The polymorphism in the caudal-related homeodomain protein Cdx-2 binding element in the human vitamin D receptor gene.

The major physiological activity of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] is the regulation of calcium absorption in the small intestine, and the level of vitamin D receptor (VDR) is an important factor in this regulation. In a previous study, we indicated-that the caudal-related homeodomain Cdx-2 played an important role in the intestine-specific transcription of the human VDR gene. In this study, the polymorphism was identified in the core sequence 5'-ATAAAAACTTAT-3' in the Cdx-2 binding site in the VDR gene promoter. In 261 Japanese women with genotyped VDR polymorphisms, 48 were genotype Cdx-A (adenine at -3731 nucleotides [nt] relative to the transcription start site of human VDR gene 5-ATAAAAACTTAT), 82 were genotype Cdx-G (guanine at -3731 nt, 5'-GTAAAAACTTAT-3'), and 131 were genotype Cdx-A/G (heterozygote). In postmenopausal Japanese women, the bone mineral density (BMD) in the lumbar spine (L2-L4) with the Cdx-G homozygote was 12% lower than that with the Cdx-A homozygote (p < 0.05). In electrophoretic gel mobility shift assay (EMSA), the oligonucleotide with Cdx-G allele markedly decreased the binding to Cdx-2 compared with that in the Cdx-A allele. The transcriptional activity of the VDR promoter with Cdx-G allele was decreased to 70% of the Cdx-A allele. In addition, in the herpes simplex virus thymidine kinase promoter, the Cdx-2 binding element with the G allele showed significantly lower transcriptional activity than that of the A allele. Thus, the polymorphism in the Cdx-2 binding site of the VDR gene (Cdx-polymorphism) would affect the expression of VDR in the small intestine. In addition, this polymorphism may modulate BMD in postmenopausal Japanese women.

Lipid profile of Helicobacter spp.: presence of cholesteryl glucoside as a characteristic feature.

The lipid and fatty acid profiles of eight Helicobacter spp. (H. nemestrinae, H. acinonyx, H. canis, Helicobacter sp. strain CLO-3, "H. rappini" [Flexispira rappini], H. pametensis, Helicobacter sp. strain Bird-B, and Helicobacter sp. strain Bird-C) and the fatty acid profiles of five additional species (H. pylori, H. felis, H. muridarum, H. mustelae, and H. fennelliae) were analyzed and compared. A heterologous fatty acid profile was observed among the Helicobacter spp., and on that basis the species could be divided into two groups. Group A had 19-carbon cyclopropane fatty acid (19:0cyc) and tetradecanoic acid (14:0) as the major fatty acids, and group B characteristically lacked the 19:0cyc and had hexadecanoic acid (16:0) and octadecenoic (18:1) acids as the major fatty acids. The species of group A are primarily gastric colonizers, and those of group B are primarily intestinal colonizers. Seven of the eight species studied showed the unusual and characteristic presence of cholesteryl glucosides (CGs), and most of these seven showed a very large amount (9.7 to 27.4% of the weight of total extractable lipid). The types of CGs and their distribution in different species were as follows: cholesteryl-6-O-acyl-alpha-D-glucopyranoside (cholesteryl-6-O-tetradecanoyl-alpha-D-glucopyranoside in H. nemestrinae and mainly cholesteryl-6-O-dodecanoyl-alpha-D-glucopyranoside in "H. rappini"), cholesteryl-alpha-D-glucopyranoside (H. nemestrinae, H. acinonyx, H. canis, Helicobacter sp. strain CLO-3, and "H. rappini"), and cholesteryl-6-O-phosphatidyl-alpha-D-glucopyranoside (H. nemestrinae, H. acinonyx, H. canis, and Helicobacter sp. strain CLO-3). Besides this, we could also detect cholesteryl acyl glucoside in H. acinonyx, cholesteryl glucoside in Helicobacter sp. strains Bird-B and -C, and cholesteryl phosphatidyl glucoside in "H. rappini" and Helicobacter sp. strain Bird-C. A selective accumulation of free cholesterol was observed in the neutral lipid fractions. On the basis of the detection of CGs in 11 of the 13 species studied so far, the presence of CGs appears to be a characteristic feature of the genus Helicobacter. In view of this and also because of a simple and rapid detection method described herein, the CGs can be used as a valuable chemotaxonomic marker.

Establishment of a mouse model of cystitis and roles of type 1 fimbriated Escherichia coli in its pathogenesis.

The role of type 1 fimbriae in promoting bladder colonization and the course of Escherichia coli cystitis were examined with type 1 fimbriated strains of clinically isolated E. coli. In the experiments of mice in vivo, intact bladder epithelium showed natural resistance to the adherence of type 1 fimbriated and non-fimbriated E. coli. However, the exfoliation of bladder superficial cells by trypsinization before the bacterial inoculation promoted the adhesion and colonization of type 1 fimbriated E. coli onto bladder epithelium. After colonization of E. coli, maximum numbers of E. coli and leukocytes were observed 3 days after inoculation. Nine days after inoculation, both of E. coli and leukocytes disappeared and the regeneration of superficial cells was observed. On the other hand, superficial cells in mice injected with phosphate-buffered saline or non-fimbriated E. coli regenerated 5 days after trypsinization. The present study demonstrated that the removal of superficial cells is essential for the adhesion and colonization of type 1 fimbriated E. coli onto bladder epithelium in vivo and a new model of E. coli cystitis in mice was established. The model which we established is valuable for histopathological, immunological, and therapeutic studies.

Membrane lipids of Mycoplasma orale: lipid composition and synthesis of phospholipids.

Lipid composition of Mycoplasma orale was examined and compared with that of horse serum added to the growth medium. Ratios of cholesterol/cholesterol ester and sphingomyelin/phosphatidylcholine were much higher in M. orale than in the horse serum, indicating the organism incorporates selectively cholesterol and sphingomyelin. A distinct difference between the lipids from the two sources was that in phospholipids of M. orale almost all (greater than 95%) of the fatty acyl residues were saturated whereas nearly half of the residues were unsaturated in horse serum phospholipids. Approximately one third of M. orale phospholipids was phosphatidylglycerol, which was synthesized by the organism as was demonstrated by 32P-labeling experiment. Its acyl residues consisted mainly of C16:0 and were efficiently labeled with 14C-palmitate but not with 14C-acetate. These results clearly indicate the de novo synthesis of phosphatidylglycerol by M. orale is through acylation with exogenous saturated fatty acids. On the other hand, all the phosphatidylcholine and sphingomyelin of M. orale were derived from the medium. The 14C-labeling experiment demonstrates that no fatty acid synthesis takes place nor exogenous fatty acid can be incorporated so efficiently as phosphatidylglycerol, suggesting that extremely high proportion of saturated fatty acyl residues in these phospholipids is the consequence of saturation directed to the acyl chains of the incorporated phospholipids.

Studies on osmotic stability of liposomes prepared with bacterial membrane lipids by carboxyfluorescein release.

The authors measured the osmotic stability of liposomes prepared with membrane lipids of bacteria, using the osmotic-shock release of entrapped carboxyfluorescein as an indicator. The sub-second physical changes of liposomes suspended in a solution of low osmotic pressure were examined by stopped flow spectrophotometry. The entrapped carboxyfluorescein was released when the liposomes burst on inflow of excess water. Liposomes prepared with the lipids of a stable Staphylococcus aureus L-form strain were more resistant to low osmotic pressure than those prepared from the wild strain of S. aureus, and liposomes prepared from Mycoplasma orale were even more resistant. Cardiolipin enhanced the lipid membrane stability in S. aureus and cholesterol in M. orale. The stability of lipid membranes to low osmotic pressure could be precisely determined by the present method.

A latex agglutination test for the detection of Mycoplasma pneumoniae in respiratory exudates: a comparative study with a commercially available DNA-probe test.

We prepared polyclonal antibody specific to Mycoplasma pneumoniae. Using this antibody, we developed a latex agglutination test (LAT) for detecting the organism in respiratory exudates as rapid diagnosis of M. pneumoniae infection. Further, LAT was compared with DNA-probe test (DP) which was the only commercially available test for the rapid detection of the organism. In LAT, both M. pneumoniae and M. genitalium give positive agglutination, but the titer of M. genitalium was significantly lower than that of M. pneumoniae. The detection limit of LAT was 2 x 10(5) CFU/ml and that of DP was 5 x 10(4) CFU/ml in vitro. It was considered that target molecules in LAT were accumulated in the pharyngeal portion of the patients, because of their long half-life at 37 C. However, ribosomal RNA which was target molecule in DP was destroyed at 37 C much sooner, and the accumulation could not be expected. Actually, positive rate in LAT was higher than that in DP among clinical specimens in which M. pneumoniae was detected by culture method. The procedure of LAT is much easier and more rapid than that of DP in which radioactive isotope is required. LAT could be the choice of test for rapid diagnosis of M. pneumoniae infection.

Application of an indirect immunofluorescence test for detection of Mycoplasma pneumoniae in respiratory exudates.

We prepared polyclonal antibody specific to Mycoplasma pneumoniae and examined the conditions influencing the ability of an indirect immunofluorescence test to detect the specific antigen in respiratory exudates. The antibody did not cross-react with normal human serum or with respiratory exudates from 10 healthy persons. Cross-reactivity of the antibody with species of mycoplasmas other than M. genitalium was fully diminished when absorbed with horse serum and yeast extract, components of the culture medium. Though the absorbed antibody cross-reacted with M. genitalium, the titer was significantly lower than when tested against M. pneumoniae. Two types of antigen-specific fluorescence were observed in clinical specimens: one is large or small fluorescent granular aggregates found in mucus, and the other is fine fluorescent particles diffused on the entire surface of small epithelial cells. Throat smears from 49 patients with serologically confirmed M. pneumoniae infections were examined by our indirect immunofluorescence method. Positive results were obtained in 42 cases, many of which were positive before a rise in serum antibody titer could be demonstrated, indicating that the method is useful for a preliminary diagnosis at an early stage of the infection.

The presence of cis-9,10-methylene hexadecanoic acid in Yersinia enterocolitica.

The fatty acids of Yersinia enterocolitica were investigated by Abbas and Card. In their report, they stated that the major fatty acids were C16:0, C16:1, C17:0 and C18:1 and branched or cyclopropane side-chain fatty acids could not be detected. We, however, found a moderate amount of cyclopropane side-chain fatty acid. We could determine that this fatty acid was cis-9, 10-methylene hexadecanoic acid by gas-liquid chromatography, gas chromatography-mass spectrometry, silver-nitrate-treated TLC and PtO2 catalyzing hydrogenation method.

An indirect immunofluorescence method for detection of Mycoplasma hominis in vaginal smears.

We developed a novel method for the detection of Mycoplasma hominis from vaginal swabs using an indirect immunofluorescence technique. It is a rapid and simple method that can be finished in only 5 hr and is more sensitive than the usual culture isolation method. The indirect immunofluorescence method was applied to vaginal smears from 193 healthy women and 33.7% gave a positive test. This value was much higher than that (11.4%) obtained from the same specimens by the culture method. When vaginal smears were subjected to Papanicolaou staining after the indirect immunofluorescence method, the specific immunofluorescence of the epithelial cells was located exactly at the sites of granular aggregates stained with Papanicolaou stain. A histological examination by Papanicolaou staining showed that the incidence of inflammation seems to be slightly higher in M. hominis-carriers than in non-carriers.

Adaptational changes of fatty acid composition and the physical state of membrane lipids following the change of growth temperature in Yersinia enterocolitica.

Yersinia enterocolitica is capable of growing in a broad range of temperatures from 4 to 45 C. How this organism alters its membrane lipids in response to the change of growth temperature is very interesting. The fatty acids of membrane lipids of cells cultured at 5, 15, 25 and 37 C were analyzed and the physical states of these membrane lipids were characterized. The major phospholipids of this bacterium were phosphatidylethanolamine, phosphatidylglycerol, cardiolipin, lysophosphatidylglycerol and lysophosphatidylethanolamine. No significant difference in phospholipid composition in response to culture temperatures was observed. It was reported in our previous paper that the major fatty acids of membrane phospholipids of Y. enterocolitica were C15:0, C16:0, C16:1, cyclopropane C17:0 and C18:0. Some differences in the fatty acid composition were, however, observed with the change of culture temperature. When the culture temperature was raised, the saturated and cyclopropane fatty acids substantially increased and the unsaturated ones decreased. A reverse phenomenon was observed when culture temperature was lowered. From the viewpoints of membrane physical state, adaptational changes were analyzed using a nylon microcapsule method. Phase transition in membrane lipids of cells grown at each culture temperature took place in the range of about 5 C below and about 10 C above the culture temperature. It is, therefore, considered that Y. enterocolitica maintains its membrane rigidity and fluidity in response to growth temperature by changing the membrane fatty acid composition.

[Study of finding the optimum dose of cefetamet pivoxil in complicated urinary tract infections].

To find the optimum dose of cefetamet pivoxil (CEMT-PI, Ro 15-8075), a new oral cephem, in the treatment of complicated urinary tract infections, we performed a randomized trial using cefaclor (CCL) as the control drug. The subjects were patients with complicated urinary tract infections associated with underlying urinary tract diseases. Patients with indwelling catheter were excluded from the analysis, as were patients with infection due to Pseudomonas aeruginosa. Patients were treated with 250 mg of CEMT-PI (CEMT-PI-L) 2 times a day, 500 mg of CEMT-PI (CEMT-PI-H)2 times a day or 500 mg of CCL 3 times a day for 7 days. The overall clinical efficacy was evaluated on the basis of the criteria proposed by the Japanese UTI Committee. Of the 103 patients evaluated for clinical efficacy, 36 patients received CEMT-PI-L, 37 patients received CEMT-PI-H and 30 patients received CCL. No significant difference in background characteristics was observed among the three treatment groups. The overall clinical efficacies at day 3 judgement were 67.9% in 28 patients treated with CEMT-PI-L, 60.0% in 30 patients treated with CEMT-PI-H and 72.0% in 25 patients treated with CCL, with no statistically significant difference. Those at day 7 judgement was 63.6% in 33 patients treated with CEMT-PI-L, 66.7% in 36 patients treated with CEMT-PI-H and 72.4% in 29 patients treated with CCL, with no statistically significant difference. The bacteriological eradication rates at day 3 judgement were 79.5% of 39 strains in the CEMT-PI-L group, 73.2% of 41 strains in the CEMT-PI-H group and 84.4% of 32 strains in the CCL group, with no statistically significant difference. Those at day 7 judgement was 78.7% of 47 strains in the CEMT-PI-L group, 85.5% of 55 strains in the CEMT-PI-H group and 94.9% of 39 strains in the CCL group, with no statistically significant difference. The overall clinical efficacy and bacteriological eradication rate at day 7 judgement for CEMT-PI-H were higher than those for CEMT-PI-L. Clinical adverse reactions were observed in one patient in each of the 3 groups, but no statistically significant difference was found. We have concluded that the optimum daily dose of CEMT-PI in the treatment of complicated urinary tract infection is 1,000 mg.

Overproduction of truncated subunit a of H+-ATPase causes growth inhibition of Escherichia coli.

Genes (uncB) for wild-type and mutant a subunits of Escherichia coli H+-ATPase (F0F1) were cloned into recombinant plasmids. The subunits were expressed under the control of a weak promoter of the unc operon at 30 degrees C and strong promoters of lambda phage at 42 degrees C. At 30 degrees C, the wild type and a truncated (Glu-269----end) a subunit complemented the defect of the a subunit mutant KF24A (Trp-111----end), whereas the other mutant subunits (Trp-111----end, Trp-231----end, Gln-252----end, and a subunit with a deletion of residues 21 to 227) did not. Three mutant subunits (Trp-231----end, Gln-252----end, and Glu-269----end) and the wild-type a subunit caused growth inhibition associated with cell elongation, an uneven distribution of membrane proteins, and an altered septum structure when they were expressed at 42 degrees C. These phenomena were not observed with the other mutant subunits, suggesting that overproduction of the middle region (between residues 111 and 230) of the a subunit causes growth inhibition.

[Improvement of the membrane filter technique for enumeration of enterococci in water].

The membrane filter technique with AC agar medium supplemented with 0.04% NaN3 and 0.00015% 2,3,5-triphenyltetrazolium chloride for enumeration of enterococci in water is described. An appropriate volume of a water sample was filtered through the membrane filter. The membrane filter was put on an AC agar plate (designated as the AC.MF technique), which was incubated at 37 C for 18 hr and further at 45 C for 24 hr. By this AC.MF technique, all the colonies grown on the membrane filters were identified as enterococci, and the count of enterococci obtained by the AC.MF technique was similar to that by the AC.MPN technique. The AC.MF technique may be useful for accurate and rapid enumeration of enterococci in water and serve as a simple method for determining the sanitary quality of water.

Lincomycin increases the half-life of beta-lactamase mRNA.

Escherichia coli K-12 strains isolates carrying plasmid pBR322 were grown in the presence of subinhibitory concentrations of lincomycin, which stimulated beta-lactamase synthesis about 2.5-fold, and the effects of the drug on the synthesis and degradation of bla mRNA were studied. The bla mRNA levels determined by 1-min pulse-labeling with [3H]uridine were significantly higher in a lincomycin-containing culture than in the control culture, indicating that stimulation of beta-lactamase synthesis is caused by an increase in the amount of bla mRNA. The enhancing effect of lincomycin was observed in strains harboring pBR322 delta P1 and pBR322 delta P3, which lacked the P1 or P3 promoter, respectively, as well as in the strain harboring pBR322. S1 nuclease analysis showed that the half-life of bla mRNA increased about 2.7-fold when lincomycin was present. These results indicate that the increase in beta-lactamase synthesis caused by lincomycin is due to an increase in the stability of bla mRNA rather than activation of its synthesis.

[A comparative study on lomefloxacin and norfloxacin in the treatment of acute uncomplicated cystitis].

To objectively evaluate the efficacy, safety and utility of lomefloxacin (NY-198), a new quinolone antibacterial agent, in the treatment of acute uncomplicated cystitis, a comparative double blind trial was performed using norfloxacin (NFLX) as the control drug. In both groups, the drug was orally administered after meals for 3 days in a dose of 100 mg t.i.d. and clinical efficacies were assessed on the 3rd day (the 1st assessment) according to the criteria by the UTI Committee in Japan. Subsequently, either the active drug (NY-198 or NFLX) or placebo was administered for 4 days and the 2nd assessment was performed on the 7th day. Further, in patients who showed excellent responses at the 2nd assessment, recurrence was examined on the 14th day (the 3rd assessment) and on around the 21st day (the 4th assessment) following a subsequent 7-day placebo treatment. 1. A total of 258 patients was treated, and among them clinical efficacies were evaluable in 207 patients (NY-198: 106, NFLX: 101) at the 1st assessment, and in 176 patients (NY-198-NY-198 which was the combination of the 1st and 2nd administrations: 47, NY-198-placebo: 43, NFLX-NFLX: 44, NFLX-placebo: 42). 2. In the evaluation of overall clinical efficacy by the committee at the 1st assessment, the overall efficacy rates in the NY-198 group and the NFLX group were 76.4% and 64.4% (excellent), respectively, or 100% and 99.0% (excellent and good), respectively. There was no statistically significant difference between the 2 groups. In the 1st assessment on effects of drugs on pain at micturition, pyuria and bacteriuria and on bacteriological response, no significant differences were observed between the 2 groups. 3. In the 2nd assessment by the committee, there were statistically significant differences among the 4 groups in the overall clinical efficacies and the effects on bacteriuria (P less than 0.05). No difference was observed between NY-198 group and NFLX group, but the groups administered with active drug for 7 days, i.e. NY-198-NY-198 group and NFLX-NFLX group were significantly superior to the groups administered with active drug for 3 days and placebo for subsequent 4 days, i.e. NY-198-placebo group and NFLX-placebo group (P less than 0.05). There were no differences in the effects on pain at micturition and pyuria among the 4 groups.(ABSTRACT TRUNCATED AT 400 WORDS)

Determination of hydrophobicity on bacterial surfaces by nonionic surfactants.

The hydrophobicity of the bacterial cell surface was determined by using nonionic surfactants. The method is based on the adsorption of nonionic surfactants at the hydrophobic sites of the cell surface. Among many nonionic surfactants, C18H37O(CH2CH2O)13H was preferred. The surfactant was added in excess to a bacterial suspension, and the suspension was mixed by sonication or mechanical stirring. The amount of surfactant remaining in the supernatant after centrifugation was determined spectrophotometrically by measuring the absorbance of tetrabromophenolphthalein ethylester. Effective dispersion of bacterial cells such as Staphylococcus aureus and Mycobacterium smegmatis was achieved by sonication in the presence of the nonionic surfactant. Adsorption measurements coincided with Langmuir's equation, indicative of monolayer adsorption. The method is useful for the determination of the hydrophobicity of various bacterial cell surfaces.

The effect of head group structure on phase transition of phospholipid membranes as determined by differential scanning calorimetry.

The phase transition characteristics of cardiolipin and phosphatidylglycerol suspensions were investigated by differential scanning calorimetry. The phase transition temperatures for dimyristoylphosphatidylglycerol, tetramyristoylcardiolipin, dipalmitoylphosphatidylglycerol and tetrapalmitoylcardiolipin were 25.0, 47.0, 40.5 and 62.2 degrees C, respectively. The phase transition temperature for a mixture of two analogous phospholipids was higher than that for phosphatidylglycerol alone, but lower than that for cardiolipin alone. It increased along with cardiolipin content. The phase transition temperature for cardiolipin was increased in the presence of divalent cations, particularly Ca2+. The results indicate that the head group of cardiolipin by itself can increase the phase transition temperature.

Characterization of a glycophospholipid of Streptococcus pyogenes-derived L-form.

A glycophospholipid was detected as a major lipid in the L-form derived from Streptococcus pyogenes. The glycophospholipid was purified and chemically analyzed. From its phosphorus:glucose:glycerol molar ratio of 1:2:2 and mass spectrum, the deacylated glycophospholipid was identified as glycerophosphoryldiglucosylglycerol. This structural assignment was confirmed by thin layer chromatography of the lipid itself, which comigrated with known phosphatidyldiglucosyldiglyceride from S. faecalis. A possible mechanism for biosynthesis of the glycophospholipid in the L-form that invokes known biosynthetic processes involved in glycophospholipid metabolism in related bacteria is proposed.