Nucleotide sequence of a full-length cDNA encoding a common precursor of platelet aggregation inhibitor and hemorrhagic protein from Calloselasma rhodostoma venom.

The nucleotide sequence of a full-length cDNA encoding the common precursor of a platelet aggregation inhibitor, rhodostomin and a hemorrhagic protein from Calloselasma rhodostoma snake venom is presented. The 1.98-kb cDNA contains an open reading frame encoding 478 amino acid residues. The complete structure of the precursor protein encoded by the cDNA is elucidated.

Involvement of both opiate and catecholaminergic receptors in the behavioural excitation provoked by thyrotropin-releasing hormone: comparisons with amphetamine.

Following direct administration of thyrotropin-releasing hormone (TRH), but not thyroid-stimulating hormone (TSH) or vehicle solution, into the lateral cerebral ventricle in rats, three main categories of behaviour were provoked: activity of the normal type--stimulation of forward locomotion, head and body rearing (as shown by an enhancement in gross movements); stereotyped activity--increased grooming and head swaying (as shown by an enhancement in fine movements); abnormal behaviour--tail elevation and piloerection (as observed grossly). The behavioural excitation caused by TRH was antagonized by pretreatment of the rats with either a narcotic receptor antagonist, naloxone, an alpha-adrenergic receptor antagonists, yohimbine, or a dopaminergic receptor antagonist, haloperidol, but not with a beta-adrenergic receptor antagonist, propranolol. Intraventricular administration of amphetamine to rats caused stimulation of forward locomotion, head and body rearing, increased grooming and sniffing. Unlike TRH, amphetamine did not produce wet-dog shakes, tail elevation and piloerection. Furthermore, the amphetamine-induced excitation was antagonized by pretreatment with a dopaminergic receptor antagonist, haloperidol, but not with either naloxone, yohimbine or propranolol. The data indicate that both opiate and catecholaminergic receptors are involved in the TRH-induced behavioural excitation, whereas dopaminergic receptors are involved in amphetamine-induced excitement in the rat.

Use of signal-distinguishable probes in differential or sequential autoradiography in hybridization analysis.

To avoid the time-consuming reprobing process in hybridization analysis, signal-distinguishable probes (32P, 35S or antigenic hapten-labeled DNA) can be added to the same hybridization mixture. After hybridization, an unambiguous result can be obtained by differential or sequential autoradiography.

A simple and rapid method for purification of oligodeoxyribonucleoside methylphosphonates.

An alternative dimethoxytrityl-on (dmt-on) method is described to purify hydrophobic oligodeoxyribonucleoside methyl-phosphonates (OM) with a phosphodiester linkage at the 5' end, instead of the conventional dmt-off method using a DEAE ion-exchange column. This method is modified from the reverse-phase method for purification of normal oligonucleotides.

Effect of liposomes on suspension stability.

The effect of positively charged liposomes on the stability of negatively charged indomethacin particle suspensions was investigated by adsorption, microelectrophoresis, sedimentation volume, and redispersibility. It was found that flocculation occurred in the neighborhood of the zero point of charge of the liposome:indomethacin suspensions. This resulted from the operation of van der Waals attraction under the condition of nil electrostatic repulsion. Correlation among the results of adsorption, microelectrophoresis, sedimentation volume, and redispersibility was good. The mixing effect of the suspension was examined by microelectrophoresis and confirmed by sedimentation volume and redispersibility. Results indicate that there was no difference between the method of dilution of the liposome:indomethacin concentrated suspension after mixing and the method of dilution of liposomes prior to mixing with the indomethacin suspension. This was due to interaction depending on the charge density of the particles.

The inhibitory action of zinc sulphate on the contractile activity of guinea-pig ileum.

The present study examines the inhibitory action of zinc sulphate (ZnSO4) on the contractile response of various agonists on guinea-pig isolated ileum. Different doses of agonists were selected to produce similar contractile activity, in order to compare the degree of inhibition produced by ZnSO4. Preincubation of ileum with ZnSO4 1 x 10(-3) or 3 x 10(-3) M for 10 min dose-dependently and significantly prevented the contraction induced by acetylcholine (1.7 x 10(-8) M), 5-HT (2.4 x 10(-6) M), histamine (5.4 x 10(-7) M) and nicotine (1.7 x 10(-6) M) but not by prostaglandin E2 (PGE2, 8.5 x 10(-9) M). The same doses of ZnSO4 reduced the twitch contraction produced by electrical field stimulation. These findings indicate that the contractile activity of PGE2 is mediated by a mechanism different from that of other agonists and of electrical field stimulation. It is likely that the contractile activity of PGE2 is acting through the receptors on the ileal muscle which are not blocked by ZnSO4 pretreatment.

Effect of stabilization temperature on the degradation of adriamycin in albumin microspheres.

The effect of stabilization temperature on the degradation of adriamycin HCl during the preparation of albumin microspheres was investigated. The degradation of adriamycin HCl by heating at various temperature with different time interval in dried adriamycin HCl powder, adriamycin HCl aqueous solution, wetted Adriablastina (adriamycin HCl with lactose) powder and Adriablastina aqueous solution was also studied. In the presence of water the degradation of adriamycin HCl was found; whereas, in the absence of water no degradation occurred. The degradation of adriamycin HCl in solution and wetted powder showed a zero order reaction. An increase in temperature increased degradation rate. The rate constant for adriamycin HCl degradation in Adriablastina solution obtained was in good agreement with that in adriamycin HCl solution. It was suggested that the presence of lactose had no interference in the degradation of adriamycin HCl. The zero-order reaction of degradation was attributed to the drug behaved like a suspension. The degradation of adriamycin HCl at various stabilization temperature during the preparation of microspheres had the same tendency as those of the adriamycin HCl solution and the wetted adriamycin HCl powder that were heated by the DSC instrument with the condition similar to the preparation of microspheres.

Utilization of patterns of biotinyl-polypeptides as a promising analytical target for bacterial identification.

The family of biotin enzymes, which are involved in carboxylation, transcarboxylation, and decarboxylation reactions in cells, is essential and conserved for bacteria. The prosthetic group biotin is stably linked to the biotinyl-polypeptide by an amide bond. Since avidin interacts with biotin in an irreversible manner, the patterns of biotinyl-polypeptides for bacterial cells could be revealed by probing them with an avidin-enzyme complex in Western blots of total cellular proteins. In this way, the family of commonly existing gene products of bacteria could be monitored and compared without the use of specific DNA probes, oligonucleotide primers, or antibodies. In this study, this approach was tested and it was found that different species of bacteria showed different patterns of biotinyl-polypeptides. Our results indicate that patterns of biotinyl-polypeptides can be a promising analytical target for bacterial identification.

Rapid verification of lambda-cDNA clones using mixed-base oligonucleotide as screening probe and sequencing primer.

A rapid procedure has been developed for the isolation and verification of cDNA clones isolated from a cDNA library based on lambda vectors. Using information of the partial amino acid sequence of a protein, synthetic mixed-base oligonucleotides are first employed as a screening probe using the plaque hybridization procedure. The cDNA inserts of the clones obtained are then directly amplified by polymerase chain reaction (PCR) using primers flanking the cloning site of the vector. Besides being used for cloning into a plasmid vector, the amplified DNA's are also subjected to nucleotide sequence analysis using the same mixed-base oligonucleotides as sequencing primers. This approach allows sequencing through the region of the known amino acid sequence for direct verification of the authenticity of the clones obtained. This procedure has successfully been used for cloning and partial characterization of the gene coding for a platelet aggregation inhibitor.

A common precursor for a putative hemorrhagic protein and rhodostomin, a platelet aggregation inhibitor of the venom of Calloselasma rhodostoma: molecular cloning and sequence analysis.

Rhodostomin is a platelet aggregation inhibitor secreted by the venom gland of Calloselasma rhodostoma. We report here the isolation of a 1.67-kilobase (kb) lambda gt11 cDNA clone using degenerate oligonucleotide probe based on a partial amino acid sequence of rhodostomin. The amino acid sequence deduced from an open reading frame of the cDNA indicates that (i) the 68-amino acid sequence of rhodostomin is located at the carboxyl terminus of the precursor polypeptide and (ii) a peroxisomal targeting sequence (ser.his.ala.) exists between the stop codon and the rhodostomin sequence of the precursor. Since the amino-terminal segment of the deduced sequence shows a high degree of identity with hemorrhagic proteins, which are zinc-metalloproteinases, found in the venom of some crotalid and viperid snakes, our results also predict the existence of at least one such hemorrhagic protein in the venom of Calloselasma rhodostoma. The derivation of a platelet aggregation inhibitor and a hemorrhagic protein from the same precursor protein is consistent with the fact that these proteins may be synergistic in function.

Molecular cloning and sequence analysis of the cDNA for ancrod, a thrombin-like enzyme from the venom of Calloselasma rhodostoma.

The 1.54 kb cDNA for ancrod, a thrombin-like enzyme, was cloned from a lambda ZAP cDNA library derived from the venom glands of Calloselasma (Agkistrodon) rhodostoma. The cDNA sequence reveals that ancrod is synthesized as a pre-zymogen of 258 amino acids, including a putative secretory peptide of 18 amino acids and a proposed zymogen peptide of 6 amino-acid residues. The amino-acid sequence of the predicted active form of the enzyme exhibits a high degree of sequence similarity to those of mammalian serine proteases (trypsin and pancreatic kallikrein) and other thrombin-like enzymes (batroxobin and flavoxobin). Key amino-acid residues (His43, Asp88, Ser182 and Asp176) that are thought to be involved in the substrate cleavage and in the substrate-binding reaction are conserved. Ancrod contains 13 cysteine residues. Based on alignment with the amino-acid sequences of trypsin and batroxobin, six disulphide bridges can be predicted to be present in the ancrod protein. The existence of a free cysteine, which changes the common sequence surrounding the Ser182 active site from Gly-Asp-Ser-Gly-Gly-Pro to Cys-Asp-Ser-Gly-Gly-Pro, is unusual for a serine protease.