[Studies on respiratory infections in primary care clinic (V). The pattern of distribution on bacteria, Mycoplasma pneumoniae and virus isolated from patients with respiratory infections, who were seen in six private clinics, and clinical efficacy of ciprofloxacin and roxithromycin].

The pattern of distribution of bacteria, Mycoplasma pneumoniae and virus isolated from the same specimen recovered from the throat swab or the sputum of 479 patients with respiratory infections who were seen in six private clinics in Sendai City of Japan during the period from October to November in 1992 (period I) and from January to February in 1993 (period II) was documented. Of the 479 patients, 234 had acute pharyngitis, 145 had acute bronchitis, 96 had influenza, 21 had acute tonsillitis, 5 had acute pneumonia and 9 had other respiratory infections. One hundred (42.4%) strains of potential pathogen and one strain of M. pneumoniae were recovered from 236 cases in period I, and 66 (27.2%) strains of potential pathogen, one strain of M. pneumonae and 73 strains of Influenza virus (30.0%: 43 of type A Hong-Kong and 30 of type B) from 243 cases in period II. Of the 166 strains, major isolates were Staphylococcus aureus (56 strains), Streptococcus pneumoniae (12 strains), Streptococcus pyogenes (15 strains), Haemophilus influenzae (17 strains), Esherichia coli (4 strains), Klebsiella spp. (35 strains), Pseudomonas aeruginosa (4 strains) and Acinetobacter spp. (23 strains). Only one strain of S. aureus was resistant to methicillin (MIC: 50 micrograms/ml). None of S. pneumoniae was resistant to 1 microgram/ml of ampicillin. Ciprofloxacin was administered to 113 cases and roxythromycin to 220 cases by doctors in charge.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of the PP2A alpha gene mutation in okadaic acid-resistant variants of CHO-K1 cells.

Okadaic acid (OA)-resistant variants of Chinese hamster ovary cells, clones CHO/OAR6-6 and CHO/OAR2-3, were isolated from a CHO-K1 culture. These variant cells were 17- to 26-fold more resistant to OA than the parental cells. The phosphorylase phosphatase activity of the variant cell extracts was 2- to 4-fold more resistant to OA than that of the parental cells in the presence of inhibitor 2, a specific inhibitor of type 1 protein serine/threonine phosphatase (PP1). Nucleotide sequencing of PP2A alpha (an isotype of PP2A catalytic subunit) cDNA demonstrated that both variants have a T-->G transversion at the first base of codon 269 (805 nt), which results in substitution of glycine for cysteine. We expressed in COS-1 cells a mutant PP2A alpha tagged with the influenza hemagglutinin epitope. The recombinant mutant PP2A alpha protein immunoprecipitated with an anti-influenza hemagglutinin antibody was more resistant than the wild type to OA, their IC50 values being 0.65 nM and 0.15 nM, and their IC80 values being 4.0 nM and 0.45 nM, respectively. The cysteine at residue 269 present only in highly OA-sensitive protein serine/threonine phosphatase catalytic subunit isozymes, PP2A alpha, PP2A beta, and PPX, is suggested to be involved in the binding of OA. CHO/OAR6-6 and CHO/OAR2-3 cells also overexpressed the P-glycoprotein, and the efflux of OA was more rapid. It is suggested that the PP2A alpha mutation in cooperation with a high level of P-glycoprotein makes the CHO-K1 variants highly resistant to OA.

Presence of p53 mutations in 3Y1-B clone 1-6: a rat cell line widely used as a normal immortalized fibroblast.

The 3Y1 cell line, established from a rat whole embryo, is widely used as a normal immortalized fibroblast. We analyzed p53 mutations in four clonal lines derived from the 3Y1 cell line; 3Y1-B clone 1-6, 3Y1-C and two clonal lines (3Y1 cl-3 and 3Y1 cl-6) which had been transformed by the human papilloma virus E6 gene. Polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) analysis and DNA sequencing showed that three clonal lines had a double mutation at codons 130 and 136 on the same allele and that the other clonal line, 3Y1 cl-3, had no mutations. 3Y1-B clone 1-6, which has been registered as the standard clonal line at the Japanese Cancer Research Resources Bank, demonstrated weak bands of the wild type allele, suggesting the existence of heterogeneous cell types in this "clonal" line. PCR-SSCP analysis of 25 subclones obtained by limiting dilution of 3Y1-B clone 1-6 cells revealed a mixture of two types of cells; 12 subclones showed only the bands of mutated allele, and 13 subclones showed both bands of the wild and mutated p53 alleles. These findings should be taken into consideration when using this cell line as a normal immortalized cell line.

Experimental models of acute and chronic sialoadenitis in the guinea pig.

Acute and chronic sialoadenitis were induced in ovalbumin-immunized guinea pigs by a single or repeated (once a day for 5 days) instillation of antigen into the parotid gland via the parotid duct. The acute sialoadenitis was characterized by infiltration of inflammatory polymorphonuclear leukocytes and the chronic one, by extensive tissue destruction together with infiltration of mononuclear leukocytes. In acute sialoadenitis, myeloperoxidase activity in the parotid gland, which was a marker of accumulation of neutrophils, was elevated, but in the chronic stage, it returned nearly to the control level. This observation is in accord with the histological findings that infiltrating cells in acute and chronic sialoadenitis were mainly polymorphonuclear and mononuclear leukocytes, respectively. Although cyclophosphamide suppressed the inflammation, both in acute and chronic sialoadenitis, indomethacin exerted its anti-inflammatory effect only in the acute stage. Our experimental models of acute and chronic sialoadenitis were easy to prepare, and had a high incidence. As the typical features of inflammatory development from acute to chronic phases were observed in these models, these models may be useful for studying the mechanism of the chronic course in immunologically induced inflammation and the effects of drugs on each phase and the chronic course of inflammation.

Mitogenic activity of Tulipa gesneriana lectins on mouse and human lymphocytes.

Tulipa gesneriana lectin-erythrocyte (TGL-E) which agglutinates mouse erythrocytes showed a potent mitogenic activity on mouse spleen cells and human peripheral blood lymphocytes, however, TGL-E had only slight mitogenic activity on mouse thymus cells. Its subunit alpha with a molecular weight (MW) of about 26,000 showed a potent mitogenic activity as did that of native lectin, but subunit beta with a MW of about 14,000 showed no activity, indicating that the mitogenic activity of TGL-E originates from subunit alpha. TGL-E stimulated T cell enriched spleen cells which passed through a nylon column, but not spleen cells from a nude mouse or spleen cells treated with anti-Thy 1.2 antibody and complement. Thus, TGL-E stimulates only mouse T cells but not B cells. The other lectin in tulip bulbs, Tulipa gesneriana lectin-yeast showed no mitogenic activity on mouse spleen, thymus cells or human paripheral blood lymphocytes.

Mutation induction by okadaic acid, a protein phosphatase inhibitor, in CHL cells, but not in S. typhimurium.

Okadaic acid (OA) is a specific and strong inhibitor of protein phosphatases 1 and 2A present in eukaryotes, and a potent promoter of carcinogenesis in mouse skin. In this study, we examined the mutagenicity of OA. OA did not induce mutations in S. typhimurium TA100 and TA98, with or without a microsomal metabolic activation system. However, it was strongly mutagenic to Chinese hamster lung (CHL) cells without a microsomal activation system, as shown using diphtheria toxin (DT) resistance (DTr) as a selective marker. Treatment of CHL cells with OA at 17.5 ng/ml induced 164 DTr mutants per 10(6) survivors. A plot of the mutation frequency against the OA concentration gave a concave curve, and the mutant frequency was calculated to be 5500/10(6) survivors/micrograms, with OA in the dose range of 10-15 ng/ml. This value was about 680 times that of ethyl methanesulfonate (EMS), and comparable to that of 2-amino-N6-hydroxyadenine, one of the strongest known mutagens. Elongation factor 2 (EF-2) obtained from 4 DTr clones was not ADP-ribosylated by DT fragment A. PCR-direct sequencing revealed that the hot spot of EF-2 for EMS mutagenesis in CHO-K1 cells, the first letter of codon 717, was not a hot spot for OA mutagenesis in CHL cells.

Mutagenic properties of 2-amino-N6-hydroxyadenine in Salmonella and in Chinese hamster lung cells in culture.

The mutagenicity of the base analogue, 2-amino-N6-hydroxyadenine (AHA), was tested in Salmonella typhimurium TA100 and TA98 and in Chinese hamster lung (CHL) cells. AHA showed very potent mutagenicity in TA100 without S9 mix, inducing 25,000 revertants/micrograms. The mutagenicity increased about 2-fold upon addition of S9 mix containing 10 microliters S9. AHA was found to be one of the strongest mutagens for TA100. Addition of S9 mix containing 100 microliters S9 induced no significant increase of revertants with AHA at amounts up to 50 ng per plate. AHA was also mutagenic for the frameshift mutant, TA98, without S9 mix, the mutagenicity for TA98 being about 1/1000 of that for TA100. When the mutagenicity of AHA was tested in CHL cells, with diphtheria toxin resistance (DTr) as a selective marker in the absence of S9 mix with a 3-h treatment of cells, DTr mutants increased dose-dependently at concentrations of 2.5-15 micrograms/ml. When cells were incubated with AHA for 24 h, a 200-fold increase in the number of DTr mutants was observed; the mutagenicity was 500-fold higher than that of ethyl methanesulfonate. This marked increase of mutagenicity by prolonged incubation may indicate that AHA induces mutations mainly after incorporation into DNA. The addition of a small amount of S9 increased the mutagenicity obtained with a 3-h treatment 2-fold, but a larger amount of S9 decreased the mutagenicity as was found with S. typhimurium TA100.

Loss of amplified genes by poly(ADP-ribose) polymerase inhibitors.

A poly(ADP-ribose) polymerase inhibitor, benzamide (BA), was found to induce flat revertants of NIH 3T3 cells that had been transformed by human Ha-ras, rat Ki-ras, rat c-raf, and human ret-II. These genes had been amplified in original transformants, but they were completely eliminated by BA. Contrary to this, endogenous activated Ha-ras in a human bladder carcinoma cell line, T24, was not eliminated by BA. The gene loss seemed to be restricted to exogenous and/or amplified sequences. BA also eliminated the amplified c-myc gene in HL-60 cells, concomitant with differentiation into granulocytes. We demonstrated that the amplified c-myc gene was not present as episomes. It is probably present as double minutes or a homogeneously staining region. Dimethylsulfoxide also induced differentiation at a concentration that did not inhibit poly(ADP-ribose) polymerase. The cell lost the c-myc gene in association with this differentiation. The amplified c-myc gene in a colon adenocarcinoma cell line, COLO 320HSR, and the amplified mdr-1 gene in an adriamycin-resistant myelogenous leukemia cell line, K562/ADM, were not eliminated by BA. Various poly(ADP-ribose) polymerase inhibitors also eliminated human Ha-ras in the NIH 3T3 transformant and the c-myc gene in HL-60 cells.

Loss of the MYC gene amplified in human HL-60 cells after treatment with inhibitors of poly(ADP-ribose) polymerase or with dimethyl sulfoxide.

In HL-60 cells, a human promyelocytic leukemia cell line, the human c-myc gene, designated MYC, is amplified about 16-fold. On differentiation of the HL-60 cells into granulocytes induced by several inhibitors of poly(ADP-ribose) polymerase [NAD+ poly(adenosine diphosphate D-ribose)ADP-D-ribosyltransferase, EC 2.4.2.30] including benzamide, nicotinamide, coumarin, and 4-hydroxyquinazoline or dimethyl sulfoxide, some MYC loss was observed. In contrast, benzoic acid, a noninhibitory analogue of benzamide, did not induce either granulocytic differentiation or loss of MYC. Loss of MYC seems to be associated with granulocytic differentiation because the time course of its loss was similar to that of appearance of nitroblue tetrazolium-positive cells, mature granulocytes, and its loss was not observed on differentiation of HL-60 cells into macrophages induced by phorbol 12-myristate 13-acetate or teleocidin. The loss of MYC is not the reason for the down regulation of MYC expression observed within 1 hr after addition of inducers, since the loss of MYC was not detected by 1-day treatment with inducers.

Studies on chemical modification of Tulipa gesneriana lectin.

Modification of lysine, tyrosine, histidine, aspartic acid and glutamic acid residues did not affect the agglutinating activity of the Tulipa gesneriana lectin (TGL). Modification of two arginine residues per subunit in the lectin with either 2,3-butanedione or phenylglyoxal led to an almost complete loss of activity. An inactive lectin modified with 2,3-butanedione recovered a full activity on dialysis against Tris-HCl buffer. The presence of 0.1 M (alpha-1----6) linked mannotriose, a potent inhibitor of the lectin, protected all the arginine residues from modification and the lectin was fully active. Circular dichroism spectroscopy showed that no significant conformational change of TGL occurred following arginine modification. A treatment of the lectin solution with N-bromosuccinimide or 2-hydroxy-5-nitrobenzyl bromide, chemical reagents for tryptophan modification, caused turbidity of the solution, accompanied with complete loss of activity. The fluorescence emission spectrum of the lectin showed a characteristic tryptophan emission with a maximum centered at 336 nm. Upon addition of manno-oligosaccharides a decrease of the fluorescence intensity was observed, indicating that the environment of tryptophan residues altered. These results suggest that arginine and tryptophan residues are importantly involved in the sugar binding of TGL.

Characteristics of specific bindings of nitrendipine and PN200-110 to various crude membranes: induction of irreversible bindings by UV irradiation.

The characteristics of the specific bindings of [3H]nitrendipine (Nit) and [3H](+)PN200-110 (PN) to crude membranes from rat skeletal, cardiac, and uterine muscle and whole brain were investigated, with special interest in the effect of UV irradiation on these bindings. The specific bindings of [3H]Nit and [3H](+)PN to these crude membranes were saturable and reversible. The specific bindings of [3H]Nit to all these membranes except crude skeletal membranes was maximum in the presence of 0.15 M NaCl plus 1 mM CaCl2 and minimal in the absence of these ions, but the specific bindings of [3H](+)PN to these crude membranes was not affected significantly by these ions. A calcium agonist and antagonists inhibited the specific bindings of [3H]Nit and [3H](+)PN to these crude membranes, the order of their inhibitory effects on specific [3H]Nit bindings being roughly Nit greater than or equal to (+)PN greater than or equal to (-)PN much greater than Bay K 8644 (Bay) greater than verapamil (Ver) greater than diltiazem (Dil). In crude skeletal membranes only, PN caused significant stereospecific inhibition. The order of inhibitions of specific [3H](+)PN bindings to these crude membranes was generally (+)PN greater than Nit greater than or equal to (-)PN greater than Bay much greater than Ver greater than or equal to Dil. In all these crude membranes, UV irradiation completely prevented decrease in the amount of specific binding of [3H](+)PN binding on addition of excess unlabeled (+)PN. These findings suggested that [3H]Nit and [3H](+)PN bind to voltage-sensitive calcium channels in crude membranes from rat skeletal, cardiac, and uterine muscle and whole brain, and that UV irradiation changes the specific bindings of [3H]Nit and [3H](+)PN from reversible to irreversible bindings.

Deletion of transfected oncogenes from NIH 3T3 transformants by inhibitors of poly(ADP-ribose) polymerase.

a1-1 cells, a transformant line obtained by transfection of NIH 3T3 cells with human c-Ha-rasT24 (hc-Ha-rasT24), were converted to morphologically normal flat cells following a 2-week culture in the presence of benzamide (BA), an inhibitor of poly(ADP-ribose) polymerase [ADP-ribosyltransferase (polymerizing); EC 2.4.2.30]. Concomitant with these morphological changes was the loss of the exogenous hc-Ha-rasT24 sequence. When cells were cultured without transfer, multiple clusters of flat revertant cells surrounded by transformed cells within single colonies of a1-1 cells were observed. This, together with the slow growth rate of flat cells in the presence of BA, indicated that flat revertants were induced rather than selected by BA. Flat cells isolated from mixed colonies completely lost the exogenous and amplified hc-Ha-rasT24 gene. In contrast, the endogenous mouse c-Ha-ras in flat revertant cells was not lost during culture with BA. Similarly, the endogenous hc-Ha-rasT24 in human bladder carcinoma T24 cells was not affected by BA. By using various chemicals, it was suggested that inhibition of poly(ADP-ribose) polymerase induces an efficient and specific loss of the exogenous transforming genes including Ki-ras, N-ras, c-raf, and ret-II.

[Clinical studies on S 6472 granule preparation (sustained-release cefaclor) in chronic respiratory tract infections].

S 6472 granule preparation (sustained-release cefaclor) was orally administered to 15 patients with chronic respiratory tract infections (2 acutely exacerbated cases of chronic bronchitis, 13 cases of secondary infections consisting of 1 case of bronchial asthma, 2 cases of bronchial asthma/pulmonary emphysema, and 10 cases of bronchiectasis) at a daily dose of 750 mg divided into 2 doses administered after breakfast and dinner, for a duration of 14 days. The drug was ineffective in 3 of the 10 cases of bronchiectasis but was effective in the other 12 cases, with a rate of efficacy of 80%. There were no side effects of abnormal laboratory findings due to administration of this drug.

In vitro activity of desacetylcefotaxime and the interaction with its parent compound, cefotaxime.

Antibacterial activities of cefotaxime and its major metabolite, desacetylcefotaxime, against 178 strains (of 10 species) were assessed in terms of minimum inhibitory concentrations (MIC50 and MIC80), and were compared with those of cefoperazone and ceftazidime. The activity of desacetylcefotaxime was several times less than that of cefotaxime against almost all of the species tested. Against Staphylococcus aureus, Morganella morganii, Enterobacter cloacae and Pseudomonas aeruginosa, the MIC80 values of desacetylcefotaxime were higher than those of cefoperazone and ceftazidime. The antibacterial potency of desacetylcefotaxime against Klebsiella pneumoniae and Pseudomonas cepacia was superior to that of cefoperazone and ceftazidime, and comparable with the activities of the latter compounds against the other 4 Gram-negative species. Partial synergy was demonstrated in the activity of cefotaxime and desacetylcefotaxime against most of the strains examined. Antagonism was observed in activity against 2 of 18 strains of M. morganii. In general, desacetylcefotaxime enhances the potency of its parent compound, cefotaxime, when they coexist. Intravenous infusion of cefotaxime 1 g over a period of 1 hour resulted in mean peak serum concentrations of 48.5 mg/L of cefotaxime and 6.5 mg/L of desacetylcefotaxime at the end of the infusion. The mean elimination half-life in beta-phase of cefotaxime was 0.8 hour and that of desacetylcefotaxime was 2 hours.