Evaluation of the association between null genotypes of glutathione-S-transferases and Behcet's disease.

Glutathione S-transferases (GST) play an important role in oxidative stress related syndromes. An imbalance of the oxidant and antioxidant systems is important in the pathogenesis of Behcet's disease (BD). The objective of this study was to evaluate the association of null genotypes of GST-M1 and GST-T1 with BD since some preliminary molecular genetic data were recently published. Ninety-four Turkish BD patients (42 male, 52 female, 37.1+/-10.4 years) and 140 healthy volunteers (70 male, 70 female, 36.8+/-11.7 years) matched for age and gender with the patients as the control group were included in the study. Distributions of GST-M1 and GST-T1 genotypes were determined by multiplexed PCR using three sets of primers for GST-M1, GST-T1, and beta-globulin genes. There was no association between BD and the frequencies of GST-M1 and GST-T1 null genotypes when compared to controls by separate analysis. However, by cross and pooled combination analysis there was a significant association between the frequencies of pooled GSTs with one or both null genotypes in BD and controls. This is the first evidence that the association between the frequencies of GST-M1 and GST-T1 null genotypes and BD might be dependent on the interaction of multiple null allele polymorphisms rather than a single null allele of GST-M1 and GST-T1.

Prostaglandin D synthase (beta-trace protein): a molecular clock to trace the origin of REM sleep?

In 1965, Zuckerkandl and Pauling proposed a novel concept that some important molecules termed semantides, which carry the information of the genes or a transcript thereof, can be used as molecular clocks to trace evolutionary history. According to this concept, enzymes are designated as tertiary semantides, following genes (primary semantides) and the mRNA (secondary semantides). Based on this idea, I propose that prostaglandin D synthase (which has been demonstrated recently as identical to the beta-trace protein present in the cerebrospinal fluid of mammals) may serve as a molecular clock to trace the origin and evolution of rapid eye movement sleep in the vertebrates.

Functional properties of the beta-globin locus control region in K562 erythroleukemia cells.

In this report, we compare the function of the human beta-globin locus control region (LCR) in three K562 erythroleukemia cell assays, including (1) a transient transfection assay for "classical" enhancer activity, (2) a colony assay that detects "productive integration events," and (3) an assay that detects the ability of LCR fragments to confer hemin inducibility on linked, stably integrated gamma-globin promoters. Various LCR fragments were inserted into an expression vector consisting of an A gamma-globin promoter driving the neomycin phosphotransferase gene (gamma-neo). Using these vectors, we determined that a 2.5-kb DNA fragment containing LCR sites I through IV (previously named mu locus activation region [mu LAR]) had activity in all three assays; of the individual LCR sites, only site II was highly active in all three assays. One region within site II, consisting of tandem AP-1/NF-E2 consensus elements, had approximately 10% as much colony assay activity as the entire mu LAR. However, this region did not have detectable activity in a transient enhancer assay in uninduced K562 cells, nor was it capable of conferring hemin inducibility on linked gamma-globin promoters in stably transfected cells. Finally, we tested the ability of the mu LAR to activate promoters (beta-globin and cathepsin G) that are not normally expressed in K562 cells. beta-neo was minimally activated by the mu LAR in transient transfection experiments. The mu LAR increased the number of stably transfected colonies produced by beta-neo, but the absolute number of beta-neo colonies, with or without the mu LAR, was approximately 10% to 20% that of gamma-neo. In contrast, a minimal cathepsin G promoter was activated by the mu LAR in K562 cells. Our results suggest that LCR functions are dependent in part on the environments and the promoters with which the LCR is tested.

Effect of human plasma proteins on spontaneous contractile activity of rat mesenteric portal vein.

This study examines the effect of various plasma proteins from man on the spontaneous contractile activity of the rat portal vein. Albumin, gamma-globulin, alpha-globulin, beta-globulin (the major plasma proteins), and immunoglobulin IgG (the major immunoglobulin present in the gamma-globulin fraction) were obtained commercially. Mesenteric portal vein strips were prepared from rats and placed in a physiological salt solution in muscle baths for the measurement of longitudinal mechanical response. Portal veins exposed to albumin or gamma-globulin showed a dose-dependent increase in the spontaneous activity, whereas those exposed to alpha-globulin or alpha- and beta-globulin together showed a dose-dependent inhibition of spontaneous activity. Immunoglobulin IgG produced a dose-dependent increase in the spontaneous activity similar to that of gamma-globulin. The increased spontaneous activity produced by albumin was not prevented by ouabain but was inhibited by phentolamine. Spontaneous contractile activity was stimulated by albumin in the chemically (6-hydroxydopamine) denervated portal vein. These findings indicate that albumin acts in a manner similar to noradrenaline. The increased spontaneous activity caused by gamma-globulin (IgG) was inhibited by ouabain or verapamil. The effect of IgG was not dependent on alpha-adrenergic, cholinergic, histaminergic, serotoninergic, or renin angiotensin systems nor was it affected by removal of the endothelium. These observations may have implications in the pathophysiology of essential hypertension.

A new hereditary persistence of fetal hemoglobin deletion has the breakpoint within the 3' beta-globin gene enhancer.

A new deletion of the beta-globin gene cluster has been characterized in two Italian brothers who are heterozygous carriers of a G gamma A gamma hereditary persistence of fetal hemoglobin (HPFH). Restriction endonuclease mapping and DNA sequencing of the region encompassing the breakpoint show that the deletion starts 3.2 kilobases (kb) upstream from the delta gene and ends within the enhancer region 3' to the beta-globin gene. Here the deletion removes one of the four binding sites for an erythroid specific transcriptional factor (NF-E1). The molecular comparison of the new deletion with others of similar size and location but associated with a delta beta-thalassemia phenotype suggests that the residual enhancer element, relocated near gamma genes, may increase the fetal hemoglobin (HbF) expression above the level observed in delta beta-thalassemia.

Function of normal and mutated gamma-globin gene promoters in electroporated K562 erythroleukemia cells.

We examined the importance of cis-acting regulatory elements of the human gamma-globin gene promoter in a cell line (K562) where this gene normally functions. A gamma-Globin promoter fragments were fused to the neomycin phosphotransferase (neoR) gene in a plasmid-based vector and transiently transfected by electroporation into K562 cells. Correctly initiated "A gamma-neo" transcripts were detected with an S1 nuclease protection assay that was internally controlled for transfection efficiency and RNA content. We first optimized the conditions for electroporation, and then determined input DNA concentrations that permitted study of gamma-promoter function in the linear range of the assay. We discovered that a gamma-globin promoter fragment extending from -299 to +36 (with respect to the transcription initiation site) was active in this transient transfection assay, and that the expression of this promoter was increased by the SV40 enhancer. Deletion of the gamma-globin promoter to position -199 did not significantly affect gamma-globin promoter function. However, deletion to -160 reduced gamma promoter strength to 70% that of control, deletion to position -130 to 19% that of control, and deletion to position -61 to 8.7% that of control. Three gamma-globin promoters containing mutations associated with hereditary persistence of fetal hemoglobin (-202 C----G, -196 C----T, and -117 G----A) were not overexpressed in the K562 cell environment, consistent with the hypothesis that these promoters are not overexpressed in fetal erythroblasts, only adult erythroid cells. This system will allow us to further dissect the roles of regulatory globin cis-acting DNA elements in fetal erythroid cells.

Effects of somatostatin on the behavior of thromboxane B2 and B-thromboglobulin in type 1 diabetes.

Somatostatin, as an adjunct to insulin in the treatment of poorly controlled type 1 diabetes, has been recently suggested. However, some authors, after injection of the polypeptide, detected a reduction in number and aggregation of platelets, others instead, reported a proaggregatory effect of the drug. To answer these questions, the plasma levels of proaggregatory thromboxane A2 and of B-thromboglobulin, marker of the platelet activation, were studied in nine control subjects and in thirteen insulin dependent diabetic patients before and during the endovenous injection, for three hours, of somatostatin (250 micrograms in bolus followed by infusion of 100 micrograms/h). In both groups, 30 min after the infusion of somatostatin, a fall of thromboxane B2, stable metabolite of thromboxane A2 and an increase of B-thromboglobulin were respectively observed. Their greatest figure was reached after 120 min and their levels did not return to basal values within the end of the observation. In diabetics, during the infusion of somatostatin, thromboxane B2 presented normal percentual falls while B-thromboglobulin showed increases which were lower than controls. In conclusion, the effect of somatostatin implied in the relative lower increase of B-thromboglobulin, seems connected to the precedent continuous activation of circulating platelets, the one, responsible for the decrease of thromboxane B2, instead, seems linked to a direct action of somatostatin, independently on the deranged metabolic conditions found in diabetic patients.

Chemotactic activity of platelet alpha granule proteins for fibroblasts.

At sites of blood vessel injury, platelets release numerous substances that may have biological activities influencing cellular responses. In this study we examined separately the chemotactic activity for fibroblasts of three highly purified proteins obtained from platelet alpha granules: platelet factor 4 (PF4), platelet-derived growth factor (PDGF), and beta-thromboglobulin (BTG). We observed that each of these proteins was strongly chemotactic for fibroblasts, with maximum chemotactic activity in each instance comparable to that observed with an optimal concentration of the control chemotactic protein, plasma fibronectin. Each protein was active at very low concentrations. The peak chemotactic activities of PF4, PDGF, and BTG occurred at 200 mg/ml, 30 ng/ml, and 6 ng/ml, respectively. Specificity of fibroblast chemotaxis to individual platelet proteins was provided by finding that anti-PF4 immunoglobulin blocked the chemotactic activity of PF4 without affecting the chemotactic activity of PDGF, while anti-PDGF immunoglobulin blocked the activity of PDGF but did not alter the capacity of PF4 to promote fibroblast chemotaxis. These results suggest that in vivo several alpha granule proteins released from platelets may affect wound healing by causing directed fibroblast migration.

beta 2-Microglobulin.

In the sixties, Ingemar Berggård discovered, isolated and characterized the low molecular weight protein beta 2-microglobulin. The relations of beta 2-microglobulin to the immune system have led to intensive research on this protein. The present work reviews the purification and characterization of human beta 2-microglobulin and animal homologues, the association of beta 2-microglobulin to other molecules, the functional studies with beta 2-microglobulin and antibodies against beta 2-microglobulin, and the evolutionary studies on beta 2-microglobulin.

Heavy chain of HLA-A and HLA-B antigens is conformationally labile: a possible role for beta 2-microglobulin.

The three-dimensional organization of HLA antigens has been investigated by spectroscopic and immunochemical techniques. Measurement of the circular dichroism shows that in papain-solubilized HLA the heavy chain as well as the previously studied light chain (beta 2-microglobulin) consists predominantly of beta-pleated sheet structures. When heavy chain is separated from the light chain under denaturing conditions and is allowed to renature, about 50% of the beta structure is lost, concomitantly with most of the alloantigenic activity. Analysis of the two acid-cleaved fragments of HLA-B7 heavy chain shows that beta structure is preferentially lost from the COOH-terminal region of the heavy chain. Exposure to denaturants per se does not inevitably result in irreversible loss of antigenic activity. However, recovery of antigenic properties does seem to depend on reassociation of the two chains. The results reported here provide further evidence for (i) the similarity of HLA antigens and immunoglobulins at the three-dimensional level and (ii) two distinct and physiologically important conformations of the HLA heavy chain, depending upon whether it is associated with the light chain.

Reaction mechanisms of beta1H globulin.

The reaction mechanisms of beta1H were studied. The generation of alternative pathway C3 and C5 convertases on the cell surface as well as in the fluid phase was inhibited by beta1H globulin. The cell preparation bearing the C3b site could bind beta1H with little effect on the C3b hemolytic activity. Bound beta1H was dissociated by the action of C3bINA and C3bINA-treated C3b bearing cell did not bind beta1H anymore. Cell-bound beta1H was also dissociated by the action of B (or Bb). From these and other results, the following conclusions were obtained. The C3b site-bearing cell could bind beta1H on the C3c region of C3b molecules facilitating the C3bINA action on C3b, and beta1H shared the same binding site with B (or Bb) inhibiting the generation of the alternative pathway convertases competitively.