Polymorphism of human glycoprotein Ib alpha results from a variable number of tandem repeats of a 13-amino acid sequence in the mucin-like macroglycopeptide region. Structure/function implications.

Four polymorphic variants of the platelet receptor for von Willebrand factor, glycoprotein Ib, have been described that differ in molecular weight on sodium dodecyl sulfate-polyacrylamide gels (Moroi, M., Jung, S. M., and Yoshida, N. (1984) Blood 64, 622-629). A recent report localized the polymorphic site to the heavily O-glycosylated region of the glycoprotein Ib alpha-chain known as the macroglycopeptide (Meyer, M., and Schellenberg, I. (1990) Thromb. Res. 58, 233-242). This region contains several tandem repeats of a mucin-like sequence, which appeared to be a likely site for polymorphic variation. We amplified genomic DNA corresponding to the macroglycopeptide from 206 individuals from four ethnic groups and identified three length variants based on the migration of the amplified DNA on denaturing polyacrylamide gels. DNA sequencing revealed that the three variants represented four alleles, two of which varied by only one base pair, a difference that did not result in an amino acid change. The three length variants differed in the number of tandem repeats of a 39-base pair sequence that results in perfect duplication of a 13-amino acid sequence that originated within a region flanked by Glu-396 and Thr-411. The smallest isoform contained one such sequence; the next largest, two repeats; and the largest, three repeats. The DNA sequence containing the tandem repeats was flanked by direct repeats typical of the target site duplications found flanking transposed DNA, suggesting a mechanism for acquisition of this region by the primordial glycoprotein Ib alpha precursor. The amino acid sequence of the repeated element that accounts for the polymorphism contained five sites for potential O-glycosylation, which together with the repeated amino acids would result in incremental differences in molecular weight of approximately 6,000 between the different isoforms. The addition of repeats to the macroglycopeptide is predicted to increase the length of this elongated glycosylated region and extend the distance between the ligand-binding domain of glycoprotein Ib and the platelet plasma membrane, an effect that would project the ligand-binding domain farther into the bloodstream. Such a change may alter the susceptibility of platelets to shear-induced activation, a process that requires an interaction between glycoprotein Ib and von Willebrand factor.     

Antiretroviral activity of mechanism-based irreversible inhibitors of S-adenosylhomocysteine hydrolase.

S-Adenosylhomocysteine hydrolase (AdoHcy-nase) is a key enzyme in transmethylation reactions. The objective of the present study was to examine the potential antiretroviral activities of novel mechanism-based irreversible AdoHcy-nase inhibitors. (Z)-4',5'-didehydro-5'-deoxy-5'-fluoroadenosine (ZDDFA), (E)-4',5'-didehydro-5'-deoxy-5'-fluoroadenosine (EDDFA), (Z)-4',5'-didehydro-5'-deoxy-5'-chloroadenosine (ZDDCA) and 5'-deoxy-5'-acetylenic adenosine (DAA) inhibited AdoHcy-nase activity with Ki values of 0.55, 1.04, greater than 10.0 and 3.30 microM, respectively. These four compounds were tested for antiviral activity in vitro against Moloney leukemia virus (MoLV) in the XC-plaque assay. MoLV replication in murine fibroblasts (SC-1) was inhibited by ZDDFA, EDDFA and DAA with IC50 values of 0.05, 0.25 and 3.30 micrograms/ml, respectively. ZDDCA did not inhibit MoLV infection at the concentrations tested. Antiviral activity correlated with the ability of the individual compounds to maintain sustained elevations in intracellular S-adenosylhomocysteine (AdoHcy) concentrations in the SC-1 cells. ZDDFA, the most potent inhibitor of AdoHcy-nase and MoLV was also the most active in maintaining sustained elevations in intracellular AdoHcy levels. The antiviral activity of ZDDFA was also examined in murine C3H1OT1/2 fibroblasts which constitutively produce MoLV. Pretreatment with ZDDFA (1.0 microgram/ml) for 24 hr inhibited virus production by 88%. Similar to the SC-1 cells, and concomitant with enzyme inhibition, there was a 300-fold increase in AdoHcy levels in ZDDFA (1.0 microgram/ml) treated C3H1OT1/2 cells. Incorporation of a [3H]methyl group from tritiated S-adenosylmethionine into total RNA in C3H1OT1/2 cells was inhibited by ZDDFA without affecting cell viability. These results suggest that mechanism-based inhibitors of AdoHcy-nase, such as ZDDFA, may have potential as antiretroviral agents.     

Cell adhesion promoting peptide GVKGDKGNPGWPGAP from the collagen type IV triple helix: cis/trans proline-induced multiple 1H NMR conformations and evidence for a KG/PG multiple turn repeat motif in the all-trans proline state

Peptide GVKGDKGNPGWPGAPY (called peptide IV-H1), derived from the protein sequence of human collagen type IV, triple-helix domain residues 1263-1277, represents an RGD-independent, cell-specific, adhesion, spreading, and motility promoting domain in type IV collagen. In this study, peptide IV-H1 has been investigated by 1H NMR (500 MHz) spectroscopy. Cis-trans proline isomerization at each of the three proline residues gives rise to a number of slowly exchanging (500-MHz NMR time scale) conformation states. At least five such states are observed, for example, for the well-resolved A14 beta H3 group, and K3, which is six residues sequentially removed from the nearest proline, i.e., P9, shows two sets. The presence of more than two sets of resonances for residues sequentially proximal to a proline, e.g., A14-cis-P15 and A14-trans-P15, and more than one set for a residue sequentially well-removed from a proline, e.g., K3, indicates long range conformation interactions and the presence of preferred structure in this short linear peptide. Many resonances belonging to these multiple species have been assigned by using mono-proline-substituted analogues. Conformational (isomer) state-specific 2D 1H NMR assignments for the combination of cis and trans proline states have been made via analysis of COSY-type, HOHAHA, and NOESY spectra. Peptide IV-H1 in the all-trans proline state ttt exists in relatively well-defined conformation populations showing numerous short- and long-range NOEs and long-lived backbone amide protons and reduced backbone NH temperature coefficients, suggesting hydrogen-bonding, and structurally informative 3J alpha N coupling constants. The NMR data indicate significant beta-turn populations centered at K3-G4, K5-G6, P9-G10, and P12-G13, and a C-terminal gamma-turn within the A14-P15-Y16 sequence. These NMR data are supported by circular dichroic studies which indicate the presence of 52% beta-turn, 10% helix, and 38% random coil structural populations. Since equally spaced KG and PG residues are found on both sides of peptide IV-H1 in the native collagen type IV sequence, this multiple turn repeat motif may continue through a longer segment of the protein. Synthetic peptide IV-H1 overlapping sequence "walk throughs" indicate that the primary biological activity is localized in the GNPGWPGAP double beta-turn domain, which contains the backbone constraining proline residues. This proline-domain conformation may suggest a collagen type IV receptor-specific, metastatic cell adhesion promoting binding domain.     

Mammalian hypotaurine aminotransferase: isethionate is not a product

We report that mammalian tissues posses hypotaurine (2-aminoethane- sulfinate) aminotransferase activity. One product of transamination, sulfinoacetaldehyde could theoretically undergo internal oxidation-reduction leading to isethionate (2-hydroxyethanesulfonate). This hypothesis was examined. No isethionate was formed $\text{in vitro}$.     

On the defence strategy of Physa fontinalis (L.), a freshwater pulmonate snail

Summary Physa fontinalis (L.) gives a characteristic, chemically mediated escape response when stimulated by the majority of British leeches and flatworms. The snail responds rapidly and consistently to contact with all the molluskivorous leeches but also to three species which may be considered harmless. However, no response was given to Erpobdella octoculata, the most abundant and widespread of the harmless leeches. The flatworms generally evoked less strong reactions. The adaptive significance of the pattern of responsiveness is discussed. A weaker shell-shaking response is elicited in conspecifics and it is shown that this antisocial behaviour leads to a relatively spaced-out dispersion pattern. A possible adaptive advantage is the reduction of risk of detection by shell-crushing fish predators, to which the snails are otherwise extremely vulnerable.     

Histone-histone interactions within chromatin. Crosslinking studies using ultraviolet light.

Irradiation of either whole cells or chromatin at 280 nm results in the covalent linkage of histones 2A and 2B, presumably at their mutual binding sites. The reaction is specific and proceeds with high yield (about 80%). Irradiation of reconstituted nucleohistone containing only H2A, H2B and DNA also yields the H2A-H2B dimer. The cross-linking event is sensitive to the conformation of the H2A-H2B pair since the histones must be bound to DNA for maximum cross-linking specificity at low ionic strength. However, the histones must first interact with each other before being deposited on the DNA, since separate addition of the histones to the DNA yields no dimer upon irradiation. If irradiation is conducted at 254 nm rather than 280 nm, DNA-histone cross-linking appears to dominate.     

The influence of a granulocytic inhibitor(s) on hematopoiesis in an in vivo culture system.

The in vivo diffusion chamber (DC) technique for mouse marrow culture was used to determine the effect of a granulocyte inhibitor on the proliferation of the pluripotent stem cell(CFU-s) and the granulocyte progenitor cell (CFU-c). Granulocyte conditioned medium was injected intraperitoneally into mice bearing DCs during the initial 48 hr of culture. The early injections of inhibitor resulted in a significantly reduced number of granulocytic progeny formed within the DCs while there was no growth inhibition of mouse fibroblasts cultured under identical conditions. The reduced cell production was due in part to a significant reduction in the self-renewal rate of the CFU-c while no apparent direct effect was observed upon the growth of the CFU-s within the same cultures. These data suggest that the granulocytic inhibitor(s) acted to reduce the proliferation within the CFU-c population and thereby diminished the amplification potential inherent in the initial cell inoculum.