Purification and characterisation of functional early pregnancy factor expressed in Sf9 insect cells and in Escherichia coli

Early pregnancy factor (EPF) is a secreted protein with growth regulatory and immunomodulatory properties. It is an extracellular form of the mitochondrial matrix protein chaperonin 10 (Cpn10), a molecular chaperone. An understanding of the mechanism of action of EPF and an exploration of therapeutic potential has been limited by availability of purified material. The present study was undertaken to develop a simple high-yielding procedure for preparation of material for structure/function studies, which could be scaled up for therapeutic application. Human EPF was expressed in Sf9 insect cells by baculovirus infection and in Escherichia coli using a heat inducible vector. A modified molecule with an additional N-terminal alanine was also expressed in E coli. The soluble protein was purified from cell lysates via anion exchange (negative-binding mode), cation exchange, and hydrophobic interaction chromatography, yielding similar to42 and 36 mg EPF from 300 ml bacterial and I L Sf9 cultures, respectively. The preparations were highly purified ( greater than or equal to99% purity on SDS-PAGE for the bacterial products and greater than or equal to97% for that of insect cells) and had the expected mass and heptameric structure under native conditions, as determined by mass spectrometry and gel permeation chromatography, respectively. All recombinant preparations exhibited activity in the EPF bioassay, the rosette inhibition test, with similar potency both to each other and to the native molecule. In two in vivo assays of immuno suppressive activity, the delayed-type hypersensitivity reaction and experimental autoimmune encephalomyelitis, the insect cell and modified bacterial products, both with N-terminal additions (acetylation or amino acid), exhibited similar levels of suppressive activity, but the bacterial product with no N-terminal modification had no effect in either assay. Studies by others have shown that N-terminal addition is not necessary for Cpn10 activity. By defining techniques for facile production of molecules with and without immunosuppressive properties, the present studies make it possible to explore mechanisms underlying the distinction between EPF and Cpn10 activity. (C) 2003 Elsevier Inc. All rights reserved.

Endothelial cell serine proteases expressed during vascular morphogenesis and angiogenesis

Many serine proteases play important regulatory roles in complex biological systems, but only a few have been linked directly with capillary morphogenesis and angiogenesis. Here we provide evidence that serine protease activities, independent of the plasminogen activation cascade, are required for microvascular endothelial cell reorganization and capillary morphogenesis in vitro. A homology cloning approach targeting conserved motifs present in all serine proteases, was used to identify candidate serine proteases involved in these processes, and revealed 5 genes (acrosin, testisin, neurosin, PSP and neurotrypsin), none of which had been associated previously with expression in endothelial cells. A subsequent gene-specific RT-PCR screen for 22 serine proteases confirmed expression of these 5 genes and identified 7 additional serine protease genes expressed by human endothelial cells, urokinase-type plasminogen activator, protein C,TMPRSS2, hepsin, matriptase/ MT-SPI, dipepticlylpepticlase IV, and seprase. Differences in serine protease gene expression between microvascular and human umbilical vein endothelial cells (HUVECs) were identified and several serine protease genes were found to be regulated by the nature of the substratum, ie. artificial basement membrane or fibrillar type I collagen. mRNA transcripts of several serine protease genes were associated with blood vessels in vivo by in situ hybridization of human tissue specimens. These data suggest a potential role for serine proteases, not previously associated with endothelium, in vascular function and angiogenesis.

A macrophage colony-stimulating factor receptor-green fluorescent protein transgene is expressed throughout the mononuclear phagocyte system of the mouse

The c-fms gene encodes the receptor for macrophage colony-stimulating factor (CSF-1). The gene is expressed selectively in the macrophage and trophoblast cell lineages. Previous studies have indicated that sequences in intron 2 control transcript elongation in tissue-specific and regulated expression of c-fms. In humans, an alternative promoter was implicated in expression of the gene in trophoblasts. We show that in mice, c-fms transcripts in trophoblasts initiate from multiple points within the 2-kilobase (kb) region flanking the first coding exon. A reporter gene construct containing 3.5 kb of 5' flanking sequence and the down-stream intron 2 directed expression of enhanced green fluorescent protein (EGFP) to both trophoblasts and macrophages. EGFP was detected in trophoblasts from the earliest stage of implantation examined at embryonic day 7.5. During embryonic development, EGFP highlighted the large numbers of c-fms-positive macrophages, including those that originate from the yolk sac. In adult mice, EGFP location Was consistent with known F4/80-positive macrophage populations, including Langerhans cells of the skin, and permitted convenient sorting of isolated tissue macrophages from disaggregated tissue. Expression of EGFP in transgenic mice was dependent on intron 2 as no lines with detectable EGFP expression were obtained where either all of intron 2 or a conserved enhancer element FIRE (the Fms intronic regulatory element) was removed. We have therefore defined the elements required to generate myeloid- and trophoblast-specific transgenes as well as a model system for the study of mononuclear phagocyte development and function. (C) 2003 by The American Society of Hematology.

Continued discovery of transcriptional units expressed in cells of the mouse mononuclear phagocyte lineage

The current RIKEN transcript set represents a significant proportion of the mouse transcriptome but transcripts expressed in the innate and acquired immune systems are poorly represented. In the present study we have assessed the complexity of the transcriptome expressed in mouse macrophages before and after treatment with lipopolysaccharide, a global regulator of macrophage gene expression, using existing RIKEN 19K arrays. By comparison to array profiles of other cells and tissues, we identify a large set of macrophage-enriched genes, many of which have obvious functions in endocytosis and phagocytosis. In addition, a significant number of LPS-inducible genes were identified. The data suggest that macrophages are a complex source of mRNA for transcriptome studies. To assess complexity and identify additional macrophage expressed genes, cDNA libraries were created from purified populations of macrophage and dendritic cells, a functionally related cell type. Sequence analysis revealed a high incidence of novel mRNAs within these cDNA libraries. These studies provide insights into the depths of transcriptional complexity still untapped amongst products of inducible genes, and identify macrophage and dendritic cell populations as a starting point for sampling the inducible mammalian transcriptome.

Isolation and characterization of a Cotesia rubecula bracovirus gene expressed in the lepidopteran Pieris rapae

Polydnaviruses are endogenous particles that are crucial for the survival of endoparasitoid wasps, providing active suppression of the immune function of the lepidopteran host in which wasp larvae develop. The Cotesia rubecula bracovirus (CrBV) is unique in that only four gene products are detected in larval host (Pieris rapae) tissues and expression of CrBV genes is transient, occurring between 4 and 12 h post-parasitization. Two of the four genes, CrV1 and CrV3, have been characterized. CrV1 is a secreted glycoprotein that has been implicated in depolymerization of the actin cytoskeleton of host haemocytes, leading to haemocyte inactivation; CrV3 is a multimeric C-type lectin that shares homology with insect immune lectins. Here, a third CrBV-specific gene is described, CrV2, which is expressed in larval P. rapae tissues. CrV2, which is transcribed in haemocytes and fat body cells, has an ORF of 963 bp that produces a glycoprotein of approximately 40 kDa. CrV2 is secreted into haemolymph and appears to be internalized by host haemocytes. CrV2 has a coiled-coil region predicted at its C-terminus, which may be involved in the formation of putative CrV2 trimers that are detected in haemolymph of parasitized host larvae.

Subordinate-manager gender combination and perceived leadership style influence on emotions, self-esteem and organizational commitment

A theoretical model was developed to investigate the relationships among subordinate-manager gender combinations, perceived leadership style, experienced frustration and optimism, organization-based self-esteem and organizational commitment. The model was tested within the context of a probabilistic structural model, a discrete Bayesian network, using cross-sectional data from a global pharmaceutical company. The Bayesian network allowed forward inference to assess the relative influence of gender combination and leadership style on the emotions, self-esteem and commitment consequence variables. Further, diagnostics from backward inference were used to assess the relative influence of variables antecedent to organizational commitment. The results showed that gender combination was independent of leadership style and had a direct impact on subordinates' levels of frustration and optimism. Female manager-female subordinate had the largest probability of optimism, while male manager teamed with a male subordinate had the largest probability of frustration. Furthermore, having a female manager teamed up with a male subordinate resulted in the lowest possibility of frustration. However, the findings show that the gender issue is not simply female managers versus male managers, but is concerned with the interaction of the subordinate-manager gender combination and leadership style in a nonlinear manner. (C) 2003 Elsevier Inc. All rights reserved.

HMG box transcription factor gene Hbp1 is expressed in germ cells of the developing mouse testis

HMG box containing protein 1 (HBP1) is a high mobility group domain transcriptional repressor that regulates proliferation in differentiated tissues. We have found mouse Hbp1 to be expressed strongly in the embryonic mouse testis from approximately 12.5 days post coitum, compared with low levels of expression in the embryonic ovary. Expression of Hbp1 is maintained in the developing testis beyond the onset of spermatogenesis after birth. Whole-mount in situ hybridisation analysis showed that expression of Hbp1 in the XY gonad is localized within the developing testis cords, the precursors of the seminiferous tubules. Expression of Hbp1 is not apparent in testis cords of gonads from homozygous We mutant embryos, which lack germ cells. In situ hybridisation analysis on cryosectioned embryonic testis indicated that Hbp1 expression resembles that of the germ cell marker Oct4. We conclude that Hbp1 is up-regulated specifically in germ cells of the developing XY gonad. The expression of Hbp1 in XY germ cells appears to correlate with the onset of mitotic arrest in these cells. (C) 2004 Wiley-Liss, Inc.

Sox8 is expressed at similar levels in gonads of both sexes during the sex determining period in turtles

A critical gene involved in mammalian sex determination and differentiation is the Sty-related gene Sox9. In reptiles, Sox9 resembles that of mammals in both structure and expression pattern in the developing gonad, but a causal role in male sex determination has not been established. A closely related gene, Sox8, is conserved in human, mouse, and trout and is expressed in developing testes and not developing ovaries in mouse. In this study, we tested the possibility of Sox8 being important for sex determination or sex differentiation in the red-eared slider turtle Trachemys scripta, in which sex is determined by egg incubation temperature between stages 15 and 20. We cloned partial turtle Sox8 and anti-Mullerian hormone (Amh) cDNAs, and analyzed the expression patterns of these genes in developing gonads by reverse transcriptase-polymerase chain reaction and whole-mount in situ hybridization. While Amh is expressed more strongly in males than in females at stage 17, Sox8 is expressed at similar levels in males and females throughout the sex-determining period. These observations suggest that differential transcription of Sill is not responsible for regulation of Amh, nor responsible for sex determination in turtle. (C) 2004 Wiley-Liss, Inc.

Detection of differentially expressed genes in synovial fibroblasts by restriction fragment differential display

Objective. To identify differentially expressed genes in synovial fibroblasts and examine the effect on gene expression of exposure to TNF-alpha and IL-1beta. Methods. Restriction fragment differential display was used to isolate genes using degenerate primers complementary to the lysophosphatidic acid acyl transferase gene family. Differential gene expression was confirmed by reverse transcription-polymerase chain reaction and immunohistochemistry using a variety of synovial fibroblasts, including cells from patients with osteoarthritis and self-limiting parvovirus arthritis. Results. Irrespective of disease process, synovial fibroblasts constitutively produced higher levels of IL-6 and monocyte chemoattractant protein 1 (MCP-1) (CCL2) than skin fibroblasts. Seven genes were differentially expressed in synovial fibroblasts compared with skin fibroblasts. Of these genes, four [tissue factor pathway inhibitor 2 (TFPI2), growth regulatory oncogene beta (GRObeta), manganese superoxide dismutase (MnSOD) and granulocyte chemotactic protein 2 (GCP-2)] were all found to be constitutively overexpressed in synoviocytes derived from patients with osteoarthritis. These four genes were only weakly expressed in other synovial fibroblasts (rheumatoid and self-limiting parvovirus infection). However, expression in all types of fibroblasts was increased after stimulation with TNF-alpha and IL-1beta. Three other genes (aggrecan, biglycan and caldesmon) were expressed at higher levels in all types of synovial fibroblasts compared with skin fibroblasts even after stimulation with TNF-alpha and IL-1. Conclusions. Seven genes have been identified with differential expression patterns in terms of disease process (osteoarthritis vs rheumatoid arthritis), state of activation (resting vs cytokine activation) and anatomical location (synovium vs skin). Four of these genes, TFPI2, GRObeta (CXCL2), MnSOD and GCP-2 (CXCL6), were selectively overexpressed in osteoarthritis fibroblasts rather than rheumatoid fibroblasts. While these differences may represent differential behaviour of synovial fibroblasts in in vitro culture, these observations suggest that TFPI2, GRObeta (CXCL2), MnSOD and GCP-2 (CXCL6) may represent new targets for treatments specifically tailored to osteoarthritis.

Auxiliary subunit regulation of high-voltage activated calcium channels expressed in mammalian cells

The effects of auxiliary calcium channel subunits on the expression and functional properties of high-voltage activated (HVA) calcium channels have been studied extensively in the Xenopus oocyte expression system, but are less completely characterized in a mammalian cellular environment. Here, we provide the first systematic analysis of the effects of calcium channel beta and alpha(2)-delta subunits on expression levels and biophysical properties of three different types (Ca(v)1.2, Ca(v)2.1 and Ca(v)2.3) of HVA calcium channels expressed in tsA-201 cells. Our data show that Ca(v)1.2 and Ca(v)2.3 channels yield significant barium current in the absence of any auxiliary subunits. Although calcium channel beta subunits were in principle capable of increasing whole cell conductance, this effect was dependent on the type of calcium channel alpha(1) subunit, and beta(3) subunits altogether failed to enhance current amplitude irrespective of channel subtype. Moreover, the alpha(2)-delta subunit alone is capable of increasing current amplitude of each channel type examined, and at least for members of the Ca(v)2 channel family, appears to act synergistically with beta subunits. In general agreement with previous studies, channel activation and inactivation gating was regulated both by beta and by alpha(2)-delta subunits. However, whereas pronounced regulation of inactivation characteristics was seen with the majority of the auxiliary subunits, effects on voltage dependence of activation were only small (< 5 mV). Overall, through a systematic approach, we have elucidated a previously underestimated role of the alpha(2)-delta(1) subunit with regard to current enhancement and kinetics. Moreover, the effects of each auxiliary subunit on whole cell conductance and channel gating appear to be specifically tailored to subsets of calcium channel subtypes.

Activation of the cAMP pathway by variant human MC1R alleles expressed in HEK and in melanoma cells

alpha-Melanocyte-stimulating hormone (alpha-MSH) activates the melanocortin-1 receptor (MC1R) on melanocytes to promote a switch from red/yellow pheomelanin synthesis to darker eumelanins via positive coupling to adenylate cyclase. The human MC1R locus is highly polymorphic with the specific variants associated with red hair and fair skin (RHC phenotype) postulated to be loss-of-function receptors. We have examined the ability of MC1R variants to activate the cAMP pathway in stably transfected REK293 cells. The RHC associated variants, Arg151Cys, Arg160Trp and Asp294His, demonstrated agonist-mediated increases in cAMP and phosphorylation of cAMP-responsive element-binding protein (CREB). Whereas the Asp294His variant showed severely impaired functional responses, the Arg151Cys and Arg160Trp variants retained considerable signaling capacity. Melanoma cells homozygous for either the Arg151Cys variant or consensus sequence both elicited CREB phosphorylation in response to alpha-MSH in the presence of IBMX. The common RHC alleles, Arg151Cys, Arg160Trp and Asp294His, are neither complete loss-of-function receptors nor are they functionally equivalent. (c) 2005 Elsevier Inc. All rights reserved.

Application of mixture models to detect differentially expressed genes

An important and common problem in microarray experiments is the detection of genes that are differentially expressed in a given number of classes. As this problem concerns the selection of significant genes from a large pool of candidate genes, it needs to be carried out within the framework of multiple hypothesis testing. In this paper, we focus on the use of mixture models to handle the multiplicity issue. With this approach, a measure of the local FDR (false discovery rate) is provided for each gene. An attractive feature of the mixture model approach is that it provides a framework for the estimation of the prior probability that a gene is not differentially expressed, and this probability can subsequently be used in forming a decision rule. The rule can also be formed to take the false negative rate into account. We apply this approach to a well-known publicly available data set on breast cancer, and discuss our findings with reference to other approaches.