Isolation and expression of a rat brain cDNA encoding glutamate carboxypeptidase II

N-acetylated alpha-linked acidic dipeptidase (NAALADase) hydrolyzes acidic peptides, such as the abundant neuropeptide N-acetyl-alpha-L-aspartyl-L-glutamate (NAAG), thereby generating glutamate. Previous cDNA cloning efforts have identified a candidate rat brain NAALADase partial cDNA, and Northern analyses have identified a family of related RNA species that are found only in brain and other NAALADase-expressing cells. In this report, we describe the cloning of a set of rat brain cDNAs that describe a full-length NAALADase mRNA. Transient transfection of a full-length cDNA into the PC3 cell line confers NAAG-hydrolyzing activity that is sensitive to the NAALADase inhibitors quisqualic acid and 2-(phosphonomethyl)glutaric acid. Northern hybridization detects the expression of three similar brain RNAs approximately 3,900, 3,000, and 2,800 nucleotides in length. In situ hybridization histochemistry shows that NAALADase-related mRNAs have an uneven regional distribution in rat brain and are expressed predominantly by astrocytes as demonstrated by their colocalization with the astrocyte-specific marker glial fibrillary acidic protein.     

Isolation of cDNA encoding full-length rat (Rattus norvegicus) poly (ADP-ribose) polymerase

Poly(ADP-ribose) polymerase (EC 2.4.2.30) is a nuclear enzyme which binds to DNA breaks and then catalyzes the covalent modification of acceptor proteins with poly(ADP-ribose). Poly(ADP-ribose) polymerase activity contributes to the recovery of proliferating cells from DNA damage and to the maintenance of genomic stability, which may be mediated by effects on chromatin structure, DNA base-excision repair and cell cycle regulation. We established the complete cDNA sequence of rat poly(ADP-ribose) polymerase by RT-PCR and direct sequencing of amplification products and compared it with that of other mammalian species. The amino acid sequence homology is strikingly high. The best conserved regions are the known functional modules of poly(ADP-ribose) polymerase.     

Molecular cDNA cloning and tissue distribution of mRNA encoding a novel ATP-binding cassette (ABC) half-transporter

After feeding a commercial rodent chow for 8 weeks, tissues from male and female rats were collected and examined for selenium content, glutathione peroxidase (GPX) activities and selenoprotein W (Se-W) levels. There were no differences (P > 0.05) between plasma selenium content, plasma GPX activity, whole blood selenium content, or whole blood GPX activity between male and female rats. There was also no gender effect on selenium concentration in muscle, brain, spleen, and skin, but selenium concentration in liver was higher (P < 0.05) in female than in male rats. Western blots indicated that the tissue distribution of Se-W was similar in male and female rats. Se-W protein level was high in testes of male rats but very low in ovaries of female rats. Muscle and skin from female rats had significantly higher (P < 0.05) Se-W levels than from male rats. Consistent with Se-W content, the Se-W mRNA levels from female skins were significantly higher (P < 0.05) than from male rats. The expression of Se-W was different in various tissues and gender influenced this regulation in some tissues.     

Isolation of a cDNA encoding Fasciola hepatica cathepsin L2 and functional expression in Saccharomyces cerevisiae

Cathepsin L2 is a major cysteine proteinase secreted by adult Fasciola hepatica. The enzyme differs from other reported cathepsin Ls in that it can cleave peptide substrates that contain proline in the P2 position. A cDNA was isolated from an expression library by immunoscreening with antiserum prepared against purified native cathepsin L2. This cDNA was sequenced and shown to encode a complete preprocathepsin L proteinase. Functionally active recombinant cathepsin L proteinase was expressed and secreted by Saccharomyces cerevisiae transformed with the cDNA. The recombinant enzyme was purified from large-scale fermentation broths using ultrafiltration and gel filtration chromatography on Sephacryl S200 HR columns. NH2-terminal amino acid sequencing showed that the cleavage point for activation of the recombinant pro-enzyme is identical to that of the F. hepatica-produced cathepsin L2. The mature active recombinant proteinase behaved similarly to the native enzyme when analysed by SDS-PAGE, immunoblotting and zymography and also cleaved peptides containing proline in the P2 position. Finally, the recombinant cathepsin L2 cleaved fibrinogen to form a fibrin clot, a property we described for F. hepatica cathepsin L2.     

Isolation and characterization of cDNA clones encoding a 32-kDa dense-granule antigen of Sarcocystis muris (Apicomplexa)

BACKGROUND AND STUDY AIMS: A second-look endoscopy is often performed to evaluate the efficacy of a prior injection therapy in patients with bleeding peptic gastric or duodenal ulcers. Although this strategy is widely established, it does not rely on unequivocal data from controlled studies. In a prospective, randomized, controlled multicenter trial we assessed the effect of programmed endoscopic follow-up examinations with eventual retreatment on the outcome of bleeding ulcers in these patients. PATIENTS AND METHODS: One hundred and five patients with gastric or duodenal peptic ulcers presenting with active (Forrest type I) or recent (Forrest type IIa and IIb) bleeding upon endoscopy within four hours after admission were included in the study. Emergency treatment consisted of the sequential injection of both epinephrine (1:10,000 v/v) and up to 2 ml of fibrin/thrombin around the ulcer base. Fifty-two patients were randomized to receive programmed endoscopic monitoring with eventual retreatment in cases of Forrest type I, IIa, or IIb ulcers beginning within 16-24 hours after the index bleed. Follow-up endoscopies were continued until the macroscopic appearance revealed a Forrest type IIc or III ulcer. Fifty-three patients in the control group were closely monitored, and only received a second endoscopy when there was clinical or biochemical evidence of recurrent bleeding. The groups did not differ with respect to age, sex, site and severity of bleeding. RESULTS: The numbers of patients with recurrent bleeding were similar whether they were endoscopically monitored or not (21% versus 17%, P=0.80 chi-squared test). In addition, there was no statistically significant difference between the two groups with respect to the number of blood units transfused, need for surgical intervention, hospital stay or number of deaths (Mann-Whitney U-test). Improving local ulcer stigmata was not related to a better outcome. CONCLUSIONS: Programmed endoscopic follow-up examinations with eventual retreatment in patients locally injected for an acute or recent hemorrhage from a gastric or duodenal ulcer did not influence their outcome when compared to patients receiving only a second endoscopic intervention upon evidence for recurrent hemorrhage. Scheduled control endoscopies cannot be recommended after an initial successful endoscopic treatment of peptic ulcer bleeding when selection of the patients for second-look endoscopy is directed by the Forrest criteria.     

The effect of pituitary adenylate cyclase activating polypeptide (PACAP) on amylase secretion from guinea pig pancreatic acini

Beta-lactamase production is one of the major mechanisms of resistance amongst bacteria especially the enteric bacilli. The purpose of this study is to assess the in-vitro activity of Sulperazon, a combination of cefoperazone and an irreversible beta-lactamase inhibitor, sulbactam, against the cefoperazone resistant isolates of aerobic gram-negative bacilli. A total of 92 such strains were tested. It was found that at a concentration of < or = 8 mg/l of sulbactam added to cefoperazone 82% of Klebsiella spp, 100% of E. coli, 100% of Enterobacter spp, 33% of Pseudomonas aeruginosa, 67% of Pseudomonas spp and 62% of Acinetobacter spp that were resistant to cefoperazone alone were susceptible to the combination. Hence it is concluded that the addition of sulbactam to cefoperazone does expand the spectrum of the in-vitro activity of cefoperazone.     

Stage-specific expression of pituitary adenylate cyclase-activating polypeptide (PACAP) mRNA in the rat seminiferous tubules

We have used in situ hybridization and Northern blot analysis with oligonucleotide probe to characterize the site of pituitary adenylate cyclase-activating polypeptide (PACAP) synthesis in the rat testis. We observed strong hybridization signal in one third of the cross-sections of the seminiferous tubules, whereas some tubules were devoid of hybridization signal, thus suggesting that PACAP mRNA is expressed in a stage-specific manner. More detailed analysis showed that PACAP mRNA was present in round spermatids at stages III-VII of the cycle. Northern blot hybridization to RNAs extracted from samples of seminiferous tubules at different stages of the epithelial cycle confirmed that expression of PACAP mRNA is restricted to specific stages of the cycle. The highest amount of PACAP mRNA was detected at stages V to early -VII of the cycle, whereas very low levels of mRNA were present at stages I-II and IX-XIV. The present results demonstrate that PACAP mRNA is expressed in the developing germ cells. This suggests that PACAP may function as a paracrine or autocrine regulatory factor for the Sertoli and germ cells, with a specific function during early spermiogenesis, shortly before the onset of nuclear elongation, at the last period of haploid gene activity.     

Cloning and sequence analysis of a cDNA encoding salmon (Onchorhynchus keta) liver transglutaminase

We isolated cDNA clones encoding a transglutaminase (TGase: EC 2.3.2.13) from a salmon (Onchorhynchus keta) cDNA library prepared from the liver. In the cDNA sequence combined, an open reading frame coding for a protein of 680 aa was found. The deduced sequence showed a considerable similarity (62.4%) to that of red sea bream TGase. By comparison of sequence similarity to other TGases, the structure of salmon TGase was like tissue type TGases, rather than membrane-associated type or plasma type TGases. As a structural feature of salmon TGase, 3 aa residues were substituted in the 25 aa sequence around the active site Cys residue, which is conserved among several tissue type TGases. The critical residues thought to form the catalytic-center triad (Cys272, His331, and Asp301) were found in the highly conserved region, but the region surrounding Tyr511, which corresponds to the residue participates in hydrogen-bond interactions of active center domain, was less similar to other TGases, except for red sea bream TGase. These finding suggests that the overall structure of fish TGase resembles tissue-type TGases, but has some unique structure.