autoradiography. Immunoprecipitates with positive and negative human antiserum were similarly analysed, using Staphylococcus aureus to absorb immunocomplexes. The larvae secrete biosynthetically labelled polypeptides into the medium, with three major polypeptides of molecular weights between 99 and 110 X 10(3) the major constituents. Both of these react strongly with human IgG in human positive sera. Many polypeptides become labelled in the larval tissue, but only one polypeptide with similar molecular weight to the ES antigens, strongly reacted with human IgG.     

Reassortant rotaviruses as potential live rotavirus vaccine candidates.

A series of reassortants was isolated from coinfection of cell cultures with a wild-type animal rotavirus and a "noncultivatable" human rotavirus. Wild-type bovine rotavirus (UK strain) was reassorted with human rotavirus strains D, DS-1, and P; wild-type rhesus rotavirus was reassorted with human rotavirus strains D and DS-1. The D, DS-1, and P strains represent human rotavirus serotypes 1, 2, and 3, respectively. Monospecific antiserum (to bovine rotavirus, NCDV strain) or a set of monoclonal antibodies to the major outer capsid neutralization glycoprotein, VP7 (of the rhesus rotavirus), was used to select for reassortants with human rotavirus neutralization specificity. This selection technique yielded many reassortants which received only the gene segment coding for the major neutralization protein from the human rotavirus parent, whereas the remaining genes were derived from the animal rotavirus parent. Single human rotavirus gene substitution reassortants of this sort represent potential live vaccine strains.     

Biotransformation of methyl tert-butyl ether by human cytochrome P450 2A6

Methyl tert-butyl ether (MTBE) is widely used as gasoline oxygenate and octane number enhancer for more complete combustion in order to reduce the air pollution caused by motor vehicle exhaust. The possible adverse effects of MTBE on human health are of major public concern. However, information on the metabolism of MTBE in human tissues is scarce. The present study demonstrates that human cytochrome P450 2A6 is able to metabolize MTBE to tert-butyl alcohol (TBA), a major circulating metabolite and marker for exposure to MTBE. As CYP2A6 is known to be constitutively expressed in human livers, we infer that it may play a significant role in metabolism of gasoline ethers in liver tissue.     

Genetic and environmental influences on human psychological differences

Psychological researchers typically distinguish five major domains of individual differences in human behavior: cognitive abilities, personality, social attitudes, psychological interests, and psychopathology (Lubinski, 2000). In this article we: discuss a number of methodological errors commonly found in research on human individual differences; introduce a broad framework for interpreting findings from contemporary behavioral genetic studies; briefly outline the basic quantitative methods used in human behavioral genetic research; review the major criticisms of behavior genetic designs, with particular emphasis on the twin and adoption methods; describe the major or dominant theoretical scheme in each domain; and review behavioral genetic findings in all five domains. We conclude that there is now strong evidence that virtually all individual psychological differences, when reliably measured, are moderately to substantially heritable.     

Natural selection and the origin and maintenance of standard genetic marker systems

Natural selection has always been assumed to be the major force of evolution, but its presence has been difficult to demonstrate. A review of the evidence for selective differences among genotypes for most human genetic polymorphisms indicates there is little of a direct nature. Indirect theoretical evidence, however, seems to support a major role for natural selection, and it does not seem to support the hypothesis that most amino acid substitutions within the human species are neutral. Among small isolates, most of the gene frequency differences are most likely due to genetic drift or the founder effect, and the principal counterbalancing force is gene flow or migration. But genetic differences among the major human subdivisions do not seem to be due to the same interacting forces. One reason for the inability to detect selection has been an oversimplified view of its operation, which assigns genotypes a constant fitness in every generation. Many recent theoretical developments of more complicated kinds of selection may lead to a resolution of the problem and suggest better interpretations of the enormous amount of data on human genetic variation that is rapidly accumulating.     

Specificity of the trypanothione-dependent Leishmania major glyoxalase I: structure and biochemical comparison with the human enzyme

Trypanothione replaces glutathione in defence against cellular damage caused by oxidants, xenobiotics and methylglyoxal in the trypanosomatid parasites, which cause trypanosomiasis and leishmaniasis. In Leishmania major, the first step in methylglyoxal detoxification is performed by a trypanothione-dependent glyoxalase I (GLO1) containing a nickel cofactor; all other characterized eukaryotic glyoxalases use zinc. In kinetic studies L. major and human enzymes were active with methylglyoxal derivatives of several thiols, but showed opposite substrate selectivities: N1-glutathionylspermidine hemithioacetal is 40-fold better with L. major GLO1, whereas glutathione hemithioacetal is 300-fold better with human GLO1. Similarly, S-4-bromobenzylglutathionylspermidine is a 24-fold more potent linear competitive inhibitor of L. major than human GLO1 (Kis of 0.54 microM and 12.6 microM, respectively), whereas S-4-bromobenzylglutathione is >4000-fold more active against human than L. major GLO1 (Kis of 0.13 microM and >500 microM respectively). The crystal structure of L. major GLO1 reveals differences in active site architecture to both human GLO1 and the nickel-dependent Escherichia coli GLO1, including increased negative charge and hydrophobic character and truncation of a loop that may regulate catalysis in the human enzyme. These differences correlate with the differential binding of glutathione and trypanothione-based substrates, and thus offer a route to the rational design of L. major-specific GLO1 inhibitors.     

Human error and the problem of causality in analysis of accidents

Present technology is characterized by complexity, rapid change and growing size of technical systems. This has caused increasing concern with the human involvement in system safety. Analyses of the major accidents during recent decades have concluded that human errors on part of operators, designers or managers have played a major role. There are, however, several basic problems in analysis of accidents and identification of human error. This paper addresses the nature of causal explanations and the ambiguity of the rules applied for identification of the events to include in analysis and for termination of the search for 'causes'. In addition, the concept of human error is analysed and its intimate relation with human adaptation and learning is discussed. It is concluded that identification of errors as a separate class of behaviour is becoming increasingly difficult in modern work environments. The influence of this change on the control of safety of large-scale industrial systems is discussed.     

Immunohistochemical detection of cytochrome P450 isoenzymes in cultured human epidermal cells

We used specific monoclonal antibodies (MAb) to human cytochrome P450 isoenzymes to determine the presence of these proteins in human epidermal cells. Two MAb (P450-5 and P450-8) recognize major forms of hepatic cytochrome P450 involved in biotransformation of xenobiotics. A third MAb, to cytochrome P450-9, is not fully characterized. The proteins were determined by the indirect immunoperoxidase technique after fixation with methanol and acetone. Biopsy materials for cultured keratinocytes, i.e., foreskin and hair follicles, contained the two major forms of cytochrome P450. In cultured keratinocytes derived from hair follicles the proteins were undetectable, whereas the keratinocytes derived from foreskin continued to express the two major forms of hepatic cytochrome P450. Cultured human fibroblasts and a human keratinocyte cell line (SVK14) showed staining similar to that of the foreskin keratinocytes. Cytochrome P450-9 was detectable only in human hepatocytes. The results indicate that, under the culture conditions applied, cultured human foreskin cells and the cell line SVK14 continue to express specific cytochrome P450 isoenzymes in culture, in contrast to hair follicle keratinocytes.     

Age dependent changes in the abundance of the major polypeptides of human lens membrane.

Changes in the abundance of the major polypeptides of human lens membrane with aging and cataractogenesis have been studied. It has been shown that with aging the relative amounts of the two major membrane polypeptides, 26,000 and 22,000 dalton components, change up to the age of approximately 40. Thereafter the relative abundance of these two polypeptides remains constant. Explanations for this alteration are discussed in relation to similar age dependent changes of the proteins of the human lens.     

Characterization of pregnancy-specific beta 1-glycoprotein synthesized by human placental fibroblasts

We have previously demonstrated that human placental fibroblasts produce a pregnancy-specific beta 1-glycoprotein (PS beta G) immunologically indistinguishable from placental PS beta G. This was confirmed by the immunocytochemical localization of PS beta G in these fibroblasts. In addition, placental fibroblasts contain all three PS beta G mRNAs of 2.3, 2.2, and 1.7 kilobases which hybridize with the three PS beta G cDNAs (PSG16, PSG93, and PSG95) identified, although at 1.4-2.5% of the levels in human term placenta. The major PS beta G species synthesized by placental fibroblasts is a 62K glycopolypeptide formed from a 58K intracellular precursor polypeptide. However, the PS beta G species found in human placenta are one major glycoprotein of 72K and two minor ones of 64K and 54K. Poly(A)+ RNA from placental fibroblasts directed the synthesis of two polypeptides of 48K and 46K (major), whereas, poly(A)+ RNA from human placenta directed the synthesis of higher levels of four polypeptides of 50 K, 48 K (major), 46 K, and 36 K. Thus, the major PS beta G species found in fibroblasts and human placenta differ. The carbohydrate side-chains are essential for the stability of fibroblast PS beta G, because PS beta G synthesis in these fibroblasts could not be detected in the presence of tunicamycin, a protein glycosylation inhibitor which did not affect PS beta G mRNA expression. Our finding that a variant PS beta G species is produced in placental fibroblasts raises the possibility that the authentic placental PS beta G species may have different functions.     

Characterization of the apolipoproteins of rat plasma lipoproteins.

Purified fractions of three major rat high-density lipoproteins (HDL) and one rat very low-density lipoprotein (VLDL) were isolated by Sephadex gel chromatography or preparative sodium dodecyl sulfate gel electrophoresis. These proteins were characterized by amino acid analysis, end-group analysis, molecular-weight determination, polyacrylamide gel electrophoresis, and circular dichroism. One of these rat proteins, of molecular weight 27 000, appears to be homologous with the human A-I protein. However, rat HDL possesses two additional major components not reported in human HDL - an arginine-rich protein of molecular weight 35 000 and a protein of molecular weight 46 000. The arginine-rich protein of the rat is similar in size and amino acid analysis to the arginine-rich protein reported in human VLDL. A major component of rat VLDL of 35 000 molecular weight appears similar or identical to the arginine-rich protein in rat HDL by every criterion employed for their characterization.     

Isolation of a receptor protein involved in attachment of human rhinoviruses

Human rhinoviruses can be classified into major and minor groups on the basis of receptor specificity. Recently, a mouse monoclonal antibody was isolated which selectively blocked the attachment of the major group of human rhinoviruses to cells. Using this monoclonal antibody, the cellular receptor for the major group of human rhinoviruses was isolated. A radioimmunoassay was developed by using the receptor antibody to specifically detect rhinovirus receptor during isolation. Solubilized receptor from detergent-treated HeLa cell membrane extracts eluted from gel filtration columns with an apparent molecular weight of 440,000. A cellular receptor protein, which had a molecular weight of 90,000 when analyzed on sodium dodecyl sulfate-polyacrylamide gels, was purified from solubilized extracts on an immunoaffinity column containing receptor antibody. Polyclonal rabbit antiserum, resulting from immunization with the isolated receptor protein, specifically blocked the attachment of the major group of human rhinoviruses and indicated that the 90-kilodalton protein plays a functional role in attachment. Prolonged exposure of HeLa cell monolayers with the receptor antibody showed no inhibition of cell growth and division.