Basic studies on group B streptococcal (GBS) culture medium.

Yeast extract and Serum are said to be necessary for the pigmentation of Group B Streptococcus (hereafter, GBS). We're examined the necessity of these for the GBS pigmentation. Firstly, we examined the following four types of culture media. (1) The medium in which serum was excluded from the New Granada medium (hereafter, called Basic medium). (2) New Granada medium (hereafter, S medium). (3) The medium in which Yeast extract was added to (1) (hereafter, Y medium). (4) The medium in which added Serum and Yeast extract were added to (1) (hereafter, SY medium). As for GBS tested (7 serotypes, 27 strains), Basic medium showed the strongest pigmentation potency, but, S and SY medium showed the weakest. About the quickness of pigmentation initiation, similar to the potency above, Basic medium was the quickest, followed by Y medium, and then S medium and SY medium. Among the media examined, there were no differences in the relationships between the quantity of GBS applied and the positive ratio of each media.>From these results, among four types of the media, Basic medium is thought to be the best one for the GBS detection.

A 7.6kb DNA region from Streptomyces kasugaensis M338-M1 includes some genes responsible for kasugamycin biosynthesis.

A 7.6kb PstI-KpnI DNA fragment including a sequence highly similar to kasugamycin acetyltransferase gene (kac) was isolated from Streptomyces kasugaensis M338-M1 and sequenced. Nine open reading frames (ORFs), designated as ORF A, B, C, D, E, F, G, H and I, were recognized in this region, although ORF A was incomplete. ORF G runs in the opposite direction to the others. The amino acid sequence deduced from ORF H showed 98% similarity to that of the kasugamycin acetyltransferase from S. kasugaensis MB273-C4, another kasugamycin (KSM) producer. Transformation of E. coli JM109 with ORF H made the strain highly resistant to KSM. The deduced amino acid sequences of the ORF A, C and D products were similar, respectively, to glucosyltransferase I from E. coli (26%), beta-alanine: pyruvate transaminase from Pseudomonas putida (32%) and dTDP-D-glucose 4,6-dehydratase (StrE) from Streptomyces griseus (37%). The strE-like ORF (ORF D) seems to be the gene responsible for formation of the 6-deoxy structure of the kasugamine moiety. ORF A and ORF C are also likely to have roles in KSM biosynthesis. Taken together, our analyses strongly suggest that this DNA region includes at least a part of the gene cluster of KSM biosynthesis.

How to decrease false-positive cases of ovarian cancer screening by transvaginal sonography.

Screening for ovarian cancer is an important step in improving ovarian cancer mortality. This is a report of screening for ovarian cancer using transvaginal sonography (TVS) on 2,095 asymptomatic women during the past four years. Ovarian tumors over 30 mm in diameter were detected in 87 women (4.2%). Fourteen tumors were morphologically classified as complex type and 73 as cystic type. Only 12 tumors were palpable on bimanual vaginal examination. Seven women with complex type tumors underwent laparotomy and their pathological diagnoses revealed 3 dermoid cysts, 3 endometrioid cysts and 1 ovarian cancer. Two women with cystic type tumors underwent laparotomy and their pathological diagnoses revealed 1 paraovarian cyst and 1 endometrioid cyst. Many ovarian tumors were detected at prolonged cycle of menstruation and 69% of them disappeared in repeated TVS. Sixty percent of ovarian cystic tumors under 50 mm in diameter also disappeared in repeated TVS. Close attention to menstrual cycle and morphologic findings is needed to decrease false-positive cases in TVS screening for ovarian cancer.

Establishment and characterization of acquired resistance to platinum anticancer drugs in human ovarian carcinoma cells.

To investigate differences in resistance to cis-diamminedichloroplatinum(II) (CDDP) and diammine (1,1-cyclobutanecarboxylato)platinum(II) (CBDCA), and their newly developed derivative, ((-)-(R)-2-aminomethylpyrrolidine (1,1-cyclobutanedicarboxylato)platinum (II) (DWA2114R), four types of resistant cell lines were established from a parental cell line (NOS2) of a serous cystadenocarcinoma of the ovary. The cross-resistance of CDDP-resistant cells (NOS2CR1 and NOS2CR2) to DWA2114R was slight (only 25% of the resistance to CDDP), and no cross-resistance was observed to anticancer drugs other than the CDDP derivative, except to camptothecin (CPT-11) in the case of NOS2CR2 cells. The cross-resistance of CBDCA-resistant cells (NOS2CBR) to DWA2114R was slight (only about 1/3 of the resistance to CBDCA), and no cross-resistance was observed among anticancer drugs other than the CDDP derivative. On the other hand, DWA2114R-resistant cells (NOS2DR) showed a high cross-resistance to CDDP, CBDCA, etoposide (VP-16), and CPT-11. Intracellular accumulations of CDDP and CBDCA were markedly reduced in NOS2CR1, NOS2CR2, and NOS2CBR cells compared to those in NOS2 cells, but were reduced only slightly in NOS2DR cells. Intracellular accumulation of DWA2114R was reduced somewhat in the four types of resistant cells. Glutathione S-transferase activity was not increased in any of the four types of resistant cells, and intracellular GSH concentration was increased only in NOS2CR2 cells (by 2.6 fold). From these results, we consider that the resistance mechanisms against CDDP and CDBCA are similar, and reduction of intracellular drug accumulations is a significant factor. Resistance to DWA2114R differed from resistance to CDDP and CBDCA in both cross-resistance spectrum and resistance mechanism, indicating that reduction in intracellular drug accumulation is not the major resistance mechanism.

Control of drug release with a combination of prodrug and polymer matrix: antitumor activity and release profiles of 2',3'-diacyl-5-fluoro-2'-deoxyuridine from poly(3-hydroxybutyrate) microspheres.

Drug release was controlled by a combination of prodrug and polymer matrix. Prodrugs of 5-fluoro-2'-deoxyuridine with different physicochemical properties were synthesized by esterification with aliphatic acids (propionate, n-butyrate, and n-pentanoate). Microspheres containing these ester prodrugs were prepared with poly(3-hydroxybutyrate) of three molecular weights (65,000, 135,000, and 450,000). The release rates from the spheres depended on both the lipophilicity of the prodrug and the molecular weight of the polymer. Regardless of the polymer, the relative release rates were propionate greater than butyrate greater than pentanoate. The release of butyrate and pentanoate from the spheres consisting of low-molecular-weight polymer (M(r), 65,000) was faster than that from the spheres of higher molecular weight (M(r), 135,000 or 450,000). A single intraperitoneal injection of spheres of the highest molecular weight polymer containing butyrate or pentanoate resulted in higher antitumor effects against P388 leukemia in mice than did free prodrugs given over a period of five consecutive days. The polymer sphere itself showed low toxicity to and good biocompatibility with mice and rats.

Immunoblotting analysis of sera from patients with candidal vaginitis and healthy females.

Antigenic components of Candida albicans were extracted from whole cells with a buffer containing SDS and 2-mercaptoethanol, and separated by SDS-polyacrylamide gel electrophoresis. The components reactive with IgG, IgA, IgM and IgE antibodies in sera from patients with (14 subjects) and without (15 subjects) C. albicans in the vagina, and from healthy females (34 subjects), were investigated by immunoblotting using immunoglobulin class-specific antibodies. Many components reacted with IgG and IgA in all sera tested; the major antigens that reacted strongly with the sera were 67, 62, 29 and 25 kDa components. Several components were observed which reacted with IgM in 63% of the sera; the 67, 62 and 25 kDa components that reacted with IgG and IgA also reacted with IgM. No components reacting with IgE were detected in any of the sera. No striking differences in antibody binding profiles to whole cell antigens were detected among the C. albicans positive and negative patients or the healthy subjects. On the other hand, IgG against extracellular proteinase was more frequently detected in the C. albicans positive patients than in the C. albicans negative group or the healthy subjects. This may suggest that vaginal infection with C. albicans contributes to a rise in anti-proteinase antibody levels.

Electrophoretic karyotypes of clinically isolated yeasts of Candida albicans and C. glabrata.

One-hundred-and-four isolates of yeast were collected from the vaginas of 97 outpatients. The isolates were identified by their characteristics in a carbohydrate assimilation test, a serological test and from their morphology. Candida albicans and Candida glabrata were the major isolates (75% and 20%, respectively). The karyotypes of the isolates were analysed by pulsed-field gel electrophoresis and almost all the karyotypes were distinguishable from one another when the band mobilities were carefully compared. Characteristics and karyotypes were not directly correlated, but seven C. albicans isolates (from six patients) had a common atypical karyotype and shared the same phenotype. These isolates are inferred to be generated by a wide genomic reorganization and mutation and the phenotypic changes may be advantageous for survival. The karyotypes of the isolates recovered from individual patients after intervals of 1-6 months were all identical except for one or two highly variable bands which were identified with an rDNA probe. This suggests that the variable bands are too variable to be useful for distinguishing strains, but from the patterns of the identical bands (i.e. except for the variable bands) we concluded that strains from individual patients do not change, at least over short periods. This, coupled with the extensive inter-isolate variability in karyotype, will be useful for Candida source determination and epidemiological studies.

Identification of saliva stains by determination of the specific activity of amylase.

The specific activity (enzyme activity/protein concentration) of amylase was determined for the identification of saliva stains. The specific activity of amylase in saliva stains rapidly decreased during the first hour but, from 1 to 28 days, this decrease was much less when the stains were kept at room temperature. Stains of various human biological materials, breast milk, nasal secretion, meconium and vaginal secretion showed comparatively high amylase activity, but the saliva stains could be differentiated by their high specific activity of amylase, over 2 I.U./mg. When saliva stains were contaminated with blood or vaginal secretions at various ratios, the specific activity of amylase decreased with increase in the ratio of contaminant, especially when the contaminant was blood. However, the specific activity of amylase was still higher than 2 I.U./mg even after one fifth volume of blood was added or after five volumes of the extract of the stains of vaginal secretions were added.

A sensitive enzyme-linked immunosorbent assay used to differentiate type AB specimen from a mixture of type A and type B specimens.

A sensitive enzyme-linked immunosorbent assay (ELISA) for detection of ABO blood substances in human body fluids was devised. The ELISA plates coated with purified human anti-A or anti-B serum effectively captured the blood substances, and these were then analysed by the combination of rabbit anti-A and goat anti-B. This capture ELISA could differentiate the type AB specimen from a mixture of the type A and the type B specimens, and the method was applied to rape cases to make the ABO typing of the criminal.

[Bacteriological and clinical evaluation of cefoxitin in the treatment of infections in 39 obstetric and gynecological patients].

A total of 39 patients was admitted to the obstetric and gynecological wards of Nagoya University Hospital and the University's 5 related hospitals during the period from July 1981 to March 1982. The efficacy of cefoxitin sodium was evaluated in the 39 patients with a wide variety of obstetric and gynecological infections. The findings are the follows: Of the 39 patients, the responses in 7 were judged "excellent", 28 "good" and 4 "poor" with the clinical efficacy rate of 89.7%. The causative pathogens in 23 of the cases were isolated from clinical materials submitted to the Institute of Anaerobic Bacteriology, School of Medicine, Gifu University. In these 23 cases, it was found that 8 were due to aerobic bacteria only (34.8%), 8 mixed aerobic-anaerobic bacteria (34.8%) and the remaining 6 anaerobic bacteria only (26.1%), i.e. a substantial percentage (60.9%) was due to anaerobic bacteria or mixed aerobic-anaerobic bacteria. Cefoxitin was active against most strains of Bacteroides spp. and was not affected by the inoculum size. On the other hand, most strains of Bacteroides spp. were resistant to cefazolin when the inoculum size was increased from 10(6)/ml to 10(8)/ml. Lincomycin was also found to be active against many fo the strains of Bacteroides spp. Cefoxitin and cefazolin were fairly active against anaerobic bacteria other than Bacteroides spp., while these same bacteria were resistant to sulbenicillin and many of them showed resistance to lincomycin. Cefoxitin was well tolerated and no untoward side effects were observed. The clinical and bacteriological results indicate that cefoxitin is a very effective antibiotic in the treatment of a wide variety of obstetric and gynecological infections and is a suitable choice as initial antibiotic therapy in these conditions.

Three-day clotrimazole therapy for vulvo-vaginal mycosis.

A self-medication therapy with clotrimazole vaginal tablets, application of two tablets at bedtime on 3 consecutive days, was carried out to find out whether vulvo-vaginal mycoses can be treated more effectively and with fewer drop-outs when treatment is simpler and shorter. Our study included forty-one patients, seventeen of whom were in their second or third trimester of pregnancy. There was no significant difference between the efficacy rates in the pregnant (82.4%) and non-pregnant (83.3%) women. In 36 infections caused by various Candida species the cure rate was 91.7%; in 8 infections due to Torulopsis glabrata it was 75%. For thirty-three women with primary infections the cure rate was 84.8% and for eight women with recurrent infections it was 75%. The cure rate for the entire group of patients was 82.9% (thirty-four out of forty-one cases). Failures were seen in six out of thirty cases, i.e. in 16.7%. In one case burning was experienced after the insertion of the vaginal tablet so that treatment was discontinued.

[Experimental and clinical studies of cefmenoxime in the field of obstetrics and gynecology].

The study group was organized to evaluate the usefulness of cefmenoxime (CMX) injection, a new synthetic cephalosporin, for the treatment of infections in the field of obstetrics and gynecology. Fundamental and clinical studies were made by the society and the following results were obtained. 1. The peak distribution of CMX's MIC for E. coli, Klebsiella sp., Enterobacter sp., Bacteroides sp. and Peptococcus sp. isolated from obstetrical and gynecological infections with relatively high frequencies area 0.1, less than or equal to 0.05, 0.2, 3.13, 1.56 micrograms/ml, respectively, with an inoculation of 10(6) cells/ml. 2. When 1 g of CMX is administered by intravenous drip infusion for 1 hour, the maximum concentrations in various tissues of female genital organs were as follows: 14.2 and 13.2 micrograms/g in ovary and oviduct, respectively, at 1.20 hours after the start of administration, and 16.9 and 26.3 micrograms/g in corpus uteri and cervix uteri, respectively, after 1 hour. As for the transfer to the exudate in the pelvic dead cavity, the peak concentration was 15.6 micrograms/ml after 2.13 hours. 3. In the clinical studies, CMX was given to 258 cases with female genital organ infections and others. As for the clinical effects, with exclusion of 3 cases in which other antibiotics are concomitantly used, responses were excellent in 76 cases, good in 162 cases and poor in 17 cases, among 255 cases in total. The efficacy rate was 93.3%. The efficacy rates by diseases were 97.1% (68/70) for intrauterine infections, 88.8% (79/89) for intrapelvic infections, 98.4% (62/63) for adnexitis, and 100% (23/23) for infections of external genital organs. As for the clinical effects on causative bacteria, the efficacy rates were 100% (19/19) for single infections due to Gram-positive bacteria, 94.8% (55/58) for single infections due to Gram-negative bacteria, and 88.2% (15/17) for single infections due to anaerobic bacteria. And its efficacy rates were 89.6% (69/77) for mixed infection cases. Side effects were observed in 2 cases (0.8%); 1 case with eruption, and 1 case with diarrhea and vomiting. As for abnormal laboratory findings, lower white blood cell count was observed in 2 cases and elevation of the values regarding hepatic functions in 9 cases. All cases were returned to the normal after the completion of the administration. Cefmenoxime showed a satisfactory clinical efficacy and a potent bacteriological effect in treatment of the infections in the field of obstetrics and gynecology, and it has been concluded that cefmenoxime will be useful addition to the antibiotics for the therapy of these infections.

[Vaginal fungi in toxemia of pregnancy (author's transl)].

In order to prevent the transfer of the fungi in maternal vagina into a neonate through transvaginal delivery, we examined the presence of vaginal fungi in the patients with toxemia of pregnancy occurring chiefly from the later stages of pregnancy and made an analysis from the quantitative aspect of fungi (number of colonies produced on Mizuno-Takada medium). The results obtained are as follows: 1) The detection rate of vaginal fungi was evidently so high as 37.1% in toxemia group compared with 27.6% in non-toxemia group (p less than 0.01). And the detection rate of vaginal Candida albicans (hereinafter: C. albicans) also proved to be higher tendency in toxemia group. 2) When the toxemia patients were divided into mild cases and severe ones for comparison, the detection rate of vaginal fungi and that of vaginal C. albicans were both higher in the severe case group. Particularly in the group which had the symptoms of toxemia at the examination time of vaginal fungi there was seen the higher rate. 3) From the quantitative aspect of vaginal fungi it is evident that there were more cases with over 51 colonies in the group showing the symptoms of toxemia at the time of fungi examination than in the group showing no such symptoms at the examination and the group of non-toxemia (p less than 0.01).