Factors regulating circulating vascular endothelial growth factor (VEGF): Association with bone mineral density (BMD) in post-menopausal osteoporosis

Vascular endothelial growth factor (VEGF) plays an important role in bone health. We investigated the factors which influence circulating VEGF and their association with bone mineral density (BMD). Two hundred and fifty two post-menopausal women aged 64.5 [9.2] years were studied. BMD was determined at the lumbar spine (LS), femoral neck (FN) and total hip (TH). Serum oestradiol and VEGF were measured. Subjects were genotyped for two polymorphic variants in the 5′ untranslated region of the VEGF gene; G(634)C and C(936)T. Positive correlations were seen between circulating VEGF and BMI (r = 0.2, p < 0.02) and oestradiol (r = 0.25, p < 0.001). Following multi-linear regression analysis, serum VEGF was associated with the G(634) polymorphism (p = 0.08) and dietary calcium intake (p = 0.02). The association with calcium intake may be mediated by PTH as suggested by the in vitro studies. Following correction for confounders, there was no association between circulating VEGF and BMD at any site. Both VEGF polymorphisms were significant predictors of LS BMD G(634)C: p = 0.017 and C(936)T: p = 0.05. Circulating VEGF may be influenced by genetic, environmental and endocrine factors. Polymorphic variants in the VEGF gene are associated with spine BMD. Further larger studies are needed.     

Soluble fibrinogen-like protein 2/fibroleukin exhibits immunosuppressive properties: suppressing T cell proliferation and inhibiting maturation of bone marrow-derived dendritic cells

Fibrinogen-like protein 2 (fgl2)/fibroleukin is a member of the fibrinogen-related protein superfamily. In addition to its established role in triggering thrombosis, it is known to be secreted by T cells. The soluble fgl2 ((s)fgl2) protein generated in a baculovirus expression system bound to both T cells and bone marrow-derived dendritic cells (DC) in a specific manner. (s)fgl2 exhibited immunomodulatory properties capable of inhibiting T cell proliferation stimulated by alloantigens, anti-CD3/anti-CD28 mAbs, and Con A in a dose-dependent manner; however, it had no inhibitory effects on CTL activity. The time- and dose-dependent inhibitory effect of (s)fgl2 on alloreactive T cell proliferation could be neutralized by a mAb against mouse fgl2. Polarization toward a Th2 cytokine profile with decreased production of IL-2 and IFN-gamma and increased production of IL-4 and IL-10 was observed in (s)fgl2-treated allogeneic cultures. Exposure of immature DC to (s)fgl2 abrogated the expression of CD80(high) and MHC class II(high) molecules and markedly inhibited NF-kappaB nuclear translocation, thus inhibiting their maturation. (s)Fgl2-treated DC had an impaired ability to stimulate allogeneic T cell proliferation. Maximal inhibition of proliferation was observed when allogeneic T cells were cultured with (s)fgl2-treated DC and (s)fgl2 protein was added in the culture. These data provide the first evidence to demonstrate that (s)fgl2 exerts immunosuppressive effects on T cell proliferation and DC maturation.     

Metabolic activation of 3-hydroxyanisole by isolated rat hepatocytes

A tyrosinase-directed therapeutic approach for malignant melanoma therapy uses the depigmenting phenolic agents such as 4-hydroxyanisole (4-HA) to form cytotoxic o-quinones. However, renal and hepatic toxicity was reported as side effects in a recent 4-HA clinical trial. In search of novel therapeutics, the cytotoxicity of the isomers 4-HA, 3-HA and 2-HA were investigated. In the following, the order of the HAs induced hepatotoxicity in mice, as measured by increased in vivo plasma transaminase activity, or in isolated rat hepatocytes, as measured by trypan blue exclusion, was 3-HA > 2-HA > 4-HA. Hepatocyte GSH depletion preceded HA induced cytotoxicity and a 4-MC-SG conjugate was identified by LC/MS/MS mass spectrometry analysis when 3-HA was incubated with NADPH/microsomes/GSH. 3-HA induced hepatocyte GSH depletion or GSH depletion when 3-HA was incubated with NADPH/microsomes was prevented by CYP 2E1 inhibitors. Dicumarol (an NAD(P)H: quinone oxidoreductase inhibitor) potentiated 3-HA- or 4-methoxycatechol (4-MC) induced toxicity whereas sorbitol (an NADH generating nutrient) greatly prevented cytotoxicity indicating a quinone-mediated cytotoxic mechanism. Ethylendiamine (an o-quinone trap) largely prevented 3-HA and 4-MC-induced cytotoxicity indicating that o-quinone was involved in cytotoxicity. Dithiothreitol (DTT) greatly reduced 3-HA and 4-MC induced toxicity. The ferric chelator deferoxamine slightly decreased 3-HA and 4-MC induced cytotoxicity whereas the antioxidants pyrogallol or TEMPOL greatly prevented the toxicity suggesting that oxidative stress contributed to 3-HA induced cytotoxicity. In summary, ring hydroxylation but not O-demethylation/epoxidation seems to be the bioactivation pathway for 3-HA in rat liver. The cytotoxic mechanism for 3-HA and its metabolite 4-MC likely consists cellular protein alkylation and oxidative stress. These results suggest that 3-HA is not suitable for treatment of melanoma.     

Evaluation of annotation strategies using an entire genome sequence

MOTIVATION: Genome-wide functional annotation either by manual or automatic means has raised considerable concerns regarding the accuracy of assignments and the reproducibility of methodologies. In addition, a performance evaluation of automated systems that attempt to tackle sequence analyses rapidly and reproducibly is generally missing. In order to quantify the accuracy and reproducibility of function assignments on a genome-wide scale, we have re-annotated the entire genome sequence of Chlamydia trachomatis (serovar D), in a collaborative manner. RESULTS: We have encoded all annotations in a structured format to allow further comparison and data exchange and have used a scale that records the different levels of potential annotation errors according to their propensity to propagate in the database due to transitive function assignments. We conclude that genome annotation may entail a considerable amount of errors, ranging from simple typographical errors to complex sequence analysis problems. The most surprising result of this comparative study is that automatic systems might perform as well as the teams of experts annotating genome sequences.     

Terminology-driven mining of biomedical literature

MOTIVATION: With an overwhelming amount of textual information in molecular biology and biomedicine, there is a need for effective literature mining techniques that can help biologists to gather and make use of the knowledge encoded in text documents. Although the knowledge is organized around sets of domain-specific terms, few literature mining systems incorporate deep and dynamic terminology processing. RESULTS: In this paper, we present an overview of an integrated framework for terminology-driven mining from biomedical literature. The framework integrates the following components: automatic term recognition, term variation handling, acronym acquisition, automatic discovery of term similarities and term clustering. The term variant recognition is incorporated into terminology recognition process by taking into account orthographical, morphological, syntactic, lexico-semantic and pragmatic term variations. In particular, we address acronyms as a common way of introducing term variants in biomedical papers. Term clustering is based on the automatic discovery of term similarities. We use a hybrid similarity measure, where terms are compared by using both internal and external evidence. The measure combines lexical, syntactical and contextual similarity. Experiments on terminology recognition and clustering performed on a corpus of MEDLINE abstracts recorded the precision of 98 and 71% respectively. AVAILABILITY: software for the terminology management is available upon request.     

Dicarboxylic amino acids and glycine-betaine regulate chaperone-mediated protein-disaggregation under stress

Active protein-disaggregation by a chaperone network composed of ClpB and DnaK + DnaJ + GrpE is essential for the recovery of stress-induced protein aggregates in vitro and in Escherichia coli cells. K-glutamate and glycine-betaine (betaine) naturally accumulate in salt-stressed cells. In addition to providing thermo-protection to native proteins, we found that these osmolytes can strongly and specifically activate ClpB, resulting in an increased efficiency of chaperone-mediated protein disaggregation. Moreover, factors that inhibited the chaperone network by impairing the stability of the ClpB oligomer, such as natural polyamines, dilution, or high salt, were efficiently counteracted by K-glutamate or betaine. The combined protective, counter-negative and net activatory effects of K-glutamate and betaine, allowed protein disaggregation and refolding under heat-shock temperatures that otherwise cause protein aggregation in vitro and in the cell. Mesophilic organisms may thus benefit from a thermotolerant osmolyte-activated chaperone mechanism that can actively rescue protein aggregates, correctly refold and maintain them in a native state under heat-shock conditions.     

The age-related resistance of rats to Plasmodium berghei infection is associated with differential cellular and humoral immune responses

In this study, we investigated how the age of rats would affect the course of infection of and the immune response to Plasmodium berghei. Both young (4-week-old) and adult rats (8-week-old) can be infected with P. berghei ANKA strain, with significantly higher levels of infected red blood cells in young rats. While 100% of young rats succumbed to infection, adult rats were able to clear blood parasites and no mortality was observed. Analysis of cellular distribution and circulating cytokines demonstrated the persistence of CD4+/CD25+ T cells and high expression of circulating interleukin-10 (IL-10) during the progression of infection in young-susceptible rats, whereas high levels of CD8+ T cells and natural killer T cells are detected in adult-resistant rats. Analysis of antibody isotypes showed that adult rats produced significantly higher levels of interferon-gamma (IFN-gamma)-dependent IgG2c antibodies than young rats during infection. Further evaluation of the role of IL-10, IFN-gamma and of immune cells showed that only the adoptive transfer of spleen cells from adult-resistant rats was able to convert susceptibility of young-susceptible rats to a resistant phenotype. These observations suggest that cell-mediated mechanisms are crucial for the control of a primary infection with P. berghei in young rats.     

Genome sequences and great expectations

To assess how automatic function assignment will contribute to genome annotation in the next five years, we have performed an analysis of 31 available genome sequences. An emerging pattern is that function can be predicted for almost two-thirds of the 73,500 genes that were analyzed. Despite progress in computational biology, there will always be a great need for large-scale experimental determination of protein function.     

The nuclear orphan receptor COUP-TFII is required for limb and skeletal muscle development

The nuclear orphan receptor COUP-TFII is widely expressed in multiple tissues and organs throughout embryonic development, suggesting that COUP-TFII is involved in multiple aspects of embryogenesis. Because of the early embryonic lethality of COUP-TFII knockout mice, the role of COUP-TFII during limb development has not been determined. COUP-TFII is expressed in lateral plate mesoderm of the early embryo prior to limb bud formation. In addition, COUP-TFII is also expressed in the somites and skeletal muscle precursors of the limbs. Therefore, in order to study the potential role of COUP-TFII in limb and skeletal muscle development, we bypassed the early embryonic lethality of the COUP-TFII mutant by using two methods. First, embryonic chimera analysis has revealed an obligatory role for COUP-TFII in limb bud outgrowth since mutant cells are unable to contribute to the distally growing limb mesenchyme. Second, we used a conditional-knockout approach to ablate COUP-TFII specifically in the limbs. Loss of COUP-TFII in the limbs leads to hypoplastic skeletal muscle development, as well as shorter limbs. Taken together, our results demonstrate that COUP-TFII plays an early role in limb bud outgrowth but not limb bud initiation. Also, COUP-TFII is required for appropriate development of the skeletal musculature of developing limbs.