Comparative analysis of prothrombin activators from the venom of Australian elapids

A key component of the venom of many Australian snakes belonging to the elapid family is a toxin that is structurally and functionally similar to that of the mammalian prothrombinase complex. In mammals, this complex is responsible for the cleavage of prothrombin to thrombin and is composed of factor Xa in association with its cofactors calcium, phospholipids, and factor Va. The snake prothrombin activators have been classified on the basis of their requirement for cofactors for activity. The two major subgroups described in Australian elapid snakes, groups C and D, are differentiated by their requirement for mammalian coagulation factor Va. In this study, we describe the cloning, characterization, and comparative analysis of the factor X- and factor V-like components of the prothrombin activators from the venom glands of snakes possessing either group C or D prothrombin activators. The overall domain arrangement in these proteins was highly conserved between all elapids and with the corresponding mammalian clotting factors. The deduced protein sequence for the factor X-like protease precursor, identified in elapids containing either group C or D prothrombin activators, demonstrated a remarkable degree of relatedness to each other (80%-97%). The factor V-like component of the prothrombin activator, present only in snakes containing group C complexes, also showed a very high degree of homology (96%-98%). Expression of both the factor X- and factor V-like proteins determined by immunoblotting provided an additional means of separating these two groups at the molecular level. The molecular phylogenetic analysis described here represents a new approach for distinguishing group C and D snake prothrombin activators and correlates well with previous classifications.

Granulocyte colony-stimulating factor modulates alpha-galactosylceramide-responsive human V alpha 24(+) V beta 11(+) NKT cells

Despite more than a 10-fold increase in T cell numbers in G-CSF-mobilized peripheral blood stem cell (PBSC) grafts, incidence and severity of acute graft-vs-host disease (GVHD) are comparable to bone marrow transplantation. As CD1d-restricted, Valpha24(+)Vbeta11(+) NKT cells have pivotal immune regulatory functions and may influence GVHD, we aimed to determine whether G-CSF has any effects on human NKT cells. In this study, we examined the frequency and absolute numbers of peripheral blood NKT cells in healthy stem cell donors (n = 8) before and following G-CSF (filgrastim) treatment. Effects of in vivo and in vitro G-CSF on NKT cell cytokine expression profiles and on responsiveness of NKT cell subpopulations to specific stimulation by alpha-galactosylceramide (alpha-GalCer) were assessed. Contrary to the effects on conventional T cells, the absolute number of peripheral blood NKT cells was unaffected by G-CSF administration. Furthermore, responsiveness of NKT cells to alpha-GalCer stimulation was significantly decreased (p < 0.05) following exposure to G-CSF in vivo. This hyporesponsiveness was predominantly due to a direct effect on NKT cells, with a lesser contribution from G-CSF-mediated changes in APC. G-CSF administration resulted in polarization of NKT cells toward a Th2, IL-4-secreting phenotype following alpha-GalCer stimulation and preferential expansion of the CD4(+) NKT cell subset. We conclude that G-CSF has previously unrecognized differential effects in vivo on NKT cells and conventional MHC-restricted T cells, and effects on NKT cells may contribute to the lower than expected incidence of GVHD following allogeneic peripheral blood stem cell transplantation.

Phobic anxiety in 11 nations: Part I: Dimensional constancy of the five-factor model

The Fear Survey Schedule-III (FSS-III) was administered to a total of 5491 students in Australia, East Germany, Great Britain, Greece, Guatemala, Hungary, Italy, Japan, Spain, Sweden, and Venezuela, and submitted to the multiple group method of confirmatory analysis (MGM) in order to determine the cross-national dimensional constancy of the five-factor model of self-assessed fears originally established in Dutch, British, and Canadian samples. The model comprises fears of bodily injury-illness-death, agoraphobic fears, social fears, fears of sexual and aggressive scenes, and harmless animals fears. Close correspondence between the factors was demonstrated across national samples. In each country, the corresponding scales were internally consistent, were intercorrelated at magnitudes comparable to those yielded in the original samples, and yielded (in 93% of the total number of 55 comparisons) sex differences in line with the usual finding (higher scores for females). In each country, the relatively largest sex differences were obtained on harmless animals fears. The organization of self-assessed fears is sufficiently similar across nations to warrant the use of the same weight matrix (scoring key) for the FSS-III in the different countries and to make cross-national comparisons feasible. This opens the way to further studies that attempt to predict (on an a priori basis) cross-national variations in fear levels with dimensions of national cultures. (C) 2002 Elsevier Science Ltd. All rights reserved.

Granulocyte-colony stimulating factor increases CD123hi blood dendritic cells with altered CD62L and CCR7 expression

Changes in blood dendritic cell (BDC) counts (CD123(hi)BDC and CD11c(+)BDC) and expression of CD62L, CCR7, and CD49d were analyzed in healthy donors, multiple myeloma (MM), and non-Hodgkin lymphoma (NHL) patients, who received granulocyte-colony stimulating factor (G-CSF) containing peripheral blood stem cell (PBSC) mobilization protocols. Low-dose G-CSF in healthy donors (8-10 mug/ kg/d subcutaneously) and high-dose G-CSF in patients (30 mug/kg/d) increased CD123(hi)BDC (2- to 22-fold, mean 3.7 x 10(6)/ L-17.7 x 10(6)/L and 1.9 x 10(6)/L-12.0 x 10(6)/ L) in healthy donors and MM but decreased CD11c(+)BDC (2- to 10-fold, mean 5.7 x 10(6)/L-1.6 x 10(6)/L) in NHL patients, on the day of apheresis, compared with steady state. After apheresis, CD123(hi)BDC counts remained high, whereas low CD11c(+)BDC counts tended to recover in the following 2-5 days. Down-regulation of CD62L and up-regulation of CCR7 on CD123(hi)BDC were found in most healthy donors and MM patients. CD49d expression was unchanged. Thus, PBSC mobilization may change BDC counts by altering molecules necessary for BDC homing from blood into tissues.

Investigation of the low-density lipoprotein receptor gene and cholesterol as a risk factor for migraine

The Low-Density Lipoprotein Receptor (LDLR) gene is a cell surface receptor that plays an important role in cholesterol homeostasis. We investigated the (TA)n polymorphism in exon 18 of the LDLR gene on chromosome 19p13.2 performing an association analysis in 244 typical migraine-affected patients, 151 suffering from migraine with aura (MA), 96 with migraine without aura (MO) and 244 unaffected controls. The populations consisted of Caucasians only, and controls were age- and sex-matched. The results showed no significant difference between groups for allele frequency distributions of the (TA)n polymorphism even after separation of the migraine-affected individuals into subgroups of MA and MO affected patients. This is in contradiction to Mochi et al. [Mochi M, Cevoli S, Cortelli P, Pierangeli G, Scapoli C, Soriani S, Montagna P. Investigation of an LDLR gene polymorphism (19p13.2) in susceptibility to migrane without aura. J Neurol Sci 2003; 213 (1-2): 7-10.] who found a positive association of this variant with MO. Our study discusses possible differences between the two studies and extends this research by investigating circulating cholesterol levels in a migraine-affected population. (C) 2004 Elsevier B.V. All rights reserved.

Origin and diversity of the Sox transcription factor gene family: genome-wide analysis in Fugu rubripes

The SOX family of transcription factors are found throughout the animal kingdom and are important in a variety of developmental contexts. Genome analysis has identified 20 Sox genes in human and mouse, which can be subdivided into 8 groups, based on sequence comparison and intron-exon structure. Most of the SOX groups identified in mammals are represented by a single SOX sequence in invertebrate model organisms, suggesting a duplication and divergence mechanism has operated during vertebrate evolution. We have now analysed the Sox gene complement in the pufferfish, Fugu rubripes, in order to shed further light on the diversity and origins of the Sox gene family. Major differences were found between the Sox family in Fugu and those in humans and mice. In particular, Fugu does not have orthologues of Sry, Sox,15 and Sox30, which appear to be specific to mammals, while Sox19, found in Fugu and zebrafish but absent in mammals, seems to be specific to fishes. Six mammalian Sox genes are represented by two copies each in Fugu, indicating a large-scale gene duplication in the fish lineage. These findings point to recent Sox gene loss, duplication and divergence occurring during the evolution of tetrapod and teleost lineages, and provide further evidence for large-scale segmental or a whole-genome duplication occurring early in the radiation of teleosts. (C) 2004 Elsevier B.V. All rights reserved.

Repressor- and activator-type ethylene response factors functioning in jasmonate signaling and disease resistance identified via a genome-wide screen of Arabidopsis transcription factor gene expression

To identify transcription factors (TFs) involved in jasmonate (JA) signaling and plant defense, we screened 1,534 Arabidopsis (Arabidopsis thaliana) TFs by real-time quantitative reverse transcription-PCR for their altered transcript at 6 h following either methyl JA treatment or inoculation with the incompatible pathogen Alternaria brassicicola. We identified 134 TFs that showed a significant change in expression, including many APETALA2/ethylene response factor (AP2/ERF), MYB, WRKY, and NACTF genes with unknown functions. Twenty TF genes were induced by both the pathogen and methyl JA and these included 10 members of the AP2/ERF TF family, primarily from the B1a and B3 subclusters. Functional analysis of the B1a TF AtERF4 revealed that AtERF4 acts as a novel negative regulator of JA-responsive defense gene expression and resistance to the necrotrophic fungal pathogen Fusarium oxysporum and antagonizes JA inhibition of root elongation. In contrast, functional analysis of the B3 TF AtERF2 showed that AtERF2 is a positive regulator of JA-responsive defense genes and resistance to F. oxysporum and enhances JA inhibition of root elongation. Our results suggest that plants coordinately express multiple repressor-and activator-type AP2/ERFs during pathogen challenge to modulate defense gene expression and disease resistance.

Cosuppression of eukaryotic release factor 1-1 in Arabidopsis affects cell elongation and radial cell division

The role of the eukaryotic release factor 1 (eRF1) in translation termination has previously been established in yeast; however, only limited characterization has been performed on any plant homologs. Here, we demonstrate that cosuppression of eRF1-1 in Arabidopsis (Arabidopsis thaliana) has a profound effect on plant morphology, resulting in what we term the broomhead phenotype. These plants primarily exhibit a reduction in internode elongation causing the formation of a broomhead-like cluster of malformed siliques at the top of the inflorescence stem. Histological analysis of broomhead stems revealed that cells are reduced in height and display ectopic lignification of the phloem cap cells, some phloem sieve cells, and regions of the fascicular cambium, as well as enhanced lignification of the interfascicular fibers. We also show that cell division in the fascicular cambial regions is altered, with the majority of vascular bundles containing cambial cells that are disorganized and possess enlarged nuclei. This is the first attempt at functional characterization of a release factor in vivo in plants and demonstrates the importance of eRF1-1 function in Arabidopsis.

Assessing computational tools for the discovery of transcription factor binding sites

The prediction of regulatory elements is a problem where computational methods offer great hope. Over the past few years, numerous tools have become available for this task. The purpose of the current assessment is twofold: to provide some guidance to users regarding the accuracy of currently available tools in various settings, and to provide a benchmark of data sets for assessing future tools.

CandiVF - Candida albicans virulence factor database

Candida albicans is a pathogen commonly infecting patients who receive immunosuppressive drug therapy, long-term catheterization, or those who suffer from acquired immune deficiency syndrome (AIDS). The major factor accountable for pathogenicity of C. albicans is host immune status. Various virulence molecules, or factors, of are also responsible for the disease progression. Virulence proteins are published in public databases but they normally lack detailed functional annotations. We have developed CandiVF, a specialized database of C. albicans virulence factors (http://antigen.i2r.a-star.edu.sg/Templar/DB/CandiVF/) to facilitate efficient extraction and analysis of data aimed to assist research on immune responses, pathogenesis, prevention, and control of candidiasis. CandiVF contains a large number of annotated virulence proteins, including secretory, cell wall-associated, membrane, cytoplasmic, and nuclear proteins. This database has in-built bioinformatics tools including keyword and BLAST search, visualization of 3D-structures, HLA-DR epitope prediction, virulence descriptors, and virulence factors ontology.

Factor structure and internal consistency of the Young Schema Questionnaire (Short Form) in Korean and Australian samples

We investigated cross-cultural differences in the factor structure and psychometric properties of the 75-item Young Schema Questionnaire-Short Form (YSQ-SF). Participants were 833 South Korean and 271 Australian undergraduate students. The South Korean sample was randomly divided into two sub-samples. Sample A was used for Exploratory Factor Analysis (EFA) and sample B was used for Confirmatory Factor Analysis (CFA). EFA for the South Korean sample revealed a 13-factor solution to be the best fit for the data, and CFA on the data from sample B confirmed this result. CFA on the data from the Australian sample also revealed a 13-factor solution. The overall scale of the YSQ-SF demonstrated a high level of internal consistency in the South Korean and Australian groups. Furthermore, adequate internal consistencies for all subscales in the South Korean and Australian samples were demonstrated. In conclusion, the results showed that YSQ-SF with 13 factors has good psychometric properties and reliability for South Korean and Australian University students. Korean samples had significantly higher YSD scores on most of the 13 subscales than the Australian sample. However, limitations of the current study preclude the generalisability of the findings to beyond undergraduate student populations. (c) 2006 Elsevier B.V. All rights reserved.

Adipose tissue engineering based on the controlled release of fibroblast growth factor-2 in a collagen matrix

Adipose tissue forms when basement membrane extract ( Matrigel (TM)) and fibroblast growth factor-2 (FGF-2) are added to our mouse tissue engineering chamber model. A mouse tumor extract, Matrigel is unsuitable for human clinical application, and finding an alternative to Matrigel is essential. In this study we generated adipose tissue in the chamber model without using Matrigel by controlled release of FGF-2 in a type I collagen matrix. FGF-2 was impregnated into biodegradable gelatin microspheres for its slow release. The chambers were filled with these microspheres suspended in 60 mu L collagen gel. Injection of collagen containing free FGF-2 or collagen containing gelatin microspheres with buffer alone served as controls. When chambers were harvested 6 weeks after implantation, the volume and weight of the tissue obtained were higher in the group that received collagen and FGF-2 impregnated microspheres than in controls. Histologic analysis of tissue constructs showed the formation of de novo adipose tissue accompanied by angiogenesis. In contrast, control groups did not show extensive adipose tissue formation. In conclusion, this study has shown that de novo formation of adipose tissue can be achieved through controlled release of FGF-2 in collagen type I in the absence of Matrigel.

Characterization of the transcriptional and functional effects of fibroblast growth factor-1 on human preadipocyte differentiation

We recently established that fibroblast growth factor (FGF)-1 promotes adipogenesis of primary human preadipocytes (phPA). In the current report, we have characterized the adipogenic effects of FGF-1 in phPA and also in a human PA strain derived from an individual with Simpson-Golabi-Behmel syndrome (SGBS PA), which exhibit an intrinsic capacity to differentiate with high efficiency. In further studies, we compared these models with the well-characterized murine 3T3-L1 preadipocyte cell line (3T3-L1 PA). FGF-1 up-regulated the adipogenic program in phPA, with increased expression of peroxisome proliferator-activated receptor-gamma in confluent PA prior to induction of differentiation and increased expression of adipocyte markers during differentiation. Moreover, phPA differentiated in the presence of FGF-1 were more insulin responsive and secreted increased levels of adiponectin. FGF-1 treatment of SGBS PA further enhanced differentiation. For the most part, the adipogenic program in phPA paralleled that observed in 3T3-L1 PA; however, we found no evidence of mitotic clonal expansion in the phPA. Finally, we investigated a role for extracellular regulated kinase 1/2 (ERK 1/2) in adipogenesis of phPA. FGF-1 induced robust phosphorylation of ERK1/2 in early differentiation and inhibition of ERK1/2 activity significantly reduced phPA differentiation. These data suggest that FGF-1 treated phPA represent a valuable in vitro model for the study of adipogenesis and insulin action and indicate that ERK1/2 activation is necessary for human adipogenesis in the absence of mitotic clonal expansion.

The mitogenic signaling pathway for fibroblast growth factor-2 involves the tyrosine phosphorylation of cyclin D2 in MCF-7 human breast cancer cells

Fibroblast growth factor-2 (FGF-2) is mitogenic for the human breast cancer cell line MCF-7; here we investigate some of the signaling pathways subserving this activity. FGF-2 stimulation of MCF-7 cells resulted in a global increase of intracellular tyrosine phosphorylation of proteins, particularly FGF receptor substrate-2, the protooncogene product Src and the mitogen-activated protein kinase (MAP kinase) cascade, A major increase in the tyrosine phosphorylation of a 30-kDa protein species was also found. This protein was identified as cyclin D2 by mass spectrometry after trypsin digestion. Immunoprecipitation of cyclin D2 and immunoblotting with anti-phosphotyrosine antibodies confirmed that the tyrosine phosphorylation of cyclin D2 was indeed induced by FGF-2 stimulation. In addition, pharmacological inhibition of Src (with herbimycin A and PP2), and of the MAP kinase cascade (with PD98059), confirmed that Src activity is required for the FGF-2-induced phosphorylation of cyclin D2 whereas MAP kinase activity is not, Thus, tyrosine phosphorylation of cyclin D2 may be a hey regulatory target for FGF-2 signaling. (C) 2000 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.