Structural characterization of the rat carboxypeptidase A1 and B genes. Comparative analysis of the rat carboxypeptidase gene family

Nucleotide sequencing of a rat carboxypeptidase B (CPB) cDNA and direct sequencing of the CPB mRNA via primer extension on pancreatic polyadenylated RNA has yielded the complete amino acid sequence of rat CPB. The rat enzyme is synthesized as a precursor species containing a large amino-terminal fragment (108 amino acids) that contributes a putative signal sequence and an activation peptide. The mature form of rat CPB is homologous to bovine CPB (77% identity); the amino acids in bovine CPB which have been previously implicated in catalysis or ligand binding are invariant in the rat orthologue. The rat CPB cDNA was used as a probe for the isolation of the rat CPB gene. Detailed characterization of three overlapping rat genomic clones demonstrated that the coding region for the rat CPB precursor is sequestered in 11 exons which are dispersed throughout 34 kilobase pairs of genomic DNA. The nucleotide sequence of a large part of the gene has been determined including that of the exons, the exon/intron boundaries, and the 5' flanking region. We also report the partial nucleotide sequence of the rat CPA1 gene. Comparative analysis of the structural organization of the rat CPB, rat CPA1, and rat CPA2 genes (Gardell, S. J., Craik, C. S., Clauser, E., Goldsmith, E. J., Stewart, C.-B., Graf, M., and Rutter, W. J. (1988) J. Biol. Chem. 263, 17828-17836) reveals that, with one exception, the number, position, and sequence composition of the exons in these three carboxypeptidase genes are conserved in spite of considerable divergence with respect to the lengths of their corresponding intervening sequences. Conserved sequences in the 5' flanking regions of the rat CPA1, CPA2, CPB, and other pancreas-specific genes have been identified.     

Transmission of Megatrypanum Trypanosomes to Cervus dama by Tabanidae1

Four fallow deer, Cervus dama, became infected with Trypanosoma (Megatrypanum) sp. by oral application of triturated guts from tabanids collected in an area with deer but without any cattle; four control calves remained negative. Upon challenge with triturated guts from tabanids from an area with pastured cattle, the four calves became infected with Trypanosoma (M.) theileri. The prepatent period in deer was five days or less. Haematopota spp. and Tabanus spp. were identified as vectors of the deer trypanosomes. It is concluded that the trypanosomes of C. dama belong to a Megatrypanum species that is not identical with T. theileri.     

Physical mapping of three fruit ripening genes：Endopolygalacturonase,ACC oxidase and ACC synthase from apple（Malus x domestica） in an apple rootstock A106（Malus sieboldii

The apple rootstock,A106(Malus sieboldii),had 17 bivalents in pollen mother cells at meiotic metaphase 1,and 17 chromosomes in a haploid pollen cell.Karyotypes were prepared from root-tip cells with 2n=34 chromosomes,Seven out of 82 karyotypes(8.5%) showed one pari of satellites at the end of the short arm of chromosome 3.C-bands were shown on 6 pairs of chromosomes 2,4,6,8,14,and 16 near the telomeric regions of short arms.Probes for three ripening-related genes from Malus x domestica:endopolygalacturonase(EPG,0.6kb),ACC oxidase(1.2kb),and ACC synthase(2kb)were hybridized in situ to metaphase chromosomes of A106.Hybridization sites for the EPG gene were observed on the long arm of chromosome 14 in 15 out of 16 replicate spreads and proximal to the centromere of chromosomes 6 and 11.For the ACC oxidase gene,hylridization sites were observed in the telomeric region of the short arm of chromosomes 5 and 11 in 87% and 81% of 16 spreads respectively,proxiaml to the centromere of chromosome 1 in 81% of the spreads,and on the long arm of chromosome 13 in 50% of the spreads. Physical mapping of three fruit ripening genes in an apple rootstock A106.Twenty five spreads were studied for the ACC synthase gene and hybridization sites were observed in the telomeric region of the short arm of chromosome 12 in 96% of the spreads.chromosomes 9 and 10 in 76% of the spreads,and chromosome 17 in 56% of the spreads.     

Latent proteinase activity of gamma-glutamyl transpeptidase light subunit.

gamma-Glutamyl transpeptidase, which is composed of two unequal subunits, exhibits proteinase activity when treated with agents such as urea and sodium dodecyl sulfate. The heavy subunit is preferentially and rapidly degraded. The enzyme also degraded bovine serum albumin in the presence of urea; however, several other proteins and model proteinase substrates were not cleaved. Treatment of the enzyme with 6-diazo-5-oxo-L-norleucine, a gamma-glutamyl analog, results in parallel loss of transpeptidase and proteinase activities indicating that the site at which gamma-glutamylation of the enzyme occurs (presumably a hydroxyl group on the light subunit) is also involved in proteinase activity. The purified light subunit, but not the heavy subunit, exhibits proteinase activity even in the absence of urea. Results suggest that dissociation of the enzyme unmasks the proteinase activity of the light subunit involving the site at which gamma-glutamylation of the enzyme occurs, and that the heavy subunit may impose transpeptidase reaction specificity by contributing the binding domains for gamma-glutamyl substrates.     

Modification of the acceptor binding site of gamma-glutamyl transpeptidase by the diazonium derivatives of p-aminohippurate and p-aminobenzoate

Hippurate and maleate have been shown to bind to the aminoacylglycine (acceptor) binding site of γ-glutamyl transpeptidase, thereby stimulating the hydrolysis of γ-glutamyl compounds at the expense of transpeptidation (Thompson, G. A., and Meister, A. (1979) J. Biol. Chem.254, 2956–2960; Thompson, G. A., and Meister, A. (1980) J. Biol. Chem.255, 2109–2113). It has now been found that a number of benzoate derivatives also bind and modulate rat kidney transpeptidase, as indicated by their ability to enhance the rate of inactivation of transpeptidase by the glutamine antagonist l-(αS, 5S)-α-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125). Furthermore, rapid loss of transpeptidase activity results upon preincubation of the enzyme with the diazonium derivatives of p-aminohippurate and p-aminobenzoate. The modified enzyme can still hydrolyze γ-glutamyl substrates but is no longer modulated by hippurate and maleate. Loss of transpeptidase activity was not associated with incorporation of radioactive label from diazotized [¹⁴C]p-aminohippurate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the modified enzyme revealed a nondissociable species, M_r 68,000, shown to result from crosslinking of the two subunits of transpeptidase (M_r 46,000 and 22,000, respectively). The crosslinking of the subunits paralleled the extent of inactivation of transpeptidation activity and both crosslinking and inactivation were prevented by treatment with the diazotized derivatives in the presence of either hippurate or maleate. These and other data indicate that the diazonium derivatives of p-aminohippurate and p-aminobenzoate interact with the acceptor binding site and produce a stable bond between amino acid residues in the vicinity of this site which, thus, appears to be located in the intersubunit contact region.     

The cytokineplast: purified, stable, and functional motile machinery from human blood polymorphonuclear leukocytes

We examined the formation of motile, chemotactically active, anucleate fragments from human blood polymorphonuclear leukocytes (PMN, granulocytes), induced by the brief application of heat. These granule-poor fragments are former protopods (leading fronts, lamellipodia) that become uncoupled from the main body of the cell and leave it, at first with a connecting filament that breaks and seals itself. The usual random orientation of such filaments can be controlled by preorientation of cells in a gradient of the chemotactic peptide, N-formylmethionylleucylphenylalanine (F-Met-Leu-Phe) (2x10(-9) M- 1x10(-8)). Cytochalsin B, 2.5-5 μg/ml, prevents fragment formation; colchicine, 10(-5) M, does not. In scanning electron micrographs, fragments are ruffled and the cell body rounded up and rather smooth. In transmission electron micrographs, fragments contain microfilaments but lack centrioles and microtubules. Like intact cells, both bound and free fragments can respond chemotactically to an erythrocyte destroyed by laser microirradiation (necrotaxis); the free, anucleate fragments may do so repeatedly, even after having been held overnight at ambient temperatures. We propse the name cytokineplast for the result of this self-purification of motile apparatus. The exodus of the motile machinery from the granulocyte requires anchoring of the bulk of the cell to glass and uncoupling, which may involve heat-induced dysfunction of the centrosome. In ultrastructural studies of the centrosomal region after heat, centriolar structure remains intact, but pericentriolar osmiophilic material appears condensed, and microtubules are sparse. These changes are found in all three blood cell types examined: PMN, eosinophil, and monocyte. Of these, the first two make fragments under our conditions; the more sluggish monocyte does not. Uncoupling is further linked to centrosomal dysfunction by the observation that colchicines-treated granulocytes (10(-5)M, to destroy the centrosome’s efferent arm) make fragments after less heat than controls. If motive force and orientation are specified mainly from the organelle-excluding leading front, then endoplasmic streaming in PMN is a catch-up phenomenon, and microtubules do not provide the vector of locomotion but rather stabilize and orient the “baggage” (nucleus, granuloplasm)—i.e., they prevent fishtailing. Moreover, constraints emanating from the centrosome may now be extended to include, maintenance of the motile machinery as an integral part of the cell.     

In vitro evaluation of native entomopathogenic fungi and neem (Azadiractha indica) extracts on Spodoptera frugiperda

The control of Spodoptera frugiperda is based on synthetic insecticides, so some alternatives are the use of entomopathogenic fungi (EF) and neem extract. The objective of the study was to evaluate in vitro effectiveness of native EF and neem extracts on S. frugiperda larvae. Six EF were identified by DNA sequencing of ITS regions from three EF (Fusarium solani, Metarrhizium robertsii, Nigrospora spherica and Penicillium citrinum). They were evaluated in concentrations of 1 × 10⁸ spores/ mL. In addition, a second bioassay was carried out evaluating only F. solani, M. robertsii and N. sphaerica and the addition of vegetable oil. On the other hand, extraction of secondary metabolites from neem seed (Azadirachta indica) was carried out by performing, mass (g) and solvent volume (mL ethanol and water) combinations, which were subjected to microwaves and ultrasound. Subsequently, these extracts were evaluated in concentrations of 3%, 4% and 5%. A survival analysis was performed for each of the bioassays. With respect to the results of the first bioassay, F. solani obtained a probability of survival of 0.476 on the seventh day, while in the second bioassay, M. robertsii obtained 0.488 survival probability. This suggests that the expected percentage of larvae that stay alive on the sixth day is 48.8%. However, in the evaluation of the neem extract the combination 1:12/70% to 4% caused 84% mortality of larvae. The use of native HE and neem extracts has potential for the control of S. frugiperda.     

The pro domain of beta-secretase does not confer strict zymogen-like properties but does assist proper folding of the protease domain

beta-Secretase (BACE) is a membrane-bound aspartyl protease that cleaves the amyloid precursor protein to generate the N terminus of the amyloid beta peptide. BACE is expressed as a precursor protein containing Pre, Pro, protease, transmembrane, and cytosolic domains. A soluble BACE derivative (PreProBACE460) that is truncated between the protease and transmembrane domains was produced by baculovirus-mediated expression. ProBACE460 was purified from conditioned media of infected insect cells using immobilized concanavalin A and immobilized BACE inhibitor, P10-P4' Stat(Val). Furin cleaves ProBACE460 between the Pro and protease regions to generate mature BACE460. The k(cat)/K(m) of ProBACE460 when assayed with a polypeptide substrate is only 2.3-fold less than that of BACE460. This finding and the similar inhibitory potency of P10-P4' Stat(Val) for ProBACE460 and BACE460 suggest that the Pro domain has little effect on the BACE active site. Exposure of ProBACE460 to guanidine denaturation/renaturation results in a 7-fold higher recovery of BACE activity than when BACE460 is similarly treated. The presence of free BACE Pro peptide during renaturation of BACE460 but not ProBACE460 increases recovery of activity. These findings show that the Pro domain in ProBACE460 does not suppress activity as in a strict zymogen but does appear to facilitate proper folding of an active protease domain.     

Characterization of plasmin-mediated activation of plasma procarboxypeptidase B. Modulation by glycosaminoglycans

Plasma carboxypeptidase B (PCB) is an exopeptidase that exerts an antifibrinolytic effect by releasing C-terminal Lys and Arg residues from partially degraded fibrin. PCB is produced in plasma via limited proteolysis of the zymogen, pro-PCB. In this report, we show that the K(m) (55 nM) for plasmin-catalyzed activation of pro-PCB is similar to the plasma concentration of pro-PCB (50-70 nM), whereas the K(m) for the thrombin- or thrombin:thrombomodulin-catalyzed reaction is 10-40-fold higher than the pro-PCB level in plasma. Additionally, tissue-type plasminogen activator triggers activation of pro-PCB in blood plasma in a reaction that is stimulated by a neutralizing antibody versus alpha(2)-antiplasmin. Together, these results show that plasmin-mediated activation of pro-PCB can occur in blood plasma. Heparin (UH) and other anionic glycosaminoglycans stimulate pro-PCB activation by plasmin but not by thrombin or thrombin:thrombomodulin. Pro-PCB is a more favorable substrate for plasmin in the presence of UH (16-fold increase in k(cat)/K(m)). UH also stabilizes PCB against spontaneous inactivation. The presence of UH in clots prepared with prothrombin-deficient plasma delays tissue-type plasminogen activator-triggered lysis; this effect of UH on clot lysis is blocked by a PCB inhibitor from potato tubers. These results show that UH accelerates plasmin-catalyzed activation of pro-PCB in plasma and PCB, in turn, stabilizes fibrin against fibrinolysis. We propose that glycosaminoglycans in the subendothelial extracellular matrix serve to augment the levels of PCB activity thereby stabilizing blood clots at sites where there is a breach in the integrity of the vasculature.