Structure and expression of the gene coding for the human serpin hLS2

We have analyzed genomic clones encoding human leuserpin 2 (hLS2). The gene covers about 14.5 kilobases and consists of 5 exons and 4 introns. The genes coding for hLS2, alpha 1-antitrypsin, alpha 1-antichymotrypsin, and rat angiotensinogen share an equivalent exon-intron structure and therefore constitute a distinct subgroup within the serpin gene family, which otherwise displays a highly variable exon-intron pattern. With the exception of a segment in the second exon, the sequence similarity of the genes coding for hLS2 and alpha 1-antitrypsin extends to all exons including one encoding the 5'-untranslated sequences. The implications of these findings with respect to the genesis of the amino-terminal heterogeneity in the serpin family are discussed.     

A new member of the plasma protease inhibitor gene family.

A 2.1-kb cDNA clone representing a new member of the protease inhibitor family was isolated from a human liver cDNA library. The inhibitor, named human Leuserpin 2 (hLS2), comprises 480 amino acids and contains a leucine residue at its putative reactive center. HLS2 is about 25-28% homologous to three human members of the plasma protease inhibitor family: antithrombin III, alpha 1-antitrypsin and alpha 1-antichymotrypsin. A comparison with published partial amino acid sequences shows that hLS2 is closely related to the thrombin inhibitor heparin cofactor II.     

“Unblocking” Serum Activity <Emphasis Type="Italic">in vitro</Emphasis> in the Polyoma System may Correlate with Antitumour Effects of Antiserum <Emphasis Type="Italic">in vivo</Emphasis>

In a variety of tumour systems, individuals carrying progressively growing neoplasms have lymphoid cells with a specific cytotoxic effect on cultured tumour cells from the same individual^1–4. Since the sera of tumour-bearing individuals have been shown to prevent tumour cell destruction by immune lymphocytes in vitro^2,5–8 and since this serum blocking activity appears early in primary and transplant tumour development^5,7, it has been suggested that the appearance of this serum blocking activity might be responsible for the progressive growth of tumours in individuals having cytotoxic lymphocytes. Counteraction of this blocking activity would thus be of primary importance in facilitating the function of an already existing or bolstered cell-mediated immunity. The serum blocking activity might be inhibited in various ways, by preventing the formation of blocking antibody or by interfering with its action (“unblocking”), as demonstrated in Moloney sarcoma regressor sera⁹. This type of serum also has a therapeutic effect on Moloney sarcomas in vivo^10,11, which has been tentatively attributed to its unblocking activity^8,9 or, possibly, to a complement-dependent cytotoxicity¹⁰. Tumour growth in the Moloney sarcoma system, however, might be due in part to continuous recruitment of neoplastic cells by virus-induced transformation and so the therapeutic effect could be due to a virus-neutralizing serum activity^9,10.     

Effects of oxygen level on metabolism and development of seedlings of Trapa natans and two ecologically related species

Seeds of the water plant Trapa natans L. (water chestnut) can germinate in strict anoxia. The seedlings show seminal roots growing upwards while shoot buds remain quiescent until O₂ becomes available. Trapa seedlings are highly tolerant to anoxia. The rate of ethanol fermentation was 21.2 μmol (g FW)⁻¹ h⁻¹, while production of lactate was negligible and lower than that of succinate. The seminal root of Trapa compares better to the rice coleoptile rather than to the rice root, both functionally and as to the metabolic response to anoxia. The anaerobic germination of Nuphar luteum L. and Scirpus mucronatus L. was also characterized by a limited developmental program.     

On the mechanism of ATP-induced shape changes in the human erythrocyte membranes: the role of ATP

In the preceding paper (Sheetz, M. and S.J. Singer. 1977. J Cell Biol. 73:638-646) it was shown that erythrocyte ghosts undergo pronounced shape changes in the presence of mg-ATP. The biochemical effects of the action of ATP are herein examined. The biochemical effects of the action of ATP are herein examined. Phosphorylation by ATP of spectrin component 2 of the erythrocyte membrane is known to occur. We have shown that it is only membrane protein that is significantly phosphorylated under the conditions where the shape changes are produced. The extent of this phosphorylation rises with increasing ATP concentration, reaching nearly 1 mol phosphoryle group per mole of component 2 at 8mM ATP. Most of this phosphorylation appears to occur at a single site on the protein molecule, according to cyanogen bromide peptide cleavage experiments. The degree of phosphorylation of component 2 is apparently also regulated by a membrane-bound protein phosphatase. This activity can be demonstrated in erythrocyte ghosts prepared from intact cells prelabeled with [(32)P]phosphate. In addition to the phosphorylation of component 2, some phosphorylation of lipids, mainly of phosphatidylinositol, is also known to occur. The ghost shape changes are, however, shown to be correlated with the degree of phosphorylation of component 2. In such experiment, the incorporation of exogenous phosphatases into ghosts reversed the shape changes produced by ATP, or by the membrane-intercalating drug chlorpromazine. The results obtained in this and the preceding paper are consistent with the proposal that the erythrocyte membrane possesses kinase and phosphates activities which produce phosphorylation and dephosphorylation of a specific site on spectrin component 2 molecules; the steady-state level of this phosphorylation regulates the structural state of the spectrin complex on the cytoplasmic surface of the membrane, which in turn exerts an important control on the shape of the cell.     

Human peptidylglycine alpha-amidating monooxygenase: cDNA, cloning and functional expression of a truncated form in COS cells

We have identified a cDNA encoding human peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) with a total length of 3748 bp by screening of a human thyroid carcinoma lambda gt11 library using two heterologous oligonucleotides to conserved regions which derived from frog skin and bovine pituitary PAM sequences. Furthermore we have identified a sequence which differs in a 321 bp deletion. COS cells transfected with a truncated form of this cDNA (lacking the putative carboxyl-terminal transmembrane domain) generated a functional PAM that showed a 20-fold increase of the activity compared to the control and was visualized by immunoblotting.     

Glycosaminoglycan-mediated leuserpin-2/thrombin interaction. Structure-function relationships

Two recently identified structural elements important for glycosaminoglycan-mediated activation of human leuserpin-2 (hLS2) were investigated in detail by functional analysis of variants secreted by transiently transfected COS cells. Highly specific requirements with respect to the nature of the involved amino acids as well as to their spatial arrangements were found to be crucial for efficient activation of hLS2 by dermatan sulfate. In contrast, binding and activation of hLS2 by heparin seem to be determined mainly by the positive charge density of the involved inhibitor segment. A dimeric repeat enriched in acidic amino acids turned out to exert a dual role with respect to structure and function of hLS2. First, in the absence of functional activators the negatively charged dimer interacts intramolecularly with the glycosaminoglycan-binding site. Second, the acidic dimer is instrumental in glycosaminoglycan-mediated activation of hLS2. The monomers constituting the acidic dimer are functionally not equivalent.     

Selective inhibition of prostaglandin biosynthesis by gold salts and phenylbutazone

Gold salts and phenylbutazone selectively inhibit the synthesis of PGF_2α and PGE₂ respectively. Lowered production of one prostaglandin species is accompanied by an increased production of the other. Selective inhibition by these drugs was observed in the presence of adrenaline, reduced glutathione and copper sulphate under conditions when most anti-inflammatory compounds inhibited PGE₂ and PGF_2α syntheses equally. It is postulated that selective inhibitors may have a different mode of action and beneficial effects may be related to the endogenous ratio of PGE to PGF required for normal function.     

Enhanced Specificity of Minimally Modified Anti-c-myc Oligonucleotides

Abstract

The sequence-specificity of antisense oligonucleotides (ODN) against c-myc mRNA was tested by Northern blot analysis. Rat smooth muscle cells were treated with antisense or control ODN against c-myc modified by the “minimal protection strategy”. At 0.3 μM concentration the ODN show a very specific reduction in c-myc mRNA levels. Use of the “minimal protection strategy” minimizes nonspecific effects as observed for all-phosphorothioate ODN containing four consecutive guanine residues.