Purification and cDNA cloning of a four-domain Kazal proteinase inhibitor from crayfish blood cells

A cDNA with an open reading frame of 684 base pairs was isolated from a library from blood cells of the crayfish Pacifastacus leniusculus. It codes for a signal sequence and a mature protein of 209 amino acids with a predicted molecular mass of 22.7 kDa. The amino acid sequence consists of four repeated stretches (45-73% identical to each other), indicating that the protein has four domains. The domains have significant sequence similarity to serine proteinase inhibitors of the Kazal family. The three first domains have a leucine residue in the putative reactive site, suggesting that the protein is a chymotrypsin inhibitor. A monomeric 23-kDa proteinase inhibitor, which by amino terminal sequencing of the mature protein was confirmed to be the cloned Kazal inhibitor, was purified from crayfish blood cells. It inhibited chymotrypsin or subtilisin, but not trypsin, elastase or thrombin. The inhibitor seemed to form a 1:1 complex with chymotrypsin or subtilisin. This protein seems to be the first described Kazal inhibitor from blood cells of any animal and the first one with four domains.     

Isolation of protein factors from opossum (Didelphis albiventris) serum which protect against Bothrops jararaca venom

The fractionation of Didelphis albiventris serum by DEAE-Sephadex A50 yields a fraction (DA2) which protects the opossum against Bothrops venom. One polypeptide (DA2-II) responsible for this protection was isolated from fraction DA2 by ion exchange chromatography and biochemically characterized. DA2-II is a 43,000 mol. wt glycoprotein with the following N-terminal sequence: LKAMDTTPPLKIKKEPVK. Pairwise comparison of the amino acid sequence with four anti-hemorrhagic factors isolated from other opossum species indicated that DA2-II possesses high similarity (60-80%) with these proteins.     

Purification, characterization, and cDNA cloning of profilin from Phaseolus vulgaris

Profilin from common bean (Phaseolus vulgaris L.) was purified to homogeneity by poly-L-Pro affinity chromatography and gel filtration. The hypocotyl and symbiotic root nodule protein was detected as a single isoform with a 14.4-kD molecular mass and an isoelectric point of 5.3. Partial amino acid and DNA sequencing of a full-length cDNA clone confirmed its identity as profilin. An antibody generated against the purified protein binds to a protein with the same molecular mass in leaves and nodules. Immunolocalization of the protein showed a diffuse distribution in the cytoplasm of hypocotyls and nodules but enhanced staining at the vascular bundles. The strong identity of the sequence among the profilins of birch, maize, and bean suggests that it may play an important role in the signal transduction mechanism of plant cells and plant-bacterial symbioses.     

Primary structure of two-chain botrocetin, a von Willebrand factor modulator purified from the venom of Bothrops jararaca

The complete amino acid sequence and location of the disulfide bonds of two-chain botrocetin, which promotes platelet agglutination in the presence of von Willebrand factor, from venom of the snake Bothrops jararaca are presented. Sequences of the alpha and beta subunits were determined by analysis of peptides generated by digestion of the S-pyridylethylated protein with Achromobacter protease I or alpha-chymotrypsin and by chemical cleavage with cyanogen bromide or 2-(2'-nitrophenylsulfenyl)-3-methyl-3-bromoindolenine. Two-chain botrocetin is a heterodimer composed of the alpha subunit (consisting of 133 amino acid residues) and the beta subunit (consisting of 125 amino acid residues) held together by a disulfide bond. Seven disulfide bonds link half-cystine residues 2 to 13, 30 to 128, and 103 to 120 of the alpha subunit; 2 to 13, 30 to 121, and 98 to 113 of the beta subunit; and 80 of the alpha subunit to 75 of the beta subunit. In terms of amino acid sequence and disulfide bond location, two-chain botrocetin is homologous to echinoidin (a sea urchin lectin) and other C-type (Ca(2+)-dependent) lectins.     

Purification and characterization of a 39,000-Da serine proteinase from the hemolymph of a solitary ascidian, Halocynthia roretzi

The melanocortin-4 receptor (MC4-R) is a G protein-coupled, seven-transmembrane receptor expressed in the brain. Inactivation of this receptor by gene targeting results in mice that develop a maturity onset obesity syndrome associated with hyperphagia, hyperinsulinemia, and hyperglycemia. This syndrome recapitulates several of the characteristic features of the agouti obesity syndrome, which results from ectopic expression of agouti protein, a pigmentation factor normally expressed in the skin. Our data identify a novel signaling pathway in the mouse for body weight regulation and support a model in which the primary mechanism by which agouti induces obesity is chronic antagonism of the MC4-R.     

Cloning, cDNA sequence analysis and patch clamp studies of a toxin from the venom of the armed spider (Phoneutria nigriventer)

The cDNAs (Tx3-2 and Pn3A) encoding precursor of toxin Tx3-2 and an isoform called Pn3A have been isolated from a library constructed from stimulated venom glands of the spider Phoneutria nigriventer. The cDNA of Tx3-2 reveals the presence of a signal peptide of 21 amino acids and of an intervening propeptide (with 16 amino acids) preceding the toxin sequence, which was followed by additional amino acid residues at the C-terminus (C-terminal peptide), implying post-translational modifications of the synthesised peptide. The deduced amino acid sequence for the mature toxin confirms the previous sequence published. In addition, by using the whole-cell patch clamp technique, we have determined that purified Tx3-2 decreases L-type currents present in GH3 cells. Finally, the presence of the cDNA Pn3A, with high sequence identity with Tx3-2, reveals the existence of a putative new toxin showing, at the cDNA level, 85.4% identity in its whole segment.     

cDNA cloning and sequence analysis of the bovine adrenocorticotropic hormone (ACTH) receptor

We isolated five independent cDNAs of nearly 3000 bp for the bovine ACTH receptor by screening adrenal cortex cDNA libraries with a PCR cloned cDNA fragment. The deduced receptor sequence includes 297 residues (M(r) = 33,258) with 81% identity with the human ACTH receptor, and shows seven hydrophobic transmembrane domains. The calculated M(r) of the receptor is smaller than the 40-45 kDa observed in crosslinking studies with labeled ACTH. Since the bovine and human receptors have two glycosylation motifs in the N-terminus, the difference may result from glycosylation of the receptor. Analysis of the sequences of both bovine and human receptors revealed a single protein kinase. A phosphorylation motif located in the third intracellular loop (Ser-209) juxtaposed to a protein kinase C phosphorylation motif (Thr-204). Thus, the involvement of protein kinase A and C pathways in ACTH action may be mediated in part by phosphorylation of the ACTH receptor at these motifs. The 3'-untranslated region of the bovine cDNA is > 2000 bp and includes two inverse repeats giving an extensive and strong secondary structure to the ACTH receptor RNA.     

Activation of single-chain urokinase-type plasminogen activator by a hemorrhagic metalloproteinase, jararafibrase I, in Bothrops jararaca venom

Urokinase-type plasminogen activator (uPA) activates plasminogen to plasmin, which is involved in the degradation of the vascular basement membrane and extracellular matrix. The present study was undertaken to examine the effects of several hemorrhagic metalloproteinases, jararafibrase (JF) I, II, III and IV, purified from Bothrops jararaca venom, on the single-chain zymogen form of uPA (scuPA). Activation of scuPA by JF I IV was estimated using a synthetic substrate for uPA (S-2444). Only JF I activated the scuPA in a time- and dose-dependent manner. SDS-PAGE analysis revealed that, after incubation with JF I, the intensity of the 55 kDa band of scuPA decreased concomitantly with increases in the intensity of the major two bands at 32 and 22 kDa under reduced and non-reduced conditions. The 32 kDa band demonstrated fibrinolytic activity in fibrin-zymographic studies. Amino-acid-sequence analysis revealed that JF I cleaved the position of 143Glu-144Leu in scuPA, indicating that JF I formed low molecular weight scuPA. From these results, it seems possible that activation of scuPA by JF I could be responsible in part for the local hemorrhage and tissue damage that are frequently observed in human victims of B. jararaca envenoming.     

Purification from Bothrops lanceolatus (fer de lance) venom of a fibrino(geno)lytic enzyme with esterolytic activity

Bothrops lanceolatus venom has high caseinolytic, phospholipasic, esterolytic and hemorrhagic activities. In spite of having no coagulant effect on plasma, this venom contains a thrombin-like enzyme. Using gel filtration and ion-exchange chromatographies, we have purified an esterolytic fraction (F-II-1a) from this venom with a protein yield of 4% and a 58% recovery in enzyme activity. SDS-PAGE in the presence of beta-mercaptoethanol showed that the enzyme is a single chain polypeptide with a MW=38,100. Immunodiffusion and immunoelectrophoresis of fraction F-II-1a against serum from horses immunized with B. lanceolatus venom and against rabbit antiserum prepared using fraction F-II-1a both showed a single immunoprecipitin line. The Km and Vmax values for TAME hydrolysis were 0.85 mM and 38.6 micromol/min/mg, respectively. The esterolytic activity was completely inhibited by PMSF (10 mM) but not by EDTA (20 mM). Fraction F-II-1a hydrolyzed the alpha and beta chains of fibrinogen. Degradation of the alpha chain occurred within 10 min while that of the beta-chain was slower. The enzyme had no effect on the gamma-chain even after 4 h of hydrolysis.     

Characterization of a fibrinogen-clotting enzyme from Trimeresurus stejnegeri venom, and comparative study with other venom proteases

It has been well documented that drug-associated cues are important for the development and expression of drug tolerance. The Pavlovian conditioning analysis of tolerance emphasizes the importance of a drug-associated cues to tolerance by equating a drug administration with a learning trial. According to this analysis, tolerance should be subject to external inhibition, the disruption of a conditional response by a novel stimulus. We previously reported that tolerance to the ataxic effect of ethanol was attenuated by a novel strobe/noise presentation (31). In this article we report evidence of a compensatory CR in rats tolerant to the ataxic effect of ethanol as tested on the tilting plane. Both the compensatory CR and tolerance were disrupted by the presentation of a novel strobe/noise stimulus providing converging evidence that the attenuation of tolerance by a novel stimulus results from external inhibition of Pavlovian conditioning. The disruption of ethanol tolerance and the conditional response mediating tolerance was also apparent when the novel omission of the strobe/noise stimulus was used as the external inhibitor in rats made tolerant to ethanol with the stimulus on. Finally, we have shown that the disruptive effect of a novel stimulus on ethanol tolerance is decreased when there is a 10-day delay between the final tolerance development session and testing, demonstrating that the interval between training and testing is important when assessing associative tolerance.     

Purification and characterization of tilapia (Oreochromis mossambicus) deoxyribonuclease I--primary structure and cDNA sequence

DNase I of tilapia (Oreochromis mossambicus) was purified to homogeneity. Tilapia DNase I is most active at pH 8.5 with Mg2+ as activator. The Ca2+/Mg2+ pair has a synergistic effect on activation. The enzyme is readily inactivated by heating above 55 degrees C, but is not inactivated by trypsin or 2-mercaptoethanol under alkaline conditions, with or without CaCl2. Its isoelectric point is 6.0. The 258-amino-acid sequence of tilapia DNase I was derived from overlapping sequences of tryptic, chymotryptic and CNBr peptides. The purified enzyme has two variants differing by a single Lys-->Arg mutation at position 125. The polypeptide chain has one disulfide bridge and one carbohydrate side chain. By mass spectrometry, the purified enzyme shows many molecular mass forms differing by Lys/Arg substitution and sugar-chain length. The major form has a molecular mass of 30,914 Da. A 1061-bp nucleotide sequence for the cDNA of tilapia DNase I, obtained by gene cloning and DNA sequencing, contains an ORF coding for a putative 26-residue transmembrane peptide and the mature DNase I polypeptide.     

cDNA cloning and expression of a novel serine protease, TLSP

A cDNA for a putative novel serine protease, TLSP, was cloned from human hippocampus cDNA with polymerase chain reaction based strategies. The putative amino acid sequence of TLSP is similar to the trypsin-type serine proteases. TLSP mRNA is expressed in keratinocytes. Overexpressed TLSP protein in neuro2a cells was detected in culture medium.     

Bothroalternin, a thrombin inhibitor from the venom of Bothrops alternatus

Bothroalternin (MW 27 kDa), a new member of the family of C-type lectins is a thrombin inhibitor which was purified from pooled B. alternatus venom by affinity chromatography on PPACK-thrombin-Sepharose, followed by size exclusion and reverse-phase on HPLC columns. Material retained on the affinity column contained proteins with apparent molecular weights ranging from 20 to 60 kDa on SDS-PAGE and inhibited aggregation of rabbit platelets induced by alpha-thrombin (IC50 = 28 microg/ml). A single band of approximately 27 kDa was recognized in Western-blot assays using polyclonal antibodies raised against bothrojaracin, a thrombin inhibitor purified from B. jararaca venom (Zingali et al., 1993). The immunological similarity of this fraction to bothrojaracin was confirmed by ELISA and competitive ELISA. Further purification by size exclusion and reverse-phase on HPLC, produced a single homogenous peak called bothroalternin. This protein was highly homologous to bothrojaracin (95% in its N-terminal sequence-for residues 1 to 25) but displaying lower inhibitory effect on thrombin induced platelet aggregation (Ic50 = 0.19 microg/ml) compared to bothrojaracin (IC50 = 0.06). Altogether, bothroalternin is a new thrombin inhibitor isolated from Bothrops alternatus venom and has been characterized as a bothrojaracin-like protein.     

Purification, characterization and molecular cloning of TGP1, a novel G-DNA binding protein from Tetrahymena thermophila

G-DNA, a polymorphic family of four-stranded DNA structures, has been proposed to play roles in a variety of biological processes including telomere function, meiotic recombination and gene regulation. Here we report the purification and cloning of TGP1, a G-DNA specific binding protein from Tetrahymena thermophila. TGP1 was purified by three-column chromatographies, including a G-DNA affinity column. Two major proteins (approximately 80 and approximately 40 kDa) were present in the most highly purified column fraction. Renaturation experiments showed that the approximately 80 kDa protein contains TGP1 activity. Biochemical characterization showed that TGP1 is a G-DNA specific binding protein with a preference for parallel G-DNAs. The TGP1/DNA complex has a dissociation constant (Kd) of approximately 2.2 x 10(-8) M and TGP1 can form supershift in gel mobility shift assays. The cDNA coding TGP1 was cloned and sequenced based upon an internal peptide sequence obtained from the approximately 80 kDa protein. Sequence analyses showed that TGP1 is a basic protein with a pI of 10.58, and contains two extensively hydrophilic and basic domains. Homology searches revealed that TGP1 is a novel protein sharing weak similarities with a number of proteins.     

Purification and characterization of cysteine proteinase from a baculovirus gene

To analyze the degradation of product proteins at the late stage of virus infection in the baculovirus expression system, a cysteine proteinase was purified from hemolymph of Bombyx mori infected with wild-type B. mori nuclear polyhedorosis virus (BmNPV). The purified cysteine proteinase preparation had two protein bands (major 35-kDa active protein and 28-kDa inactive protein) on SDS-PAGE. Based on the N-terminal amino acid sequences of them, it was found that both proteins originated in the cysteine proteinase gene of BmNPV. The purified cysteine proteinase had an optimum pH at 4.0, and also had activities at neutral pHs. When recombinant luciferase was used as a natural substrate, it was degraded rapidly by the cysteine proteinase at the physiological pH of hemolymph. These results suggest that the cysteine proteinase from a BmNPV gene participates in the degradation of foreign protein expressed by the baculovirus system.     

Amino acid sequence of piratoxin-I, a myotoxin from Bothrops pirajai snake venom, and its biological activity after alkylation with p-bromophenacyl bromide

OBJECTIVE: To investigate possible differences in cytomegalovirus (CMV) strain distribution between the eye and blood in AIDS patients with CMV retinitis. METHODS: CMV DNA sequences from aqueous humour and peripheral blood leukocytes (PBL), obtained from 13 AIDS patients with CMV retinitis, were compared. DNA was isolated and the CMV IE-1 sequence (part of the immediate early-1 gene) and the a-sequence (located in the a-region) were amplified by polymerase chain reaction (PCR). The PCR products of the a-sequence were analysed by Southern blotting for amplified fragment-length polymorphisms. The level of divergence between the a-sequences of aqueous humour- and PBL-derived CMV was studied in two patients by cloning these sequences followed by sequence analysis. RESULTS: CMV DNA could be detected in all aqueous humour samples and in 10 out of 13 paired blood samples. In the 10 patients, with CMV DNA detectable in both aqueous humour and PBL, seven cases showed differences between the amplified products of both compartments. Sequence analysis in two patients revealed that the aqueous humour and PBL of the same patient can harbour both identical, similar and highly divergent CMV a-sequences. CONCLUSION: These results indicate that despite the haematogenous spread of CMV, the eye, being a relatively shielded organ, may contain CMV strains different from those found in the blood.     

Isolation and characterization of a myotoxic phospholipase A2 from the venom of the arboreal snake Bothriechis (Bothrops) schlegelii from Costa Rica

PURPOSE: To evaluate the cytotoxicity of immunotoxin 4197X-ricin A (4197X-RA) and its ability to inhibit protein synthesis and human lens epithelial cell (LEC) proliferation on the inner surface of the lens capsule. SETTING: Houston Biotechnology, Inc., The Woodlands, Texas. METHODS: A cell culture system was established using human LECs as a model for the proliferation of remnant LECs that occurs during posterior capsule opacification (PCO) after extracapsular cataract extraction. The LEC culture system was also used in vitro for testing compounds that might inhibit this process in vivo. Human LECs were cultured on the surface of the original lens capsule fixed to collagen. Variability was reduced by dissecting each lens capsule into equivalent halves and exposing the segments to immunotoxin 4197X-RA. RESULTS: Protein synthesis and LEC proliferation were almost completely inhibited at relatively low 4197X-RA concentrations after short exposure. The inhibitory effects persisted up to 3 weeks after withdrawal of the immunotoxin and after several media exchanges. CONCLUSION: Immunotoxin 4197X-RA may help prevent PCO after primary cataract surgery.     

Isolation: analysis and properties of three bradykinin-potentiating peptides (BPP-II, BPP-III, and BPP-V) from Bothrops neuwiedi venom

Several methods of external and internal fixation are used in urgent situations to lessen intrapelvic bleeding associated with unstable pelvic fractures. Pelvic stabilization limits pelvic expansion and thereby restricts the space for potential blood loss. This study compared several fixation methods using cadaveric pelves to determine which method best prevents pelvic expansion. Three methods of internal fixation and three methods of external fixation were compared. Anteroposterior fixation provided the greatest control against pelvic expansion; however, it is clinically impractical for emergency use. Therefore, external fixation provided the most reliable control of pelvic expansion in the emergency setting.