Stimulatory effect of menaquinone-7 (vitamin K2) on osteoblastic bone formation in vitro.

Menaquinone-7, which is vitamin K2 (menatetrenone) with seven isoprene units, is highly contained in the fermented soybean. The effect of menaquinone-7 (MK-7) on osteoblastic bone formation was investigated. Femoral-diaphyseal and metaphyseal tissues of young male rats (4 weeks old) were cultured for 48 h in a medium containing either vehicle or MK-7 (10(-7)-10(-5) M). Calcium content, alkaline phosphatase activity, and deoxyribonuclic acid (DNA) content in the diaphyseal and metaphyseal tissues was significantly increased in the presence of MK-7 (10(-6) and 10(-5) M). The effect of MK-7 in increasing the diaphyseal and metaphyseal calcium content and alkaline phosphatase activity was completely prevented in the presence of cycloheximide (10(-6) M), an inhibitor of protein synthesis. Moreover, osteoblastic MC3T3-E1 cells after subculture were cultured for 24 h in a serum-free medium containing MK-7 (10(-7)-10(-5) M). Protein content, alkaline phophatase activity, osteocalcin and DNA content in the cells was significantly increased in the presence of MK-7 (10(-6) and 10(-5) M). The effect of MK-7 in increasing protein content, alkaline phosphatase activity, and osteocalcin production in the cells was completely blocked by cycloheximide. This study demonstrates that MK-7 has an anabolic effect on bone tissue and osteoblastic MC3T3-E1 cells in vitro, suggesting that the compound can stimulate osteoblastic bone formation.

Therapeutic effects of cefluprenam (CFLP) on polymicrobial infections associated with Enterococcus faecalis in rat pyometra model.

Enterococcus faecalis plays an important role as one of the pathogens in polymicrobial infections. We evaluated the efficacy of cefluprenam (CFLP) using a polymicrobial pyometra of a model rat. Rats were infected with a mixed intrauterine inoculation of E. faecalis plus either Bacteroides fragilis or Prevotella bivia (minimal inhibitory concentration of CFLP: E. faecalis, 3.13 mug/ml; B. fragilis, 3.13 mug/ml; P. bivia, 3.13 mug/ml). Immediately after inoculating 10(5) cfu/rat of each organism, CFLP (either 40 mg/kg, i.v., q.i.d. for 5 days or 80 mg/kg, i.v., b.i.d. for 5 days) was administered. The intrauterine inflammatory change and bacterial count in the treated group were compared with those in the non-treated control group. CFLP significantly (P < 0.01) decreased the bacterial counts except for B. fragilis in the regimen of 80 mg/kg, b.i.d. However, the regimen of 40 mg/kg, q.i.d. significantly (P < 0.05) reduced the bacterial counts more than did that of 80 mg/kg, b.i.d. CFLP proved to demonstrate a good tissue concentration above 3 mug/g for 1 h. These results suggest that CFLP in a more divided dose is efficacious for the treatment of polymicrobial infections associated with E. faecalis in pyometra.

Resolution and characterization of polymorphic DNA by SSCP and chemical cleavage methodologies.

A variety of techniques have been developed to detect single-base changes for the two different purposes. One is the detection of mutational events without phenotypic selection, and another is the rapid and conventional identification of mutations such as the specific base changes related to activation of oncogene, genetic diseases, etc. In this study, the utility of the two methods, single strand conformation polymorphism (SSCP) and chemical cleavage, was explored using 13 E. coli lacI- mutations cloned onto M13 phase. The 167 base region encompassing mutations was amplified by PCR as dsDNA. Following denaturation, these PCR products were analyzed by non-denaturing polyacrylamide gel electrophoresis (SSCP) and the separation of the ssDNA fragment carrying the altered sequence from the original sequence was found to be dependent on the location and type of the change. Hetero duplexes of changed/original sequences were also prepared by hybridization of the above PCR products. Mismatched C and T bases were modified by hydroxylamine and osmium tetroxide, respectively, and subsequently treated with piperidine to analyze the cleaved DNA fragments on a polyacrylamide gel (Chemical Cleavage). The cleavage efficiency was also found to be influenced by the type of mismatch and its surrounding sequence. Such observed characteristics should contribute to a better appreciation for these types of mutational systems, which in turn should lead to insight into the mechanisms of mutagenesis.

[The effects of stimulus duration, component duration, and schedule of stimulus presentation on the observing response in rats].

Seven rats were exposed to a procedure where responses on an observing lever (observing responses) changed the schedule on a food lever from a mixed schedule to a schedule with a discriminative or uncorrelated stimulus for a fixed duration: Observing responses produced a stimulus associated with a variable-ratio 50 (S+) or a stimulus associated with extinction (S-) in the S+/S- condition, an S- in the S- only condition, and an S+ in the S+ only condition, while they produced two uncorrelated stimuli in the uncorrelated condition. Experiment 1 employed two stimulus durations (30 or 6 s) and two schedules of stimulus presentation (a variable-interval 30 s or a fixed ratio 1), and examined observing response rates in the S+/S-, S- only, S+ only, and uncorrelated conditions. Experiment 2 employed two component durations (80 or 300 s), and examined observing response rates in the S+/S- and S- only conditions. In both experiments, rates of observing responses were maintained in both S+/S- and S+ only conditions, but decreased in the S- only and uncorrelated conditions. These results indicate that an S- is not a conditioned reinforcer irrespective of manipulated variables.

Effects of discrete-trial and free-operant procedures on the acquisition and maintenance of successive discrimination in rats.

Rats were trained on a successive discrete-trial discrimination between two tonal stimuli to examine the effects of availability of a lever during intertrial intervals. In the discrete-trial condition, in which a lever was removed from the chamber during intertrial intervals, 10-s trials were initiated by the presentation of both discriminative stimulus and lever. In the free-operant condition, in which a lever was present during both trials and intertrial intervals, 10-s trials were initiated only by the presentation of a discriminative stimulus. Experiment 1 employed 50-s intertrial intervals and demonstrated that discriminative performances were acquired faster and maintained better in the free-operant conditions than in the discrete-trial conditions. Experiment 2 employed 5-s intertrial intervals and showed that poor discriminative performances in the discrete-trial conditions were improved. These results indicate that the presentation of a lever to start a trial can overshadow or mask the control by a discriminative stimulus and thereby obstruct the acquisition and maintenance of discriminative performances. Furthermore, the overshadowing or masking effects are strengthened as a function of the duration of intertrial intervals.

[Pharmacokinetic and clinical studies on ceftriaxone in the field of obstetrics and gynecology].

Ceftriaxone (CTRX) was studied regarding its penetration into the adnexa uteri and uterine tissues, as well as its utility and safety in the treatment of patients with obstetric and gynecologic infections. The results obtained are summarized below. 1. When 1 g of CTRX was administered by intravenous bolus injection, Cmax in tissues of adnexa uteri and uterus ranged from 42.2 to 80.5 micrograms/g, Tmax ranged from 0.42 to 0.81 hour, and the AUC ranged from 314.9 to 606.9 micrograms.hr/g. Thus, drug penetration into these tissues was good. 2. Clinical efficacy of CTRX was evaluated in 29 obstetric and gynecological patients. The clinical efficacy was good in all cases. 3. Bacteriological effects of CTRX were very good, and 90% of the organisms isolated before treatment were eradicated. 4. Laboratory testing revealed an occurrence of mild eosinophilia in 1 case.

[Pharmacokinetic, bacteriological and clinical studies on cefuzonam in the field of obstetrics and gynecology].

The distribution of cefuzonam (CZON, L-105) into the uterus and uterine adnexa was investigated and the usefulness and the safety of CZON in obstetric and gynecological infections were studied. The results are summarized as follows: 1. Following one shot intravenous injection of CZON 1 g, a good distribution of the drug into tissues of uterus and uterine adnexa was observed, with Cmax values of 15.7-33.9 micrograms/g, Tmax of 7.3-34.0 minutes and AUC values of 18.7-35.3 micrograms X hr/g. 2. In all of the 30 cases of obstetric and gynecological infections treated, CZON was evaluated effective. 3. Bacteriologically, 93.9% of total bacteria that had been isolated were eliminated by the administration of the drug. 4. Against all the strains of bacteria isolated before the treatment and replaced bacteria, MIC50, MIC80 and MIC90 of CZON were 0.20 micrograms/ml, 12.5 micrograms/ml and 25 micrograms/ml, respectively, showing low values. 5. Subjective and objective findings and clinical laboratory test values during and after the trial showed no side effects associated with CZON.

[Experimental study of the mechanism of peritoneal dissemination--with special reference to scanning electron microscopic observations].

Most malignant ovarian tumors metastasize by peritoneal dissemination and have a poor prognosis. Few studies have been made to determine how free ovarian tumor cells act on the peritoneum and become lodged and proliferate therein and much still remains to be clarified concerning this issue. In the present study, we transplanted 2 X 10(6) cells of JOHYL-1 strain established from an atypical dysgerminoma into the peritoneal cavity of nude mice observed serially morphological changes in the peritoneum resulting from tumor cell dissemination both by light microscopy and SEM. The findings thus obtained may be summarized as follows: From 5 to 7 days after intraperitoneal transplantation of tumor cells, mesothelial cells of the peritoneum began to swell, with the intercellular boundaries becoming distinct. Microvilli of the mesothelial cells increased in number, forming a mesh-like structure, and in some places were found to be in direct contact with a tumor cell. From 10 days after tumor cell transplantation onwards the mesothelium showed enlarged intercellular spaces in some places, and at these sites tumor cells were seen to have adhered. On other sites mesothelial cells were being partially lost. From 11 days after being transplanted, the tumor cells started proliferating and infiltrating in the muscle layer. Concerning the site of tumor cell implantation, evidence was obtained showing that tumor cells adhered to intercellular spaces of mesothelial cells, and/or in defects formed on connective tissue by a loss of mesothelial cells. The study thus demonstrated a salient usefulness of the JOHYL-1 ascites type cell line, with a reliably great capacity for growth in vivo, as an experimental model for the study of peritoneal dissemination of tumor cells.

[Pharmacokinetic and clinical studies of imipenem/cilastatin sodium in the field of obstetrics and gynecology].

Imipenem/cilastatin sodium (MK-0787/MK-0791) was studied for its penetration into the adnexa uteri and uterine tissue, as well as for its clinical efficacy in the treatment of patients with obstetric and gynecologic infections. The following results were obtained. When 500 mg/500 mg of MK-0787/MK-0791 was administered by an intravenous drip infusion, peak levels of MK-0787 in tissues of adnexa uteri and uterus ranged from 14.6 micrograms/g to 25.8 micrograms/g, Tmax ranged from 0.55 hour to 0.98 hour, and the AUC ranged from 25.6 micrograms X hr/g to 45.2 micrograms X hr/g. Thus, the penetration of the drug into these tissues was good. Clinical efficacy of MK-0787 was evaluated in 30 patients in the field of obstetrics and gynecology. The clinical efficacy was excellent or good in all patients. Bacteriological effects of MK-0787/MK-0791 were very good, and 90% of the organisms detected before the treatment were eradicated. The antimicrobial activity of MK-0787 was tested against pathogens isolated before, during and after the treatment. Mean MIC80 values of MK-0787 were 0.39-0.78 micrograms/ml against all Gram-positive bacteria, 0.20-0.39 micrograms/ml against all Gram-negative bacteria, and less than or equal to 0.10-0.20 micrograms/ml against all anaerobic bacteria. The antimicrobial activity of MK-0787 appeared very good. No side effects or abnormal laboratory findings were observed except a slight elevation of S-GPT in 1 patient.

[Biochemical activity in peritoneal effusion of ovarian malignancy].

This study was provided biochemical findings on ascitic fluid following the treatment of patients with ovarian tumors. The results were as follows: LDH and LDH isozyme. The ascitic fluid from patients with germ cell tumors showed an LDH1 value of 38.8% with an H/M ratio of 2.82, which were significantly higher than those found in the benign tumor group (LDH1, 15.2%: H/M ratio, 1.05). In cases of adenocarcinomas, the LDH5 value was 27.2%, compared to 15.7% in cases of benign tumors. Of 28 patients with adenocarcinoma, 15 patients (53.6%) were positive for this elevation. The decline in total LDH activity and elevation of H/M ratio were observed as a general tendency in patients satisfactorily responding to treatment. ALP and ALP isozyme. The malignant tumor group displayed higher values for both total ALP activity and HSAP in ascitic fluid, as compared with the benign tumor group. In the malignant tumor group, alpha 2 and alpha 2-beta regions were prominent in the isozyme pattern and the isozymes of the same regions remained even after inactivation by heat. The total activity decreased significantly in the responders to treatment. TP, alpha 1-AT, alpha 1-AG, alpha 2-M and IgG. The ascitic fluid levels of alpha 1-AT, alpha 2-M and IgG did not significantly differ among the group of patients with germ cell tumors, the group of patients with ovarian gonadal stroma and the group of patients with benign tumors. In the adenocarcinoma group the alpha 1-AT and alpha 2-M levels were higher than those in the benign tumor group. The ascitic fluid alpha 1-AG level and alpha 1-AG/TP ratio were elevated in tumors of germ cell origin and in adenocarcinomas; they tended to correlate with the degree of malignancy of the tumor. The results of the present study suggest that simultaneous measurement of the ascitic fluid levels of LDH isozymes, ALP isozymes, alpha 1-AG, alpha 1-AT, alpha 2-M and IgG may play a potential adjunctive role in providing information for diagnostic differentiation between benign and malignancy, estimation of the histologic type and extent of neoplastic growth, evaluation of the therapeutic response and prediction of the clinical progress.

[Radiation-induced differential responses of uterine adeno- and squamous cell carcinoma spheroids in dose-survival relationships].

Radiation-induced dose-survival relationships of human uterine adeno and squamous carcinoma cells grown as spheroid were studied, comparing them with monolayer cells. HEC-59 and SKG-3a cell lines were used. The spheroids were produced by Yuhas's method. In monolayer culture, D0,Dq and n values in the Hit-theory were as follows: HEC-59 (D0 = 1.6Gy, Dq = 0.6Gy, n = 1.5) and SKG-3a(D0 = 1.3Gy, Dq = 0.4Gy, n = 1.4). Survival curves of both cell lines as spheroids demonstrated biphasic curves after acute irradiation. HEC-59 cells grown as spheroids 300 microns in diameter did not have a great second component. SKG-3a cell, however, did apparently have a large amount of the second component when a 6Gy dose was exceeded. The shoulder in the dose-survival curves of both cells grown as spheroids became greater than monolayer cells up to 3Gy dose. It was therefore proven that spheroids possess similar characteristics to solid tumors. This suggests that spheroids are useful in studying radiation-induced effects in solid tumors.

Effects of Ca2+ and calmodulin on contraction of chemically skinned muscle fibers from pregnant rat uteri.

The effects of Ca2+ and calmodulin on contraction of saponin-treated (chemically skinned) uterine smooth muscle fibers of pregnant rats were examined. Ca2+ sensitivity, defined as the pCa required for half maximum activation of force production, was found to change with the progress of pregnancy; low in the early and middle stages and high in the later stages of pregnancy. The overall change of Ca2+ sensitivity was about pCa 1.5 during the period of pregnancy. The effect of calmodulin on contraction was also found to be dependent on the stages of pregnancy. Calmodulin was effective on the augmentation of the tension rather than the change in Ca2+ sensitivity, and this augmentation was large in the early and middle stages of pregnancy. The amount of calmodulin, which eluted out of uterine muscle cells during saponin treatment, was large in the early and middle stages of pregnancy. The results indicate that the contractile response of the uterine muscle cells during the period of pregnancy seems to be controlled by both the changes in Ca2+ sensitivity and in the amount of free calmodulin in uterine muscle cells.

Effects of sexual steroid hormones treatment on Ca2+ sensitivity of chemically skinned uterine muscle fibers from ovariectomized rats.

We considered the possible relationship between the serum levels of progesterone and estradiol-17 beta, which were controlled to that of several stages in normal pregnant rats, and the Ca2+ sensitivity, defined as the pCa required for half maximum activation of force production, of chemically skinned uterine muscle fibers from ovariectomized rats. The Ca2+ sensitivity was negligibly influenced by the levels of these hormones.

[Preclinical and clinical studies on aztreonam in the field of obstetrics and gynecology].

Penetration of aztreonam (AZT) into the uterus and the adnexal tissues and usefulness and safety of AZT for obstetric and gynecologic infections were studied with the following results. By one shot intravenous injection of AZT 1 g, the uterus and the adnexal tissues showed favorable penetration with Cmax 27.0-48.5 micrograms/g, AUC 29.4-84.9 micrograms X hr/g and Tmax 0.10-0.44 hours. MIC50, MIC80 and MIC90 of AZT for Gram-negative bacteria measured prior to administration were very low being 0.10 micrograms/ml, 0.20 micrograms/ml and 1.56 micrograms/ml, respectively. Clinical effect of AZT for 30 infection cases in obstetrics and gynecology was evaluated according to an overall efficacy criteria resulting in "good" for all the cases. With regard to microbiological effect, 90.9% of the pathogens isolated prior to the administration were eliminated by AZT. During and after the administration of AZT, side effect due seemingly to AZT was not observed in subjective and objective symptoms and laboratory values.

[Clinical trial of cefpiramide in the gynecological field].

Cefpiramide (CPM) was administrated intramuscularly to 27 cases of gynecological infections to evaluate its clinical efficacy and safety and the following results wee obtained. CPM was effective to all the cases of gynecological infections, and excellent was seen in 14 cases and good was seen in 13 cases. CPM eliminated 75% of clinical isolates. Laboratory tests were performed to blood samples and functions of liver and kidney before and after CPM treatment. Elevation of GPT was observed in 2 cases but required no treatment.

[A study on factors influencing survival in stage I ovarian cancer].

Early diagnosis is difficult in ovarian cancer and the prognosis is unfavorable even in stage I case. A total of 131 cases with stage I ovarian carcinoma were treated at our institution, 73 were stage Ia, 13 were stage Ib, 45 were stage Ic and 39 were LPM. To define the criteria for selection of therapy, we retrospectively reviewed the stage I ovarian cancer. The results were as follows: The best survival rate was demonstrated in 96% of LPM cases followed by 73% of endometrioid adeno-carcinoma. Cases with rupture of capsule and with ascites at surgery influenced the 5 year survival rate. We observed no difference in survival rate between the conservative operation (unilateral salpingo-oophorectomy) group and the complete operation (bilateral salpingo-oophorectomy and total hysterectomy) group. Those who had received postoperative chemotherapy showed the longest survival rate of 86%. Evaluation of deaths; 9 of 21 with stage Ic died within 12 months after surgery, and 4 of 7 with stage Iai between 1 and 3 years. The analysis has shown the importance of adequate postoperative treatment and of strict follow-up to guard against recurrence of malignancy in patients with ruptured tumor capsules as well as in those with ascites.

[Fundamental and clinical studies on cefpimizole in the field of obstetrics and gynecology].

Cefpimizole (AC-1370) was studied for its transference into adnexa uteri and uterine tissues as well as for its effects and safety on gynecological infections. The results obtained are as follows: Peak levels of AC-1370 were obtained in the antecubital vein and uterine artery at 10 minutes, in the tissues of adnexa uteri and uteri about 30 minutes after one shot intravenous injection of AC-1370 1 g, and relatively high concentrations were maintained for several hours. In the treatment of 30 cases of gynecological infections, the clinical efficacy of AC-1370 was assessed as effective in all cases. As for the bacteriological effects of AC-1370, 77.6% of isolated organisms were eradicated and 90% of all cases were effective. Side effects and abnormal laboratory findings due to AC-1370 were not observed during and after administration.