SC-49992--a potent and specific inhibitor of platelet aggregation

Fibrinogen binding is required for platelet aggregation and subsequent thrombus formation. SC-49992 (SC), an RGDF mimetic, is a potent and specific inhibitor of the binding of fibrinogen to its receptor on activated platelets, glycoprotein IIb/IIIa (IC50 0.7 microM). SC was more potent (1-5 microM) than either RGDS, RGDF or the gamma chain dodecapeptide in blocking platelet aggregation to a variety of agonists in both dog and human platelet rich plasma. SC was more potent as an inhibitor of GP IIb/IIIa on platelets than it was against other integrin and non-integrin receptors, including the RGD-dependent vitronectin receptor and other non-RGD-dependent integrins such as CDII/CD18. SC had little effect on ristocetin induced agglutination. SC blocked ex vivo collagen induced aggregation in dogs and collagen induced thrombocytopenia in rats. These data suggest that elimination of the Arg-NH2 and the Arg-Gly amide bond of RGDF provided increased inhibitory potency and specificity. This structural modification may be of value in the development of other more potent RGDF mimetics for the inhibition of platelet aggregation.     

Cloning, sequencing, and heterologous expression of rat methionine synthase cDNA

Methionine synthase catalyzes cobalamin-dependent methyl transfer reaction from 5-methyltetrahydrofolate to homocysteine, forming methionine. Rat methonine synthase cDNA was cloned and analyzed by RT-PCR, 3'- and 5'-RACE techniques. The cDNA consists of a 0.3-kb upstream untranslated region, a 3.8-kb coding region, and a 0.4-kb downstream untranslated region. The open reading frame encoded a polypeptide of 1,253 amino acid residues with a calculated molecular weight of 139,162. This molecular weight was in good agreement with the observed one (143,000) of the purified rat liver enzyme. The deduced amino acid sequence was 53, 92, and 64% identical with those of the Escherichia coli, human, and presumptive Caenorhabditis elegans enzymes, respectively. All the fingerprint sequences, forming parts of the cobalamin- and S-adenosylmethionine-binding sites, were completely conserved in the rat methionine synthase. A high-level expression of catalytically active enzyme in insect cells was done by infection with a baculovirus containing the rat methionine synthase cDNA.     

Cloning, characterization, and tissue expression pattern of mouse tuftelin cDNA

Tuftelin is a protein that has been suggested to function during enamel crystal nucleation. Published sequences for bovine tuftelin cDNA and genomic clones proposed different reading frames that radically affected the derived amino acid sequence of the tuftelin carboxyl-terminus. We have isolated and characterized a full-length mouse cDNA clone and a partial porcine cDNA clone that include the region of the proposed frame-shift. The mouse tuftelin clone is 2572 nucleotides in length, exclusive of the poly(A+) tail. Translation from the 5'-most ATG yields a protein of 390 amino acids with an isotope-averaged molecular mass of 44.6 kDa and an isoelectric point of 5.9. Comparison of the bovine, mouse, and porcine cDNAs supports the revised bovine tuftelin amino acid sequence and suggests that the bovine tuftelin translation initiation codon be re-assigned to a more 5' ATG. Re-assigning the translation initiation codon lengthens the tuftelin protein by 52 amino acids, 51 of which are identical between bovine and mouse. At the carboxyl-terminus, the revised bovine and the mouse sequences match at 39 of the final 42 amino acid positions, compared with 2 identities with the originally published bovine reading frame. Northern blot analysis reveals that tuftelin is not ameloblast-specific but is expressed in multiple tissues, including kidney, lung, liver, and testis. Two tuftelin RNA messages, of 2.6 and 3.2 kb, were detected. DNA sequence characterization of an RT-PCR amplification product confirmed expression of tuftelin in kidney, and identified an alternatively spliced mouse tuftelin mRNA lacking exon 2.     

Cloning and expression of human deoxyguanosine kinase cDNA

A human cDNA sequence homologous to human deoxycytidine kinase (dCK; EC 2.7.1.74) was identified in the GenBank sequence data base. The longest open reading frame encoded a protein that was 48% identical to dCK at the amino acid level. The cDNA was expressed in Escherichia coli and shown to encode a protein with the same substrate specificity as described for the mitochondrial deoxyguanosine kinase (dGK; EC 2.7.1.113). The N terminus of the deduced amino acid sequence had properties characteristic for a mitochondrial translocation signal, and cleavage at a putative mitochondrial peptidase cleavage site would give a mature protein size of 28 kDa. Northern blot analysis determined the length of dGK mRNA to 1.3 kbp with no cross-hybridization to the 2.8-kbp dCK mRNA. dGK mRNA was detected in all tissues investigated with the highest expression levels in muscle, brain, liver, and lymphoid tissues. Alignment of the dGK and herpes simplex virus type 1 thymidine kinase amino acid sequences showed that five regions, including the substrate-binding pocket and the ATP-binding glycine loop, were also conserved in dGK. To our knowledge, this is the first report of a cloned mitochondrial nucleoside kinase and the first demonstration of a general sequence homology between two mammalian deoxyribonucleoside kinases. Our findings suggest that dCK and dGK are evolutionarily related, as well as related to the family of herpes virus thymidine kinases.     

Cloning, sequencing and expression of a cDNA encoding mammalian valyl-tRNA synthetase

The incus of the right ear from 4 growing mongrel dogs was surgically disarticulated and left in the middle ear space. The external auditory canal was then filled with teflon paste and sutured. After 6 weeks (D-6 group) and 13 weeks (D-13 group) the animals were sacrificed. The right experimental incus and the left control one were embedded in methyl methacrylate and sectioned in single 50-microns-thick sections according to the principal axis of the two processes. On the microradiographs of each section we evaluated the thickness of the body and of both processes and the percentage area of the primary channels of the secondary osteons and that of the appositional bone tissue. The thickness of the body and of the two processes was more pronounced in all the experimental incuses, in which 6% (in D-6) and 8% (in D-13) of the total area were occupied by new appositional woven bone. In the body of the D-13 group, 9% of the pre-existing bone was substituted by secondary osteons. The results indicate that the incus react to the variations of mechanical stimuli.     

cDNA cloning and expression analysis of the murine ribonuclease L inhibitor

This study was designed to test the hypothesis that the immune changes seen during in vivo whole body hyperthermia are mediated by elevations in the plasma concentrations of either catecholamines, growth hormone or beta-endorphins. Eight healthy volunteers were immersed in a hot water bath (WI; water temperature 39.5 degrees C) for 2 h during which their rectal temperature rose to 39.5 degrees C. In a single blind, randomized, cross-over study the stress hormone effects were blocked one at a time by administration of propranolol, somatostatin or naloxone; the results were compared to those obtained during saline infusion (control). Blood samples were collected before, at the end of 2 h of WI (body temperature 39.5 degrees C), and 2 h later. Hormone blockade did not abolish the hyperthermia-induced recruitment of natural killer (NK) cells to the blood, and no influence was observed on the percentages or concentrations of any other subpopulations of blood mononuclear cells, except that the number of cluster designation (CD)3+ cells slightly increased after hyperthermia only in the propranolol experiment. Furthermore, the NK cell activity, both unstimulated and interferon-alpha or interleukin-2 stimulated, did not differ from the control situation. It is of interest, however, that somatostatin partly abolished the hyperthermia induced increase in the neutrophil number. Based on these data and previous results showing that growth hormone infusion increases the concentration of neutrophils in the blood, it is suggested that growth hormone is at least partly responsible for hyperthermia induced neutrocytosis.     

Cloning and high-level expression of alpha-galactosidase cDNA from Penicillium purpurogenum

The cDNA coding for Penicillium purpurogenum alpha-galactosidase (alphaGal) was cloned and sequenced. The deduced amino acid sequence of the alpha-Gal cDNA showed that the mature enzyme consisted of 419 amino acid residues with a molecular mass of 46,334 Da. The derived amino acid sequence of the enzyme showed similarity to eukaryotic alphaGals from plants, animals, yeasts, and filamentous fungi. The highest similarity observed (57% identity) was to Trichoderma reesei AGLI. The cDNA was expressed in Saccharomyces cerevisiae under the control of the yeast GAL10 promoter. Almost all of the enzyme produced was secreted into the culture medium, and the expression level reached was approximately 0.2 g/liter. The recombinant enzyme purified to homogeneity was highly glycosylated, showed slightly higher specific activity, and exhibited properties almost identical to those of the native enzyme from P. purpurogenum in terms of the N-terminal amino acid sequence, thermoactivity, pH profile, and mode of action on galacto-oligosaccharides.     

Cloning and expression of the cDNA encoding an alternative oxidase gene from Aspergillus niger WU-2223L

A cDNA fragment encoding the mitochondrial alternative oxidase, the enzyme responsible for cyanide-insensitive and salicylhydroxamic acid (SHAM)-sensitive respiration, from the citric acid-producing fungus Aspergillus niger WU-2223L was cloned and expressed in Escherichia coli as a host strain. Synthetic primers were designed from the conserved nucleotide sequences of the alternative oxidase genes from higher plants and a yeast. The 210-bp DNA fragment was amplified by PCR with these primers using chromosomal DNA of WU-2223L as a template, and was employed to screen a cDNA library of A. niger. One full-length cDNA clone of 1.2 kb was obtained, and was sequenced to reveal that the clone contained an open reading frame (ORF-AOX1) encoding a polypeptide of 351 amino acids. The predicted amino-acid sequence exhibited 50%, 55%, and 52% homology to the alternative oxidases of Hansenula anomala, Neurospora crassa and Sauromatum guttatum, respectively. In the 5'-terminus region of the ORF-AOX1, a mitochondrial targeting motif was found. The whole open reading frame of ORF-AOX1 was ligated to plasmid pKK223-3 to construct the expression vector pKAOX1. The E. coli transformant harboring pKAOX1 showed cyanide-insensitive and SHAM-sensitive respiration, and expression was increased approximately two-fold by the addition of IPTG. These results indicated that the ORF-AOX1 encodes an alternative oxidase of A. niger.     

Imidazole derivatives, platelet aggregation inhibitors

Study of the platelet aggregation inhibition has shown the efficiency of compounds with imidazole ring alone or fused. All the compounds resulting of the molecular design starting from these structures has an in vitro activity. We have been able to discuss the correlation existing between activities, toxicities and structures with a particular emphasis to the lipophilicity.     

Cloning and sequencing an ovine interleukin-4-encoding cDNA

We have cloned a cDNA containing the complete coding sequence of ovine(ov) interleukin 4 (IL4) by the polymerase chain reaction using primers based on the 5' and 3' untranslated regions of the human IL4 gene. RNA was isolated from phorbol myristate acetate- and calcium ionphore A23187-stimulated mesenteric lymph node cells. The ovIL4 cDNA is 535 bp in length and contains an open reading frame of 408 nucleotides (nt) coding for a 15.1-kDa IL4 precursor of 135 amino acids (aa). Cleavage of the putative signal peptide of 22 aa yields the mature form of 13.2 kDa. Analysis of the mature aa sequence shows two potential N-linked glycosylation sites and six Cys residues. Ovine and bovine IL4 are shorter than human, mouse and rat IL4, because of a 51-nt deletion in the coding region. Comparison of the predicted aa sequence shows that ovIL4 shares 92, 57, 37 and 42% identity with the bovine, human, mouse and rat IL4s, respectively.     

Effect of a competitive inhibitor of platelet aggregation on experimentally induced laminitis in ponies

OBJECTIVES: To determining whether inhibition of platelet aggregation prevents development of carbohydrate overload-induced alimentary laminitis. ANIMALS: 22 healthy adult ponies. PROCEDURES: Acute laminitis was induced by oral administration of corn starch/wood flour to 16 ponies, 8 of which were treated with a synthetic analogue of the platelet fibrinogen receptor antagonist peptide (RPR) RGDS (arginine-glycine-aspartic acid-serine) 110885; 6 ponies served as negative controls. Blood was collected before and at 4, 8, 12, 24, 28, and 32 hours after administration of carbohydrate overload, and PCV, total plasma protein concentration, platelet count, activated clotting time, whole blood recalcification time, spontaneous platelet aggregation, ex vivo platelet aggregation responses, in vivo platelet activation, and platelet-neutrophil aggregates were evaluated. RESULTS: Of 16 ponies given carbohydrate, 6 of 8 untreated ponies developed laminitis and 0 of 8 ponies treated with RPR 110885 developed laminitis. The RPR 110885 treatment attenuated the increase in platelet-neutrophil aggregates observed in untreated ponies. CONCLUSIONS: Platelets are involved in the pathogenesis of equine alimentary laminitis. CLINICAL RELEVANCE: Platelet aggregation inhibitors may be useful for prevention or treatment of laminitis, or both.     

Cloning, expression and characterisation of the cDNA encoding human hepatic squalene synthase, and its relationship to phytoene synthase

The reaction catalysed by squalene synthase (SQS) shows many similarities to that performed by another polyisoprene synthase, phytoene synthase (PhS). By identifying sequences conserved between yeast SQS (ySQS) and PhS, we have cloned a 2-kb cDNA (hSQS) encoding human SQS, a protein of 417 amino acids with a predicted M(r) of 48,041, which has only limited homology to ySQS. When expressed in E. coli, the hSQS cDNA directed the production of active enzyme. Two hSQS mRNA species of 2.0 and 1.55 kb have been identified which differ in their 3' untranslated sequences. The two mRNAs are present in roughly equal amounts in heart, placenta, lung, liver, kidney and pancreas, but the 2-kb mRNA predominates in brain and skeletal muscle. In HepG2 cells, both mRNAs are induced 2-4-fold by the 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor, lovastatin. In contrast, Northern blot analysis of rat tissues reveals only a 2.0-kb mRNA, which is considerably up-regulated in vivo by lovastatin.