[Host-vector systems in filamentous fungi].
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Biomedical subjects
Publications and source records attributed to K Gomi.
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We studied the identification and susceptibility of clinically isolated yeast-like fungi at Showa University Hospital from April 1988 to March 1989. Clinically significant of yeast-like fungi were observed in 7.1% of specimens from outpatients, 13.0% of inpatients. In both outpatients and inpatients, yeast-like fungi were isolated mainly from sputum and urine. But, one third of them were considered as non-pathogenic and not identified. The species of isolates were, Candida albicans 57%, Candida tropicalis 14% and Candida glabrata 8% in both inpatients and outpatients, and these species shared most part. The isolation frequency of Candida parapsilosis was higher in blood and cerebrospinal fluid (CSF) specimen than the others. The susceptibility test by agar dilution method indicated most of the isolates were susceptible to Amphotericin B and Miconazole (MIC less than or equal to 25 micrograms/ml). There was no difference in MIC between predominantly isolated fungi and commonly isolated fungi. Notably, isolates from blood and CSF showed a significant high tolerance against Amphotericin B and Miconazole than from the other specimens. The MICs of Fluconazole were shown to be very high (greater than 100 micrograms/ml) in normal Sabouraud agar, were decreased dose-dependently by human sera in the medium. These findings indicated the component(s) of sera enhanced the anti-fungal activity of Fluconazole.
The efficacy, safety and usefulness of BMY-28100 for the treatment of bacterial pneumonia were compared with those of cefaclor (hereinafter referred to as CCL) in a double-blind study. The daily dosages were 750 mg for BMY-28100 and 1,500 mg for CCL, divided into 3 administrations daily. These drugs were administered orally for at least 14 days. A total of 172 cases were enrolled in this study. Of these, cases which deviated from the protocols were excluded from evaluations. Thus, clinical efficacy was evaluated in 124 cases, adverse reactions were evaluated in 160 cases, and abnormal laboratory test values were evaluated in 146 cases. The following results were obtained. 1. Efficacy rates ("good" or better responses) in bacterial pneumonia cases as evaluated by the subcommittee were 81.7% (49/60) in the BMY-28100 group and 89.1% (41/46) in the CCL group, thus no significant difference was found between the 2 groups. 2. Efficacy rates ("good" or better responses), as evaluated by investigators, in the same bacterial pneumonia cases which were subjected to the evaluation by the subcommittee were 83.3% (50/60) in the BMY-28100 group and 88.9% (40/45) in the CCL group, thus no significant difference between the 2 groups was found also. 3. Bacteriological response rates in bacterial pneumonia cases were 86.2% (25/29) in the BMY-28100 group and 85.7% (18/21) in the CCL group with no significant difference between the 2 groups. 4. Incidences of subjective/objective clinical adverse symptoms were 3.5% (3/85) in the BMY-28100 group and 1.3% (1/75) in the CCL group, and no significant difference was observed between the 2 groups. No significant difference was also found between the 2 groups in incidences of abnormal laboratory test values, as abnormalities were found in 21.1% (16/76) of the cases in the BMY-28100 group and 25.7% (18/70) in the CCL group. 5. As for overall usefulness of the drug in bacterial pneumonia cases, utility rates ("useful" or better evaluations) as evaluated by the subcommittee were 83.6% (46/55) in the BMY-28100 group and 90.5% (38/42) in the CCL group, and the rates as evaluated by investigators in cases judged as evaluable by the subcommittee were 78.3% (47/60) and 82.2% (37/45), respectively. There were no significant differences between the 2 groups. The utility rates as evaluated by investigators in cases in which diseases were diagnosed as bacterial pneumonia or lung abscess by investigators were 78.3% (47/60) in the BMY-28100 group and 82.2% (37/45) in the CCL group.(ABSTRACT TRUNCATED AT 400 WORDS)
We have identified a minimum functional domain of human thrombomodulin for anticoagulant activity using deletion analysis. Four mutants were constructed by site-directed deletion mutagenesis to delete one or more epidermal growth factor (EGF)-like structures from the domain of human thrombomodulin containing six repeated EGF-like structures. These deletion mutants were expressed transiently in COS-1 cells, and their protein C-activating cofactor activities in the culture medium were examined. One mutant protein, E456, which contains the fourth, fifth, and sixth EGF-like structures expresses apparent cofactor activity. However, neither E456-N24 (24 NH2-terminal-residue deletion) nor E456-C16 (16 COOH-terminal-residue deletion) have cofactor activity. E456 was partially purified and its anticoagulant effects on plasma clotting time and platelet aggregation examined. E456 expressed almost the same anticoagulant activities as D123 which contains six consecutive EGF-like structures of thrombomodulin. It was concluded that E456 is the minimum functional domain for both protein C-activating cofactor activity and anticoagulant activity.
The purpose of this study was to investigate the effects of hybrid material compounded with periodontal ligament cells on human teeth and porous hydroxyapatite. As the first step, we tried to produce hybrid materials compounded with periodontal ligament cells and porous hydroxyapatite, and to observe the attachment and invasion of periodontal ligament cells porous hydroxyapatite. Human periodontal ligament cells which had been scraped off the root with a disposable blade were cultured at 37 degrees C in alpha-MEM plus 10% fetal calf serum. The culture cells were attached to the porous hydroxyapatite in vitro. After 3 and 14 days of incubation, the specimens were fixed in gluteraldehyde and examined using SEM. Other specimens were embedded in paraffin and successive specimens were prepared for to remodelling of the attachment and invasion. Many cells attached to the P-HAP granules, and some cells invaded the P-HAP granules. The results suggest that when these hybrid materials are implanted in bone defects, the cells remain in the defect for a certain period, and exhibit the functions and characteristics of itself.
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Highly purified human recombinant interferon-gamma (ReIFN-gamma) was radioiodinated with 125I-Bolton-Hunter reagent and used to characterize the receptor for ReIFN-gamma. 125I ReIFN-gamma specifically and saturably bound four human cells tested, FL, WISH, Daudi, and HL-60, which were reported to have ReIFN-gamma binding sites. Scatchard analysis of the binding data of FL cells revealed the presence of 5200 binding sites per cell and a Kd value of 2.1 X 10(-10) M. Although the binding of 125I ReINF-gamma was inhibited by unlabelled natural IFN-gamma and ReIFN-gamma, it was not inhibited by unlabelled human ReIFN-alpha and ReIFN-beta, suggesting that receptors for (Re)IFN-gamma were different from those for IFN-alpha or IFN-beta. However, ReIFN-gamma displaced the binding of 125I ReIFN gamma 3 to 5 times more effectively than IFN-gamma. Internalization of 125I ReIFN-gamma bound to the cell surface receptor was observed at 37 degrees C. Pretreatment of FL cells with unlabeled ReIFN-gamma caused a concentration-dependent down-regulation in the ReIFN-gamma receptor, which was specific for IFN-gamma and reversible. From these studies, we concluded that although the ReIFN-gamma is not identical to putative IFN-gamma, the recombinant form binds to the same binding sites for IFN-gamma and does not bind to the binding sites for IFN-alpha or beta.
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The synergism of anticellular and antiviral activities of recombinant human interferon-gamma (ReIFN-gamma) and recombinant human interferon-beta (ReIFN-beta) was examined in vitro using human melanoma SK-MEL-28 cells. Some differences were detected in the kinetics of anticellular activity between both IFNs, namely the inhibitory effect of ReIFN-beta occurred earlier than that of ReIFN-gamma. Significant synergism was detected in the anticellular activity of both IFNs when growth curves and isobolograms were examined. A difference between ReIFN-gamma and ReIFN-beta was also detected in antiviral activity. The antiviral activity of ReIFN-gamma against vesicular stomatitis virus (VSV) was significantly weaker than that of ReIFN-beta, even though both IFNs exhibited almost equivalent antiviral activities against Sindbis virus. However, ReIFN-gamma and ReIFN-beta exhibited synergistic antiviral activities against both VSV and Sindbis virus. The analysis of cell cycle distribution by flow cytometry revealed that there were some differences in the distribution pattern between cells treated with ReIFN-gamma alone, ReIFN-beta alone, or ReIFN-gamma and ReIFN-beta in combination. ReIFN-beta induced a prolongation or accumulation of S phase, whereas the effect of ReIFN-gamma was cycle-nonspecific. The combination of ReIFN-gamma and ReIFN-beta induced a decrease of G1 phase and an increase of G2M phase. These results suggest that ReIFN-gamma and ReIFN-beta used in combination were more effective in inhibiting the growth of human tumor cells and the proliferation of viruses than IFN used individually.
The antitumor activity of recombinant human interferon-gamma (ReIFN-gamma) and recombinant human interferon-beta (ReIFN-beta) against human osteosarcoma G-292 cells was compared in vitro and in vivo. Both IFNs inhibited the growth of G-292 cells cultured in vitro and transplanted into nude mice in vivo. In in vivo experiment, both IFNs injected i.t. were more effective at inhibiting the growth of G-292 cells than systemic administration such as i.v., i.p. or s.c. In comparing the various routes of systemic administration of both IFNs, i.v. injection was slightly more effective. Both IFNs exhibited more significant growth inhibition when they were administered every day for 10 d, as compared with a single or intermittent administrations, suggesting that the antitumor activity of both IFNs was time-dependent. Significant difference was not detected in the in vivo antitumor activity between both IFNs when they were compared on the basis of their antiviral units. However, the time dependency for antitumor effect of ReIFN-beta was more significant than that of ReIFN-gamma. ReIFN-gamma and ReIFN-beta used in combination exhibited synergistic antitumor activity both in vitro and in vivo, though the in vivo synergism of both IFNs was not so effective as that of in vitro: These results indicate that both ReIFN-gamma and ReIFN-beta are effective at inhibiting the growth of human osteosarcoma in vivo.
A 58-year-old female who had a serum cholinesterase activity four times higher than normal is described. Investigation of her family revealed that five out of seven tested had high level of serum cholinesterase activity. The family distribution of the increased cholinesterase suggests that the inheritance is transmitted in a autosomal dominant manner. Analysis of the isoenzyme of serum cholinesterase of five cases including the patient showed that none of them had extracomponent (C5) of the cholinesterase. Examination of anti-cholinesterase antibodies in her serum was negative. Molecular weight of her serum cholinesterase by HPLC analysis was not different from that of a healthy subject. It was speculated that the overproduction of usual components (C1, C2, C3, C4), decreased clearance of cholinesterase by a certain mechanism(s), and/or the presence of cholinesterase which has more active catalysis could be responsible for her and her family's hypercholinesterasemia.
We studied the mechanisms of resistance to interferon (IFN) in two human melanoma cell lines, SK-MEL-28/beta cells resistant to human recombinant beta-interferon (ReIFN-beta) and SK-MEL-28/gamma cells resistant to human recombinant gamma-interferon (ReIFN-gamma). SK-MEL-28/beta cells were resistant to both ReIFN-beta and IFN-alpha, but not ReIFN-gamma or natural gamma-interferon (IFN-gamma), and SK-MEL-28/gamma cells were resistant to both ReIFN-gamma and IFN-gamma, but not ReIFN-beta or IFN-alpha. SK-MEL-28/s cells, sensitive to both ReIFN-beta and ReIFN-gamma, had receptors for ReIFN-beta and ReIFN-gamma. Furthermore ReIFN-beta and IFN-alpha shared the common receptors as judged by competition assays, while the receptors for ReIFN-gamma were independent in SK-MEL-28/s cells. The Kd values of binding of both IFNs to SK-MEL-28/beta or SK-MEL-28/gamma cells were essentially equivalent to those to SK-MEL-28/s cells. Both labeled IFNs were internalized rapidly into all cell lines at 37 degrees C. No significant difference in internalization was found between IFN-sensitive and -resistant cells. Each cell line expressed HLA-AB and Mr 97,000 protein antigens, the expressions of which were enhanced by ReIFN-beta and ReIFN-gamma. Partial resistance to the enhancement of both antigens by both IFNs was seen in the resistant cells. Finally ReIFN-gamma, but not ReIFN-beta, induced a Mr 56,000 protein in SK-MEL-28/s cells. This effect of ReIFN-gamma was also detected in SK-MEL-28/beta cells, but not SK-MEL-28/gamma cells. These results provide some information concerning the nature of IFN-resistant cells. The receptors had no significant correlation with the resistance, while the expression of HLA-AB or Mr 97,000 protein antigens or the induction of proteins had some correlation with the resistance.
Human recombinant interferon-gamma (ReIFN-gamma) and human natural interferon-gamma (IFN-gamma) showed significant differences in antiviral and anticellular activities. Namely, ReIFN-gamma exhibited antiviral activity against Sindbis virus and anticellular activity against HeLa S3 cells at significantly lower concentrations as compared with IFN-gamma at short exposure times of between 1 and 4 hr. This difference of activities was explicable in terms of different binding activities of the two IFNs to the receptors. The binding of 125I-ReIFN-gamma to the receptors was competitively decreased in a dose dependent manner by unlabeled ReIFN-gamma, whereas the competition by unlabeled IFN-gamma was significantly weaker. The results of pretreatment of the cells with unlabeled ReIFN-gamma or IFN-gamma suggested that the binding of IFN-gamma to the receptors was slower than that of ReIFN-gamma. ReIFN-gamma and IFN-gamma exhibited greater colony formation-inhibitory activity against HeLa S3 cells, as compared with IFN-alpha or ReIFN-beta. ReIFN-gamma also inhibited DNA, RNA and protein syntheses of HeLa S3 cells more potently than ReIFN-beta. However, the 2'-5' oligoadenylate (2'-5' A) synthetase activity in ReIFN-gamma-treated cells was significantly lower than that in ReIFN-beta-treated cells, suggesting that 2'-5' A synthetase does not play a major role in the anticellular activity of ReIFN-gamma, at least against HeLa S3 cells.
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We developed a new method for the determination of CRP by latex immunoassay which measures the increase in optical density as a result of latex agglutination due to antigen-antibody reaction. This latex agglutination photometric immunoassay can be used for quantitative analysis of various biological substances. We have established the best conditions for CRP determination for the instrument employed here (LA system) which allows rapid and accurate to low concentration measurement. The measurement range of the system is from 0.01-3 mg/l (non-diluted method), which when compared to the lower detection limit of RIA, 3 micrograms/l, it has the same order of magnitude. It is more sensitive than radial immunodiffusion which has the lower detection limit of 2 mg/l and has the similar value to that of radio-electroimmunodiffusion, 10 micrograms/l. Furthermore, the usual method used in the clinical laboratories, the capillary microprecipitation method, has the lower detection limit of approximately 10 mg/l. The method presented here is adequately sensitive to measure the low concentration of CRP among healthy individuals which has not been possible except by using RIA. The normal value derived from 106 healthy individuals by this method is found to be 0.2 +/- 0.2 mg/l (mean +/- 2SD).
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