Biomedical subjects
Publications and source records attributed to H Fukuhara.
A low-affinity glucose transporter gene of Saccharomyces cerevisiae was cloned by complementation of the rag1 mutation in a strain of Kluyveromyces lactis defective in low-affinity glucose transport. Gene sequence and effects of null mutation in S. cerevisiae were described. Data indicated that there are multiple genes for low-affinity glucose transport.
To evaluate the efficacy of drugs for treatment, we tried to make an animal model of Pneumocystis carinii (P. carinii) pneumonia. Specific pathogen free (SPF) rats immunosuppressed with corticosteroid were intratracheally inoculated with P. carinii. Six weeks after the inoculation, the lung sections of infected rat lung showed increased numbers of P. carinii in the alveoli and thickening of alveolar septa with mononuclear cell infiltration. From 7 to 9 weeks after inoculation, the intensity of infection became more severe, and some rats died at this period. Then, to evaluate drug efficacy in this model, we finished the drug therapy by the 6th week and used the number of P. carinii in the lung and the inflammation score as an indicator of drug efficacy. In this P. carinii pneumonia animal model, both sulfamethoxazole-trimethoprim clinically established anti P. carinii drug and interferon-gamma which is one of the lymphokines mainly produced by T lymphocytes indicated therapeutic and synergistic efficacy against P. carinii pneumonia.
In most yeast species, the mitochondrial DNA (mtDNA) has been reported to be a circular molecule. However, two cases of linear mtDNA with specific termini have previously been described. We examined the frequency of occurrence of linear forms of mtDNA among yeasts by pulsed-field gel electrophoresis. Among the 58 species from the genera Pichia and Williopsis that we examined, linear mtDNA was found with unexpectedly high frequency. Thirteen species contained a linear mtDNA, as confirmed by restriction mapping, and labeling, and electron microscopy. The mtDNAs from Pichia pijperi, Williopsis mrakii, and P. jadinii were studied in detail. In each case, the left and right terminal fragments shared homologous sequences. Between the terminal repeats, the order of mitochondrial genes was the same in all of the linear mtDNAs examined, despite a large variation of the genome size. This constancy of gene order is in contrast with the great variation of gene arrangement in circular mitochondrial genomes of yeasts. The coding sequences determined on several genes were highly homologous to those of the circular mtDNAs, suggesting that these two forms of mtDNA are not of distant origins.
The terminal structure of the linear mitochondrial DNA (mtDNA) from three yeast species has been examined. By enzymatic digestion, alkali denaturation, and sequencing of cloned termini, it was shown that in Pichia pijperi and P. jadinii, both termini of the linear mtDNA were made of a single-stranded loop covalently joining the two strands, as in the case of vaccinia virus DNA. The left and right loop sequences were in either of two orientations, suggesting the existence of a flip-flop inversion mechanism. Contiguous to the terminal loops, inverted terminal repeats were present. The mtDNA from Williopsis mrakii seems to have an analogous structure, although terminal loops could not be directly demonstrated. Electron microscopy revealed the presence, among linear molecules, of a small number of circular DNAs, mostly of monomer length. Linear and circular models of replication are considered, and possible conversion mechanisms between linear and circular forms are discussed. A flip-flop inversion mechanism between the inverted repeat sequences within a circular intermediate may be involved in the generation of the linear form of mtDNA.
The RAG1 gene of Kluyveromyces lactis encodes a low-affinity glucose/fructose transporter. Its transcription is induced by glucose, fructose, and several other sugars. The RAG4, RAG5, and RAG8 genes are trans-acting genes controlling the expression of the RAG1 gene. We report here the characterization of one of these genes, RAG5. The nucleotide sequence of the cloned RAG5 gene indicated that it encodes a protein that is homologous to hexokinases of Saccharomyces cerevisiae. rag5 mutants showed no detectable hexokinase or glucokinase activity, suggesting that the sugar kinase activity encoded by this gene is the only hexokinase in K. lactis. Both high- and low-affinity transport systems of glucose were affected in rag5 mutants. The defect of the low-affinity component was found to be due to a block of transcription of the RAG1 gene by the hexokinase mutation. In vivo complementation of the rag5 mutation by the HXK2 gene of S. cerevisiae and complementation of hxk1 hxk2 mutations of S. cerevisiae by the RAG5 gene showed that RAG5 and HXK2 were equivalent for sugar-phosphorylating activity but that RAG5 could not restore glucose repression in the S. cerevisiae hexokinase mutants.
A case of acetaminophen-induced pneumonitis is described. A 63-year-old woman took 2350 mg of acetaminophen for fever. She subsequently developed a non-productive cough and dyspnea. Chest X-ray revealed diffuse reticulonodular shadows in bilateral lung field and she was admitted to our hospital. PaO2 was 45.0 Torr and PaCO2 35.7 Torr while breathing room air. Bronchoalveolar lavage examination showed an increase in the percentage of lymphocytes and a decrease of CD4/CD8 ratio. Microscopic examination of a transbronchial lung biopsy specimen showed alveolar septal thickening. The lymphocyte stimulation test was positive for acetaminophen, with a stimulation index of 237%, while other drugs used were negative. Because drug-induced pneumonitis was suspected, all drugs were stopped and she was administered methylprednisolone. Consequently her symptoms, laboratory data and chest X-ray findings improved. Based on these findings, we diagnosed this case as acetaminophen-induced pneumonitis. To our knowledge, there has been only one previously reported case of acetaminophen-induced pneumonitis.
Cefpodoxime (CPDX-PR) was evaluated clinically in respiratory tract infections. The results obtained are summarized as follows; 1. The total number of the patients who were treated with CPDX-PR was 61, out of whom 53 cases were evaluated for clinical efficacy and 55 cases were investigated for the safety of the drug. CPDX-PR was given orally twice a day at 100-200 mg for 5-21 days. 2. Clinical efficacies were excellent in 9 patients, good in 36, fair in 4 and poor in 4. The overall clinical efficacy was 84.9%. In particular, CPDX-PR showed satisfactory efficacy for acute respiratory infections and mild chronic respiratory infections, with efficacy rates of 88.6% (31/35) and 100% (8/8), respectively. 3. No adverse reactions was observed, but slight and transient elevation of BUN was noted. In conclusion, it has been confirmed that CPDX-PR is an excellent and safe drug for the treatment of the respiratory tract infections.
Forty patients with chronic respiratory infections were randomly assigned to 2 groups to compare the effect of once daily administration of 300 mg each and 3 times daily administration of 600 mg each of ofloxacin (OFLX). Twenty patients were administered with 300 mg OFLX a day and 18 cases received 600 mg. The number of underlying diseases in the 300 mg group was greater than that in the 600 mg group. The ratios of general amelioration of clinical symptoms were 80.0% in the 300 mg group and 88.9% in the 600 mg group. For bacteriological effects, the eradication rate was 80.0% in the 300 mg group and it was 84.6% in the 600 mg. The incidence of side effects in the 300 mg group was 0% and that of the 600 mg group was 5.6% (1 patient) but the symptom was mild. The incidence of abnormal laboratory test results was 15.0% in the 300 mg group and it was 11.2% in the 600 mg group, but all of these abnormalities were slight and transient. The safety rates in the 300 mg and the 600 mg groups were 95.0% and 94.4%, respectively. Efficacy rates in the 300 mg and the 600 mg groups were 80.0% and 88.9%, respectively. There was no statistically significant difference in all the results between the 2 treatment groups, and the both treatments were highly effective. From the above results, we consider that once daily administration of 300 mg is a useful therapy in respiratory tract infections.
The bacteriology of empyema fluid and the clinical background of 23 cases from July 1987 through July 1992 were studied. Nineteen cases were male and 4 female, with a mean age of 59.6 years (range; 33 to 84 y.o.). There were 15 cases of community-acquired infection and 8 of nosocomial infection. Acute pneumonia and/or lung abscess developed into empyema in 19 cases. Sixteen cases had associated predisposing conditions, such as diabetes, chronic bronchitis, disorders with dysphagia, and excess alcohol intake. Forty-one strains were isolated from empyema in 22 cases. The predominant organisms, in order of prevalence, were "Streptococcus milleri" group (11 strains), Peptostreptococcus spp. (6), Prevotella spp. (6), Fusobacterium spp. (5) and other viridans streptococci (3). The majority of streptococcal infections, which were primarily caused by "S. milleri" group, were mixed with anaerobes and/or aerobes/facultatives. These results demonstrate that oral streptococci, especially "S. milleri" group, and anaerobes play a significant role as pathogens in empyema.
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A 7965 bp DNA segment from the right arm of chromosome III of Saccharomyces cerevisiae, encompassing the sup61 and RAD18 genes, was sequenced. Four new open reading frames were found in this DNA fragment. One of them, YCR103, is 51% homologous with the G10 gene product of Xenopus laevis.
A 10,095 base pair DNA fragment from the right arm of chromosome III of Saccharomyces cerevisiae has been sequenced and analysed. It encompasses the silent mating-type locus HMR. Both HMRa1 and HMRa2 genes, as well as their flanking regulatory regions, have been identified. Three new open reading frames longer than 80 amino acid residues were found in this fragment. One of them (YCR137) shows features compatible with a membranous localization and a transporter function. The other two do not show a similarity with any known gene. A new gene coding for tRNA(thr)al (ACU) has been identified. It is located in a region coding for several delta sequences.
A DNA fragment that can complement the leu2 mutation of Saccharomyces cerevisiae was cloned from the genomic library of Kluyveromyces lactis. The nucleotide sequence revealed an open reading frame of 362 codons, 75% homologous to S. cerevisiae LEU2 gene. The upstream region contained a CCGGAACCGG sequence identical to the site of leucine-specific control of LEU2. Further upstream, there is a partial open reading frame homologous to rat ribosomal protein L7.
In most strains of Kluyveromyces lactis, respiratory function is not required for growth on glucose. However, some natural variant strains are unable to grow when respiration is blocked by specific inhibitors (Rag- phenotype). This phenotype is due to an allelic variation of the chromosomal gene RAG1. The sensitive variants have a recessive allele rag1. The RAG1 gene has been cloned by complementation of a rag1 strain from a genomic bank derived from a Rag+ strain. The nucleotide sequence of the cloned gene indicated that the RAG1 product was a sugar transporter protein. The amino acid sequence deduced from the gene structure contained the 12 hydrophobic segments typical of a transmembrane protein, and showed a high degree of homology with the GAL2 (galactose permease) and HXT2 (a high-affinity glucose transporter) proteins of Saccharomyces cerevisiae. In a rag1 null mutant, as in the natural rag1 variant, uptake of glucose at high external glucose concentrations was impaired. The RAG1 protein appears to correspond to a low-affinity glucose transporter. Transcription of the RAG1 gene, which was undetectable when cells were grown in glycerol, was induced by glucose. It is concluded that respiration-dependent growth on glucose of the Rag- variant strains is due to a defect in this inducible glucose transport system.
The RAG1 gene encodes a membrane protein involved in the low-affinity glucose/fructose transport system of the yeast Kluyveromyces lactis. Analysis of steady-state mRNA levels analysis and quantitation of expression by beta-galactosidase from RAG1-lacZ fusions assays revealed that the RAG1 gene was poorly expressed in cells grown under gluconeogenesis conditions, but was induced more than ten-fold when they were grown on various sugars. These sugars included glucose, fructose, mannose, sucrose, raffinose, as well as galactose. Nucleotide sequence and deletion analysis of the 5' flanking region of the RAG1 gene showed that an essential cis-acting element required for induced transcription of the RAG1 gene resided between -615 and -750 from the coding sequence. This region contained a 22 bp purine stretch, and a pair of 11 bp direct repeat sequences. The 11 bp repeats harbor a CCAAT motif, a consensus sequence for binding of the yeast and mammalian HAP2/3/4-type protein complex. The transcription of the RAG1 gene was dramatically affected by three unlinked mutations, rag4, rag5 and rag8. We discuss the possible roles of RAG4, RAG5 and RAG8 gene products in the expression of the RAG1 gene, as well as the importance of the inducible RAG1 gene in the fermentative growth of K. lactis.
A new plasmid was found in the yeast Kluyveromyces waltii. This high-copy-number plasmid, named pKW1, is a double-stranded circular DNA plasmid of 5619 bp. It has several features characteristic of the 2 mu-type plasmids: presence of two inverted repeats and four open reading frames, as well as the interconversion of two isomeric forms. However, the nucleotide sequence shows little homology with known yeast plasmids. An ARS function was localized within a segment of 545 bp near one of the inverted repeats. Chimeric plasmids carrying this segment efficiently transformed K. waltii. A strain of K. waltii cured of the plasmid (cir degree) was also obtained. In the pKW1 sequence, a functionally neutral region was found at which foreign DNA can be inserted with little effect on plasmid stability. Such constructions carrying the full sequence of pKW1 replicated autonomously in a cir degree host and were particularly stable. pKW1-derived full-sequence plasmids also transformed K. thermotolerans, but not K. lactis.
The mechanism of aggregation of Eikenella corrodens 1073 with E. corrodens aggregating factor (EcAF) which was purified from submandibular-sublingual (SM-SL) saliva was investigated. Heating (100 degrees C, 10 minutes) or protease treatment of E. corrodens cells abolished the aggregating activity. The aggregation was inhibited by adding N-acetyl-D-galactosamine (GalNAc) and saccharides which contain a galactose residue at the non-reducing end. The aggregating activity was sensitive to EDTA and was restored by Ca2+ but not by Mn2+ or Mg2+. Neuraminidase treatment of EcAF increased their ability to aggregate. E. corrodens, suggesting that the sialic acids on EcAF interfere with aggregation. These data suggest that the aggregation of E. corrodens 1073 with EcAF is mediated by specific interactions between a bacterial cell surface lectin-like substance and a complementary GalNAc-like receptor. EcAF also aggregated 16 strains of oral bacteria including periodontopathic bacteria such as Porphyromonas (Bacteroides) gingivalis 381 and Actinobacillus actinomycetemcomitans ATCC29522; however, those aggregations were not inhibited by GalNAc. Therefore, EcAF appears to have more than two types of bacterial binding site and plays important roles in accumulation of dental plaque by forming a complex network of plaque bacteria including periodontopathic strains.
In order to determine the optimal dose of cefepime (CFPM) for respiratory tract infections, a dose finding study was conducted in patients with chronic respiratory tract infections, and the clinical properties of the drugs were compared with those of ceftazidime (CAZ). Each drug was administered by intravenous drip infusion at the dose of 2 g/day of CFPM, 4 g/day of CFPM or 2 g/day of CAZ for 14 days. 1. The overall efficacy rates evaluated by the committee were 82.6% (19/23) for the CFPM 2 g/day group, 85.0% (17/20) for the 4 g/day group and 79.3% (23/29) for the CAZ 2 g/day group, with no statistically significant difference among the three groups. 2. The overall efficacy rates evaluated by the attending physicians were 91.3% (21/23) for the CFPM 2 g/day group, 78.9% (15/19) for the CFPM 4 g/day group and 75.9% (22/29) for the CAZ 2 g/day group, with no significant difference among the three groups. 3. Bacteriological eradication rates were 88.2% (15/17) for the CFPM 2 g/day group, 68.8% (11/16) for the CFPM 4 g/day group and 63.2% (12/19) for the CAZ 2 g/day group, with no statistically significant difference among the three groups. 4. The incidences of adverse reactions were 3.8% (1/26) for the CFPM 2 g/day group, 9.1% (2/22) for the CFPM 4 g/day group and 3.4% (1/29) for the CAZ 2 g/day group, with no significant difference among the three groups. The incidences of abnormal laboratory findings were 37.5% (9/24) for the CFPM 2 g/day group, 15.0% (3/20) for the CFPM 4 g/day group and 3.4% (1/29) for the CAZ 2 g/day group. There was a significant difference among the three groups. 5. Utility rates assessed by the committee were 81.8% (18/22) for the CFPM 2 g/day group, 76.2% (16/21) for the CFPM 4 g/day group and 75.9% (22/29) for the CAZ 2 g/day group. Utility rates assessed by attending physicians were 90.9% (20/22), 78.9% (15/19) and 72.4% (21/29), respectively. There was no significant difference among the three groups. From the above results, it is concluded that the optimal dosage of CFPM is 2 g/day for chronic respiratory tract infections.