The inhibition of tube formation in a collagen-fibrinogen, three-dimensional gel by cleaved kininogen (HKa) and HK domain 5 (D5) is dependent on Src family kinases

Cleaved high molecular weight kininogen (HKa), as well as its domain 5 (D5), inhibits migration and proliferation induced by angiogenic factors and induces apoptosis in vitro. To study its effect on tube formation we utilized a collagen-fibrinogen, three-dimensional gel, an in vitro model of angiogenesis. HKa, GST-D5 and D5 had a similar inhibitory effect of tube length by 90+/-4.5%, 86+/-5.5% and 77+/-12.9%, respectively. D5-derived synthetic peptides: G440-H455 H475-H485 and G486-K502 inhibited tube length by 51+/-3.7%, 54+/-3.8% and 77+/-1.7%, respectively. By a comparison of its inhibitory potency and its sequences, a functional sequence of HKa was defined to G486-G496. PP2, a Src family kinase inhibitor, prevented tube formation in a dose-dependent manner (100-400 nM), but PP3 at 5 microM, an inactive analogue of PP2, did not. HKa and D5 inhibited Src 416 phosphorylation by 62+/-12.3% and 83+/-6.1%, respectively. The C-terminal Src kinase (Csk) inhibits Src kinase activity. Using a siRNA to Csk, expression of Csk was down-regulated by 86+/-7.0%, which significantly increased tube length by 27+/-5.8%. The addition of HKa and D5 completely blocked this effect. We further showed that HKa inhibited Src family kinase activity by disrupting the complex of uPAR, alphavbeta3 integrin and Src. Our results indicate that the anti-angiogenic effect of HKa and D5 is mediated at least in part through Src family kinases and identify a potential novel target for therapeutic inhibition of neovascularization in cancer and inflammatory arthritis.     

The effect of dimethyl sulfoxide on cholesterol and bile acid metabolism in rats

The effect of DMSO on cholesterol and bile acid metabolism was studied in rats. Male Sprague-Dawley rats were randomly assigned to one of two groups and given either tap water or 2% DMSO (v/v) in tap water to drink for 9 days. Both food (stock rat diet) and water were available ad libitum. Animals in both groups gained weight equally throughout the study. They also had similar liver weights (g/100 g body wt) at the end of the study (control: 5.0 +/- 0.1 (N = 6) vs DMSO: 4.9 +/- 0.1 (N = 6]. The activity of hepatic cholesterol 7 alpha-hydroxylase (pmole/mg/min), the rate-limiting enzyme of bile acid biosynthesis, was significantly (P less than 0.005) reduced in the treated animals (control: 9.7 +/- 1.0 (N = 6) vs DMSO: 4.3 +/- 0.7 (N = 6)). Plasma cholesterol (mg/dl) was significantly (P less than 0.005) elevated in the treated animals (control: 90 +/- 3 (N = 6) vs DMSO: 107 +/- 4 (N = 6)), a finding consistent with the reduced CH-7 alpha hydroxylase activity in this group. DMSO treatment did not affect either microsomal cholesterol content or hepatic glutathione content. Thus, this study has shown that DMSO treatment per se can affect cholesterol and bile acid metabolism. However, the precise mechanisms whereby DMSO exerts the observed effects are not known.     

Analysis of mosquito bloodmeals using RFLP markers

An important variable in the amplification of arthropod vector-borne diseases is the degree of contact between human hosts and mosquito vectors. To analyze this interaction, a DNA based method was developed to differentiate human bloodmeals from other sources in the mosquito Anopheles stephensi (Diptera: Culicidae) Liston. A portion of the host mitochondrial DNA cytochrome B genes were PCR amplified and classified to the species level based on their restriction fragment length polymorphism (RFLP). The cytochrome B sequences showed sufficient interspecific polymorphism to distinguish between human, cow, sheep, chicken, and guinea pig hosts. XhoI could distinguish human from other vertebrates whereas TaqI alone could separate the others. The importance of these results in epidemiological studies of malaria and other vector borne diseases is discussed.     

Study of the mouse sortilin gene: Effects of its transient silencing by RNA interference in TM4 Sertoli cells

Using databases of the mouse genome in combination with a sequence deduced from a mouse sortilin cDNA originated in our laboratory, we found the sortilin gene to map to a region of chromosome 3. The mouse sortilin gene contains 19 short exons separated by introns of various sizes. The study elucidated the exon-intron boundaries. Some introns extend over more than 24 kb. In the cytoplasmic domain of the translation product, we found a dileucine motif and three other motifs known to constitute the active sorting signal of the mannose 6-phosphate receptor (M6P-R). We also tested the hypothesis that sortilin is involved in the sorting of prosaposin (SGP-1) to the lysosomes. Prosaposin was initially identified in Sertoli cells, found in large amounts in the lysosomal compartment and implicated in the degradation of residual bodies released by the spermatids during spermiation. Interestingly, the targeting of prosaposin to the lysosomes is independent of the M6P-R. This investigation demonstrated that sortilin was required for the trafficking of prosaposin to the lysosomes in TM4 cells. The requirement of sortilin was shown using a siRNA probe to block the translation of sortilin mRNA. Sortilin-deficient cells were not able to route prosaposin to the lysosomal compartment but continue to transport cathepsin B, since this hydrolase uses the M6P-R to be routed to the lysosomes. These results indicate that sortilin appears to be involved in the lysosomal trafficking of prosaposin.     

Characterization of human HtrA2, a novel serine protease involved in the mammalian cellular stress response.

Human HtrA2 is a novel member of the HtrA serine protease family and shows extensive homology to the Escherichia coli HtrA genes that are essential for bacterial survival at high temperatures. HumHtrA2 is also homologous to human HtrA1, also known as L56/HtrA, which is differentially expressed in human osteoarthritic cartilage and after SV40 transformation of human fibroblasts. HumHtrA2 is upregulated in mammalian cells in response to stress induced by both heat shock and tunicamycin treatment. Biochemical characterization of humHtrA2 shows it to be predominantly a nuclear protease which undergoes autoproteolysis. This proteolysis is abolished when the predicted active site serine residue is altered to alanine by site-directed mutagenesis. In human cell lines, it is present as two polypeptides of 38 and 40 kDa. HumHtrA2 cleaves beta-casein with an inhibitor profile similar to that previously described for E. coli HtrA, in addition to an increase in beta-casein turnover when the assay temperature is raised from 37 to 45 degrees C. The biochemical and sequence similarities between humHtrA2 and its bacterial homologues, in conjunction with its nuclear location and upregulation in response to tunicamycin and heat shock suggest that it is involved in mammalian stress response pathways.     

Phylogenetic relationships of Iranian Allium sect. Allium (Amaryllidaceae,Allioideae) as inferred from nrDNA ITS,cpDNA rps16 and trnL–F sequences

Allium is a particularly species rich (more than 800 species) and economically important genus, with numerous taxonomic problems at all levels of classification. In this study, we try to uncover the phylogenetic relationships of the common leek Allium ampeloprasum based on selected samples of this species and its putative relatives in A. sect. Allium from Iran. The silica‐dried leaf samples of 56 accessions representing 23 species of Allium were sequenced; 53 sequences of nrDNA ITS, 35 sequences of plastid rps16 and 52 sequences of trnL–F were generated and additional accessions were extracted from GenBank in order to cover all recognized main lineages in the genus. Maximum Parsimony and Bayesian Inference generated similar trees, but the placement of A. ampeloprasum and its relatives differed slightly between the nuclear and the plastid phylogenies. In the nrITS tree, A. ampeloprasum is retrieved as a highly supported clade with A. iranicum, while in the combined plastid tree A. ampeloprasum formed a highly supported clade with A. vineale. This supports the hypothesis of a possible hybrid origin of A. ampeloprasum. Allium iranicum formed a clade in the plastid tree, but was resolved as paraphyletic in the nrITS tree, probably due to presence of multiple non‐concerted copies of nrITS. Close relationships are suggested between the following species: A. aznavense and A. wendelboi with A. talyschense, A. erubescens and A. rotundum with A. scorodoprasum and A. abbasii with A. phanerantherum.     

Synthesis of 1-(2-naphthylsulfonyl)pyrazole-C-glycosides

2-Naphthylsulfonylhydrazine was reacted with aromatic aldehydes or aldehydo sugars to give the corresponding hydrazones which undergo Michael addition reactions with malononitrile or ethyl cyanoacetate to form pyrazole derivatives.     

Applicability of Heavy-Metal Phytoextraction in United Arab Emirates: An Investigation of Candidate Species

Phytoremediation of contaminated calcareous desert land in the United Arab Emirates has been investigated. Soils from 12 northern UAE sites, suspected of metal contamination, were acid-extracted and analyzed by ICP-OES for Co, Cr, Cu, Fe, Mn, Ni, Pb, and Zn. Twenty-two plants naturally growing at contaminated sites were sampled and analyzed for their uptake of Co, Cr, Cu, Mn, Ni, Pb, and Zn and eight commercially available plants, grown under controlled conditions, were also studied for their phytoextraction capabilities. The concentration of available Cr was found to be 1300 ± 150 mg/kg in the soil of the Ajman Industrial Zone and 80 ± 10 mg/kg of Pb was found at Bithna. Among the plants investigated, Portulaca oleracea and Iresine herbstii showed potential for Cr(VI) and Pb(II) accumulation, respectively, with bioconcentration factors (BCF) greater than unity. Atriplex halimus accumulated Co(II), Cr(III), and Cu(II) each with a BCF > 1.