Molecular cloning of a GABA receptor subunit from Laodelphax striatella (Fallén) and patch clamp analysis of the homo-oligomeric receptors expressed in a Drosophila cell line.

A cDNA encoding a gamma-aminobutyric acid (GABA) receptor subunit was cloned from the small brown planthopper Laodelphax striatella. The L. striatella GABA receptor subunit was found to have high amino acid sequence similarity to the bd-type splice variant of the Drosophila GABA receptor Rdl subunit and several other GABA receptor subunits, with identities of over 70%. The cDNA was inserted into the expression vector pAc5.1-lac-Hygro. Clonal cell lines stably expressing homo-oligomeric L. striatella GABA receptors were generated by transfecting the vector into D.mel-2 cells. Expression of functional GABA receptors in the cell lines was demonstrated by whole-cell patch clamp recordings. GABA induced inward currents with an EC(50) value of 29 microM and a Hill coefficient of 1.7. The GABA-evoked responses reversed close to the Nernst equilibrium potential for chloride ions. The amplitudes of agonist-induced currents were found to be in the order muscimol (100 microM) >/= GABA (100 microM) > isoguvacine (100 microM) > cis-4-aminocrotonic acid (CACA) (100 microM) > 5-(4-piperidyl)-3-isoxazolol (4-PIOL) (1 mM). Antagonists such as fipronil (100 nM), 4'-ethynyl-4-n-propylbicycloorthobenzoate (EBOB) (100 nM), dieldrin (100 nM) and SR95531 (gabazine) (1 microM) suppressed GABA-induced currents. The functional expression of a GABA receptor from an agricultural pest presents a unique opportunity to discover new molecules active at this important target site.

Induction of apoptosis by cyclooxygenase-2 inhibitors in prostate cancer cell lines.

Prostaglandins are thought to play an important role in the proliferation of prostate cancer and are highly expressed in prostate cancer tissue. Cyclooxygenase-2 (COX-2), or prostaglandin endoperoxide synthase, is a key enzyme in the conversion of arachidonic acid into prostaglandin. In several cancers, COX-2 contributes to the proliferation and metastasis of cancer cells. To assess the role of COX-2 in prostate cancer, we investigated whether the inhibition of COX-2 affected the proliferation of prostate cancer cells. The human prostate cancer cell lines, LNCaP and PC 3, and a normal prostate stromal cell line (PrSC) were treated with COX-2 inhibitors NS 398 and Etodolac. The proliferation rate of the cell lines was examined using 3(4,5-dimethylethiazoly 1-2-) 2,5-diphonyl tetrazolium bromide (MTT) assays. A DNA fragmentation assay was also used for proof of apoptosis. COX-2 inhibitors could suppress the proliferation of LNCaP and PC 3 cells. In contrast, PrSC was not affected by COX-2 inhibitors. These suppressive effects occurred in a time- and dose-dependent manner. One of mechanisms responsible for cell death was apoptosis. COX-2 seems to play a significant role in the progression of prostate cancer. COX-2 may be a therapeutic target for prostate cancer. Since COX-2 inhibitors suppress proliferation and induce apoptosis in prostate cancer cells, and have no effect in normal prostate stromal cells, COX-2 inhibitors will be useful for the treatment of prostate cancer.

Reversible dimerization of 20 kilodalton human growth hormone (hGH).

A noncovalent dimer of the 22 kilodalton human growth hormone (22 K-hGH) is known to have diminished somatogenic activity compared with monomeric 22 K-hGH. In the present study, we examined the biological activity and physicochemical behaviour of a noncovalent dimer of the 20 kilodalton human growth hormone (20 K-hGH), an isoform of 22 K-hGH. Analysis of the equilibrium between monomeric and associated forms revealed that the associated 20 K-hGH was present in the dimeric form in aqueous solution. The kinetics of dimerization in rat plasma followed the theory of dissociation-association equilibrium, and more than 99% of 20 K-hGH molecules existed as a monomer in the equilibrium state at the physiological hGH concentration. Analysis of the pharmacokinetics showed that the ratio of the administrated dimer in rat circulation decreased from 43% to less than 4% in 2 h. A preparation of noncovalent dimeric 20 K-hGH had essentially the same degree of biological potency as that of a monomer in both in vitro and in vivo bioassays. In conclusion, dimerization of 20 K-hGH is reversible both in vitro and in vivo and a noncovalent dimer can function as a pharmaceutically active component of a 20 K-hGH preparation, in contrast to a 22 K-hGH preparation.

Comparative study of the N-glycans of human monoclonal immunoglobulins M produced by hybridoma and parental cells.

Cell-cell hybridization is one method of establishing cell lines capable of producing an abundance of antibodies. In order to clearly characterize antibodies produced by hybridomas, the influence of cell-cell hybridization on the glycosylation of produced antibodies should be studied. In this report, we describe structural changes of the N-glycans in immunoglobulin M (IgM) produced by a hybridoma cell line termed 3-4, which was established through hybridization of an IgM-producing Epstein-Barr virus transformed human B-cell line termed No. 12, and a human myeloma cell line termed P109. We analyzed the structures of sugar chains on the constant region of the mu-chain of IgMs produced by parental No. 12 cells and hybridoma 3-4 cells. In both parental cells and hybridoma cells, the predominant structures at Asn171, Asn332, and N395 were fully galactosylated biantennary complex types, with or without core fucose and/or bisecting GlcNAc. However, the amount of bisecting GlcNAc was markedly decreased in the hybridoma cells. Therefore, the activity of UDP-N-acetylglucosamine:beta-D-mannoside beta-1,4-N-acetylglucosaminyltransferase (GnT-III) responsible for the formation of bisecting GlcNAc was measured in parental cells and hybridoma cells. No. 12 cells showed some GnT-III activity, whereas P109 cells showed no such activity. The corresponding level of activity observed in hybridoma 3-4 cells was much lower than that in No. 12 cells. The above results demonstrated a reduction in the intracellular activity of GnT-III in the hybridoma cells, which was largely due to the influence of P109 cells. Moreover, the sugar chain structures of IgMs produced by the cells reflected the level of GnT-III activity.

[Sensitivity of Helicobacter pylori to amoxicillin and clarythromycin with special reference to eradication therapy].

We isolated strains of Helicobacter pylori from gastric mucosa of patients with peptic ulcer before and after eradication therapy, and studied their sensitivity to amoxicillin (AMPC) and clarythromycin (CAM). Of 85 strains of H. pylori isolated before therapy, MIC90 was 0.025 microgram/ml and no strains were resistant to AMPC. On the other hand, MIC90 of CAM was 0.05 microgram/ml and seven (8.2%) were already resistant to CAM. The H. pylori strains from eight cases of failed eradication therapy with lansoprazole + AMPC remained AMPC sensitive. However H. pylori strains from nineteen cases (82.6%) out of 23 of failed eradication therapy with lansoprazole + CAM became CAM resistant. The situation was similar for the cases of failed eradication therapy with lansoprazole + AMPC + CAM. Drug sensitivity tests prior to eradication therapy are to be recommended. A disc method may be used as a simple alternative to MIC measurement.

[Intraocular neovascularization].

To investigate the mechanism of intraocular neovascularization, we studied how vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) are expressed in the ocular tissues under hypoxic conditions. Prior to proliferation of vascular endothelial cells resulting in neovascularization, the retinal tissues such as pericytes, retinal glial cells, ganglion cells, and ciliary epithelium react directly to hypoxia expressing VEGF and/or IL-8 and stimulate endothelial cell proliferation in a paracrine manner. We demonstrated that transcription factor activator protein-1 (AP-1) is activated for expression of VEGF messenger ribonuculeic acid (mRNA) and in a similar way nuclear factor kappa B (NF-kappa B) is activated for expression of IL-8 mRNA. However, hypoxia-induced expression of VEGF and/ or IL-8 is only one aspect of the complicated processes in intraocular neovascularization. We hope that further detailed analysis of the mechanism will make it possible to inhibit and treat clinically intraocular neovascularization in the near future.

[A study on MRSA infections and replacement of bacteria. Efficacy of vancomycin-ceftazidime combination therapy].

We analyzed the results of bacteriological tests on patients with MASA infections admitted to Osaka Prefectural Hospital, for the past 10 years. The conclusions obtained are as follows. 1. In our hospital, MRSA infections accounted for 50% or more of all Staphylococcus aureus infections in 1983 and 1984 (in the first half of the 1980's), markedly decreased to about 10% after 1988, because of the preventive measures taken against nosocomial infections and initiation of anti-MRSA treatment. During the latter period, use of antibiotics including third generation cephems was not especially restricted. 2. Glucose non-fermentative Gram-negative rods (GNF-GNR) or yeasts co-isolated with S. aureus were more frequent in MRSA infected patients, than those in MSSA infected patients. 70 to 80% of GNF-GNR were P. aeruginosa. 3. Sensitivities of MRSA to drugs were studied. VCM was most active, followed by arbekacin and rifampicin. Effectiveness against co-isolated GNF-GNR was high with ofloxacin, netilmicin and ceftazidime (CAZ). Therefore, is expected that, to prevent the replacement of opportunistic infections, combination therapies using vancomycin and CAZ would be effective.

Amino acid sequences around exofacial proteolytic cleavage sites of band 3 from bovine and porcine erythrocytes.

1. Amino acid sequences of bovine and porcine band 3, an erythrocyte anion transporter, were determined. 2. The sequence of bovine band 3 was positioned to residues 519-599 (the numbering is based on human band 3), in which probably 6 residues were unidentified. 3. Binding site of DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonate), a potent anion transport inhibitor, was identified as Lys-539 in the bovine case. 4. A loop (residues 551-567), which provides exofacial proteolytic cleavage sites, contains only 53% homology between human and bovine, whereas the residues flanking it on either side are > 84% homologous. 5. Furthermore, the loop of porcine band 3 was indicated to consist of a 6 or 7-residues short peptide as compared with those of other species.

HAF, hepatoma aggregation factor produced by Streptomyces sp. strain No. A-6143.

We searched for a new cell aggregation factor for hepatoma AH109A cells, and found one we called HAF in the culture filtrate of Streptomyces sp. strain No. A-6143 isolated from a soil sample. HAF was purified by salting-out with ammonium sulfate. DEAE-cellulose column chromatography, gel filtration on Sephadex G-100, and hydroxylapatite column chromatography, HAF was glycoprotein which had a molecular weight of about 73,000. HAF was stable from pH 6 to 8 at 37 degrees C and up to 40 degrees C at pH 8.0 and the aggregation activity of HAF was maximum around pH 8 at 30 degrees C. The activity was not influenced by some saccharides, but it was inhibited by EDTA and EGTA: moreover HAF activity was restored by the addition of calcium ions. HAF aggregated hepatoma AH136B and COS-7 cells as well as hepatoma AH109A cells, but it was inert to other cancer cells and human erythrocytes. These properties proved that HAF is completely different from other aggregation factors for cancer cells so far reported.

[A case of mesenteric panniculitis of the transverse colon].

A case of mesenteric panniculitis in a 55-year-old female was reported. Image diagnosis of this rare clinical entity, which is characterized by a nonspecific inflammatory process involving the adipose tissue of the mesentery, was considered. We treated this patient conservatively and this led to remission.

[Treatment and prevention of infections with cefoxitin sodium in patients with biliary tract infections and obstructive jaundice].

UNLABELLED: A total of 24 patients who was hospitalized in the Internal Medicine Wards of Yamaguchi University attached Hospital and the university's 3 related hospitals were administered with cefoxitin. The breakdown of the patients treated with cefoxitin was 7 with cholecystitis, 7 with choledochitis and the remaining 10 for the prevention of infections with obstructive jaundice. Daily doses of 2-6 g of cefoxitin were administered for 6-40 days by intermittent intravenous drip infusion in divided doses. RESULTS: 1. Of 14 patients with biliary tract infections, 10 (71.4%) responded favorably with cefoxitin. 2. Of 10 patients with obstructive jaundice used for the prevention of infections, 8 (80%) responded favorably with cefoxitin. 3. No untoward side effects were observed. 4. Cefoxitin proved to be a safe and effective antibiotic in the treatment of biliary tract infections and for the prevention of infections in patients with obstructive jaundice.