Upregulation of angiotensin II AT1 receptors in the mouse nucleus accumbens by chronic haloperidol treatment

The distribution of angiotensin II AT1 and AT2 receptor subtypes were mapped in the mouse brain by in vitro autoradiography. Along with a differing distribution of AT1 and AT2 receptors in the hind brain compared to the rat, moderate densities of AT1 receptors were observed in dopamine-rich regions, namely the caudate putamen and nucleus accumbens, previously observed in the human, but not rat or rabbit. Considering our previous anatomical and functional studies demonstrating an interaction between brain angiotensin II and dopaminergic systems, the effect of chronic treatment with the dopamine antagonist, haloperidol, on AT1 and AT2 receptor levels was investigated in the mouse brain. Haloperidol treatment for 21 days resulted in an increase in angiotensin II AT1 receptor levels in the nucleus accumbens, accompanied by an increase in dopamine D2 receptors, but no change in dopamine D1 receptors. Striatal AT1 receptors did not alter with treatment, nor did AT1 or AT2 receptors in a number of brain regions not associated with dopaminergic systems, such as the median preoptic nucleus, paraventricular hypothalamic nucleus, and nucleus of the solitary tract. The present study suggests that brain angiotensin II-dopamine interactions extend beyond the known effects on the nigrostriatal dopaminergic system, to the mesocorticolimbic dopaminergic system.     

Improvement of the production of foreign proteins using a heterologous secretion vector system in Bacillus subtilis: effects of resistance to glucose-mediated catabolite repression

To improve protein production, a heterologous secretion vector system was constructed with the aid of the amyR2 region. The operator sequence (amyO) of the amyR2 region on the secretion vector was changed through site-directed mutagenesis to eliminate carbon-source-mediated catabolite repression. Three substitutional (AG, G5, G10), one deletional (delta HH), and one insertional (AGHF) mutant promoters were obtained. The expression level and the degree of catabolite repression of amyR2 and the mutant promoters were examined with a single copy system using an integrational promoter probe vector, pDH32. Under glucose-free culture conditions, expression levels from all mutant promoters except HH were 1.4 to 1.5 fold higher than that from amyR2. While the expression of the amyR2 promoter was repressed by 90% in the presence of 2% glucose, expression levels of the mutant promoters were repressed by only 1% to 50%. To evaluate the advantage of the mutant promoters in production of foreign proteins by the heterologous secretion system, beta-lactamase and human pancreatic secretory trypsin inhibitor (hPSTI) were expressed by the mutant promoters. When B. subtilis LKS87 was used as a host strain, the production of the target proteins using the respective mutant promoter was increased by about 1.5 fold under glucose-free culture conditions. Under the high glucose culture conditions, secretion of target proteins produced from the mutant promoters increased 1.5 to 2 fold, whereas those by the amyR2 promoter were reduced to between 50% and 60%. The additive effect of degUh mutation on protein production was not observed under high glucose culture conditions. In addition, such culture conditions inhibited proteolytic degradation of secreted target proteins after the stationary growth phase even in B. subtilis LKS88 (degUh mutant). Thus, our results indicated that the mutant promoters, which are resistant to glucose-mediated catabolite repression, are very useful for over-production of foreign proteins under the high glucose culture conditions using the heterologous expression-secretion system in B. subtilis.     

Retrospective comparison of infusional 5-fluorouracil, doxorubicin, and mitomycin-C (modified FAM) combination chemotherapy versus palliative therapy in treatment of advanced gastric cancer

About one-third of patients with gastric cancer are unresectable at the time of diagnosis. Their median survival is < 6 months, with a grave prognosis. The purpose of this study was to assess the efficacy of a modified FAM (mFAM) regimen in advanced gastric cancer. We retrospectively reviewed the clinical records of 409 advanced gastric cancer patients who had not received curative surgery. Among 409 patients, 202 patients were treated with an mFAM regimen (infusional 5-FU + doxorubocin + mitomycin-C), and 207 patients received no chemotherapy (control group). No differences were found in clinical parameters between the two groups. The 1-year survival rates were 34.1% for the mFAM-treated group and 22.5% for the control group (p = 0.0135). In subset analysis, a higher 1-year survival rate was demonstrated in patients with mFAM and palliative surgery. Of the 154 evaluable patients in the mFAM-treated group, the response rate was 17.5%. In these patients, median response duration was 30 weeks, and progression-free survival was 23 weeks. Overall toxicity of mFAM regimen was relatively tolerable and reversible. In conclusion, FAM combination chemotherapy, which has been used as a standard therapy, prolonged survival after modification of the administration schedule and combination with palliative surgery. A prospective randomized study is warranted to confirm this conclusion from our retrospective study.     

The potential for immunoconjugates in lymphoma therapy

Contemporary combination chemotherapy offers curative treatment for 30% to 50% of patients with advanced-stage, aggressive non-Hodgkin's lymphomas (NHL). However, conventional therapies cure few patients with indolent lymphomas or relapsed lymphomas of any histology. Myeloablative chemoradiotherapy with bone marrow or stem cell transplantation can provide pro-longed disease-free survival for a minority (20% to 50%) of patients with relapsed NHL, but new treatment approaches are clearly needed. In recent years, several groups of investigators have provided preliminary evidence suggesting that monoclonal antibodies (mAbs), in unmodified form or conjugated to toxins, drugs, or radioisotopes, may offer another effective therapeutic modality for patients with relapsed lymphomas. This article reviews current immunotherapy of NHL using antibody immunoconjugates.     

Identification and characterization of novel antimicrobial decapeptides generated by combinatorial chemistry

Novel combinatorial libraries consisting of simplified amino acid sequences were designed to screen for peptides active against the Candida albicans membrane. A novel decapeptide, KKVVFKVKFK, that had a unique primary amino acid sequence was identified in this work. This peptide irreversibly inhibited the growth of C. albicans and showed a broad range of antibacterial activity but no hemolytic activity. Circular dichroism spectra revealed that the predominant secondary structure of this peptide strongly depended on the membrane-mimetic environments; the peptide preferred to form an amphipathic alpha-helical structure in the presence of 50% trifluoroethanol, while it preferred to adopt a distorted alpha-helical structure in the presence of sodium dodecyl sulfate micelles. Experiments in which dye was released from vesicles indicated that this novel antimicrobial peptide killed microorganisms through the action on the membrane as its primary target. Replacement of amino acids in this active decapeptide on the basis of information from the libraries could provide unique information about factors affecting its antimicrobial activity such as its secondary structure, net positive charge, and hydrophobicity.     

Production of monoclonal antibodies specific to Clostridium botulinum type B neurotoxin

Four monoclonal antibodies were produced for use in a rapid method to detect Clostridium botulinum type B neurotoxin. Cells of mouse myeloma cell line SP2/0 were fused with splenocytes of immunized BALB/c mice. An immunoblot assay of semipurified commercial neurotoxins of C. botulinum types A, B, C, D, E, and F was used to show specificity. All the monoclonal antibodies reacted with type B neurotoxin but did not cross-react with the other types. The monoclonal antibodies, separately and combined, did not neutralize the toxin in mice, and all showed specificity to the whole neurotoxin molecule and the heavy-chain component by immunoblot. No evidence of specific binding to the hemagglutinin molecule was noted. When tested against concentrated cultured supernatants of C. botulinum types A, B, E, and F, the 4 monoclonal antibodies reacted only against type B strains. They will be incorporated into a rapid assay with other specific monoclonal antibodies to detect C. botulinum neurotoxins from pure cultures or suspect foods.     

Determination of ginsenoside Rf and Rg2 from Panax ginseng using enzyme immunoassay

We have developed an enzyme immunoassay (EIA) to quantify trace amounts of ginsenoside Rf (Rf), one of the glycosides of protopanaxatriol from Panax ginseng. A carrier protein of bovine serum albumin (BSA) was coupled to the carbohydrate component of Rf using the periodate oxidation method. Antibodies were raised in rabbits using Rf-BSA conjugate as the immunogen and competitive indirect EIA was used for the determination of Rf. The working range was 0.01-10 ng per assay. The anti-Rf antiserum cross-reacted with ginsenoside Rg2 (105%), which is also a component of Panax ginseng and has a very similar chemical structure to Rf. These results suggest that the anti-Rf antiserum could also be used for the quantitation of ginsenoside Rg2 as well as ginsenoside Rf. In a comparison of EIA and HPLC the linear regression equation and correlation coefficient for the two methods were y(EIA) = 1.31x (HPLC)-11.48 and 0.98, respectively.