Evidence for new phosphorylation sites for protein kinase C and cyclic AMP-dependent protein kinase in bovine heart 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

Bovine heart 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2) was phosphorylated by incubation with [gamma-32P]MgATP and cyclic AMP-dependent protein kinase (PKA) or protein kinase C (PKC). After digestion with chymotrypsin, the phosphorylation sites for the two protein kinases were identified by peptide mapping, and microsequencing. Evidence for new phosphorylation sites for PKA (Ser-483) and PKC (Ser-84 and Ser-466) was obtained.     

Assignment of the dystonia-parkinsonism syndrome locus, DYT3, to a small region within a 1.8-Mb YAC contig of Xq13.1.

A YAC contig was constructed of Xq13.1 in order to sublocalize the X-linked dystonia-parkinsonism (XDP) syndrome locus, DYT3. The contig spans a region of approximately 1.8 Mb and includes loci DXS453/DXS348/IL2R gamma/GJB1/CCG1/DXS559. For the construction of the contig, nine sequence-tagged sites and four short tandem repeat polymorphisms (STRPs) were isolated. The STRPs, designated as 4704#6 (DXS7113), 4704#7 (DXS7114), 67601 (DXS7117), and B4Pst (DXS7119) were assigned to a region flanked by DXS348 proximally and by DXS559 distally. Their order was DXS348/4704 #6/4704 #7/67601/B4Pst/DXS559. They were applied to the analysis of allelic association and of haplotypes in 47 not-obviously-related XDP patients and in 105 Filipino male controls. The same haplotype was found at loci 67601 (DXS7117) and B4Pst (DXS7119) in 42 of 47 patients. This percentage of common haplotypes decreased at the adjacent loci. The findings, together with the previous demonstration of DXS559 being the distal flanking marker of DYT3, assign the disease locus to a small region in Xq13.1 defined by loci 67601 (DXS7117) and B4Pst (DXS7119). The location of DYT3 was born out by the application of a newly developed likelihood method for the analysis of linkage disequilibrium.     

CHARACTERIZATION OF HUMAN RECOMBINANT INTERLEUKIN 2 BINDING TO HEPARIN AND HEPARAN SULFATE USING AN ELISA APPROACH

We have developed an enzyme-linked immunosorbent assay (ELISA) approach for the study of interactions between cytokines and glycosaminoglycans. This involves, as solid phase, a synthetic heparin–bovine serum albumin (BSA) complex in which the heparin is coupled via its reducing terminus to the protein using sodium cyanoborohydride. We have investigated the sensitivity and specificity of this experimental technique, employing antithrombin (AT III) and fibroblast growth factor 2 (FGF-2) as well-characterized heparin binding proteins. Using this ELISA method, we have established that human recombinant interleukin (IL-2) binds to heparin in a concentration-dependent manner. Soluble heparin competes for the binding of IL-2 to the complex with 50% inhibition at 5 μg/ml. This IC₅₀value provides an estimate of the binding constant of around 0.5 μM. This value is at least two orders of magnitude larger than that for the binding of IL-2 to its dimeric and trimeric cell surface receptors, but similar to that for binding to the IL-2 receptor β polypeptide acting alone. Our ELISA shows that in addition to soluble heparin, fuciodan also competes for IL-2 binding, but chondroitin sulfate and dermatan sulfate are inactive. Of six heparan sulfates tested, only one highly sulfated preparation competed for IL-2. The interaction between IL-2 and heparin-like glycosaminoglycans is likely to be an important mechanism for retaining IL-2 close to its sites of secretion, thus giving rise to localized concentration gradients in the tissues.     

The potential for heparin and its derivatives in the therapy and prevention of HIV-1 infection

Heparin is one of several sulphated polysaccharides which potently inhibit replication of the human immunodeficiency virus type 1 (HIV-1) in cultures of CD4;ve human cells. The EC50 value is around 5 μg ml-1. We have demonstrated that heparin binds to recombinant gp120, the envelope glycoprotein of HIV-1, at a site termed the V3 loop, or principle neutralizing domain, which consists of a disulphide-bridged loop of 32–35 amino acids particularly enriched with basic residues. Using a series of chemically modified heparins we have shown that there is structural specificity in the anti-HIV activity of heparin. Heparin is routinely used clinically as an anticoagulant, and has proved essentially non-toxic and well tolerated. Low anticoagulant derivatives of heparin which retain high anti-HIV-1 activities in vitro may be generated by several routes. Such preparations are ideal candidates for clinical investigation as potential novel therapeutic agents for use in combination with other drugs in the management of AIDS and HIV infection. This revised version was published online in November 2006 with corrections to the Cover Date.     

Multiple tandem 18-kb sequences clustered in the region of the acute promyelocytic leukemia breakpoint on chromosome 17

This paper describes the cloning of an 18-kb sequence present in approximately 30 copies on chromosome 17. Most of these are clustered in the region of the breakpoint associated with acute promyelocytic leukemia (APL). These copies map both above and below the breakpoint, and pulsed field gel analysis indicates that the majority of these sequences lie within a region of approximately 2 megabases. The organization of these sequences appears to be that of large imperfect palindromes.     

Effect of immune system imagery on secretory IgA

This study was an investigation of the effects of physiologically-oriented mental imagery on immune functioning. College students with normal medical histories were randomly selected to one of three groups. Subjects in Group 1 participated in short educational training on the production of secretory immunoglobulin A. They were then tested on salivary IgA, skin temperature, and the Profile of Mood States (POMS) before and after listening to a 17-minute tape of imagery instructions with specially composed background "entrainment" music designed to enhance imagery. Subjects in Group 2 (placebo controls) listened to the same music but received nor formal training on the immune system. Group 3 acted as a control and subjects were tested before and after 17 minutes of no activity. Treatment groups listened to their tapes at home on a bi-daily basis for six weeks. All groups were again tested at Weeks 3 and 6. Secretory IgA was analyzed using standard radial immunodiffusion techniques. Repeated measures analyses of variance with planned orthogonal contrasts were used to evaluate the data. Significant overall increases (p less than 0.05) were found between pre- and posttests for all three trials. Groups 1 and 2 combined (treatment groups) yielded significantly greater increases in sIgA over Group 3 (control) for all three trials. Group 1 (imagery) was significantly higher than Group 2 (music) in antibody production for Trials 2 and 3. Symptomatology, recorded by subjects at Weeks 3 and 6, was significantly lower for three symptoms (rapid heartbeat, breathing difficulty, and jaw clenching), favoring both treatment groups over the control group.     

A trypsin-sensitive, heat-labile, N-ethylmaleimide-sensitive factor in adipocyte post-microsomal supernatant which affects the assay of adipocyte glycerol phosphate acyltransferase activities.

Addition of adipocyte 100 000 g post-microsomal supernatant to assays of glycerol phosphate acyltransferase in isolated mitochondria or microsomal fractions decreased activity at lower concentrations of palmitoyl-CoA. At higher concentrations of palmitoyl-CoA, activation was observed on addition of post-microsomal supernatant. The effect of post-microsomal supernatant to decrease activity at lower [palmitoyl-CoA] was abolished by heating or by trypsin treatment, and was also abolished by addition of N-ethylmaleimide to assays or by pretreatment of post-microsomal supernatant with N-ethylmaleimide. The stimulatory effect seen at higher [palmitoyl-CoA] was not sensitive to heat or trypsin treatment. The effect of post-microsomal supernatant at lower [palmitoyl-CoA] cannot be attributed to palmitoyl-CoA hydrolase activity. It was found that brief treatment of adipocyte mitochondria with low concentrations of trypsin was an effective way to remove contaminating microsomal glycerol phosphate acyltransferase activity. Adipocyte post-microsomal supernatant was more effective than an equivalent quantity of liver post-microsomal supernatant protein in decreasing adipocyte microsomal glycerol phosphate acyltransferase activity. The effects of the supernatants from both tissues were decreased by flavaspidic acid. Semi-purified Z-protein fraction from rat liver did not mimic the effect of adipocyte post-microsomal supernatant to decrease glycerol phosphate acyltransferase at lower [palmitoyl-CoA]. Post-microsomal supernatants obtained from noradrenaline-treated adipocytes were less effective than those from control cells in decreasing glycerol phosphate acyltransferase activity in microsomal fractions at lower [palmitoyl-CoA]. It is suggested that adipocyte cytosol may contain an acyl-CoA-binding protein or proteins differing from Z-protein in some respects. The physiological significance of the findings is briefly discussed.     

Resistance of factor XIII to degradation or activation by plasmin

The effect of plasmin on the subunit polypeptides of factor XIII has been investigated. purified human plasma (a2b2) and platelet (a2) zymogens and the enzyme (a2) were incubated with plasmin at plasmin: factor XIII ratios of 0.03-0.5 casein units per mg protein. Under conditions in which plasmin readily digested fibrinogen and casein, it had no effect on either a2b2 or a2. There was no evidence for cleavage of peptide bonds in the zymogens, and all the potential catalytic activity was retained after prolonged incubation. Similarly a2*, either in the presence or absence of b subunit, was also unaffected by plasmin incubation. 90% of the activity was recovered after incubation of factor XIII with plasmin. b subunit was also not degraded. Additionally, no evidence was obtained that plasmin could activate factor Xiii. These results indicate that in purified systems there is no significant interaction between plasmin and factor XIII.