Biomedical subjects

T Kojima

Publications and source records attributed to T Kojima.

At least 361 records · Page 20Linked to original sources

PubMed1996

ErbB-2 expression is correlated with poor prognosis for patients with osteosarcoma.

BACKGROUND: It has been reported that the c-erbB-2 protooncogene is frequently amplified and overexpressed in many types of cancers, except sarcomas and hematological malignancies. METHODS: Expression of ErbB-2 in the tumors of 26 patients with conventional osteosarcoma was evaluated by immunoblotting. DNA from osteosarcoma tissues that expressed ErbB-2 were analyzed by Southern blot hybridization to examine gross rearrangement of the gene. The DNA was also surveyed for the presence of genetic mutation in the transmembrane domain of ErbB-2 by polymerase chain reaction-single-stranded DNA conformation polymorphism analysis. In addition, possible correlation of ErbB-2 expression with gender, age, histopathologic subtype, and response to chemotherapy was analyzed. Survival analysis was performed by the Kaplan-Meier test using the approximate chi-square statistic for the log-rank test. RESULTS: The ErbB-2 protein was detected in 11 of 26 osteosarcoma tissues (42%) by immunoblot analysis. Expression of ErbB-2 was confirmed by immunohistochemical studies using specific anti-ErbB-2 monoclonal antibody. However, neither amplification of the c-erbB-2 gene nor evidence of significant genetic mutation was found in these osteosarcomas. Expression of ErbB-2 examined by immunoblotting was most strongly correlated with early pulmonary metastases (P < 0.05). Among the entire group of 26 patients in this study, Kaplan-Meier life table survival of the patients with apparent ErbB-2 expression was significantly worse than that of the patients with little ErbB-2 expression (P < 0.01). CONCLUSIONS: In 42% of the osteosarcomas, the tumor cells expressed ErbB-2. Expression of ErbB-2 was strongly correlated with early pulmonary metastasis and poor survival rate for the patient. These data suggest that ErbB-2 plays a significant role in aggressive tumor growth and in the promotion of metastatic potential in osteosarcomas. ErbB-2 in the osteosarcoma tissues would be a useful prognostic marker for patients.

Adolescent↗

PubMed1996

Long-term cryopreserved allogeneic nervous and vascular tissue transfers in monkeys.

Four adult Japanese monkeys were used. Donor tissues were harvested from the radial artery, saphenous vein and median nerve, 10 cm in length, and cryopreserved at -196 degrees C in a stepwise manner for long-term preservation for more than 6 months. After rapid thawing of the tissue, the allogeneic tissue transfer was performed of the radial artery to the radial artery, the saphenous vein to the ulnar artery and the median nerve to the median nerve between male and female monkeys. After one year follow-up, angiography was performed and the transferred tissue was harvested for scanning electron microscopic examination and histological examination. As a result, the allogeneic artery in the radial artery was completely patent with no stenosis, whereas there was obstruction of the allogeneic vein in the ulnar artery. Good axonal recovery of the median nerve was observed on histological examination. The cellular structure of neurovascular tissue can be maintained even after long-term cryopreservation. The antigenicity of tissue may be reduced significantly by being frozen, and cryopreserved allogeneic artery can be transferred successfully without the use of immunosuppressive agents. In this report, clinical aspects and the great potential of allogeneic neurovascular tissue transfer, especially in combination with arterial transfer, using cryopreservation are discussed.

Animals↗

PubMed1996

Effect of heat stress on development in vitro and in vivo and on synthesis of heat shock proteins in porcine embryos.

The present study was conducted (1) to examine the effect of an acute increase in ambient temperature on the development of porcine day 6 embryos in culture and after transfer to recipient gilts, and (2) to analyze intracellular production of heat shock proteins (hsps). The viability of porcine day 6 embryos following a temporary acute elevation in ambient temperature (at 42 degrees-45.5 degrees C and for 10-180 min) was examined. Synthesis of 70 kDa hsp (hsp70) and 90 kDa hsp (hsp90) was determined by SDS-PAGE and Western blot analysis in porcine day 6 embryos subjected to heat stresses. Nonheat-stressed embryos were considered as control. Significantly higher numbers of viable nuclei were observed in treatment groups of 42 degrees C-10 min (236.6 +/- 71.4; P < 0.05) and 43 degrees C-30 min (276.8 +/- 89.4; P < 0.005) compared to control (173.9 +/- 53.9). The 42 degrees C-180 min group (158.0 +/- 27.1 microns) had a greater increase in diameter after 24 hr in culture following heat stress compared to control (82.5 +/- 47.3 microns), while heat stress with 43 degrees C for > or = 60 min, 44 degrees-44.5 degrees C for > or = 30 min, or 45 degrees-45.5 degrees C for > or = 10 min impaired their survival, as assessed by differences in number of viable nuclei. The embryos subjected to heat stresses under the conditions of 42 degrees C-180 min, 43 degrees C-10 min, 43 degrees C-30 min, 44 degrees C-10 min, or 45 degrees C-10 min developed to normal piglets after transfer to recipient gilts. Overall pregnancy rate was 75% (6/8), and farrowing rate 62.5% (5/8). Of heat-stressed embryos transferred, 59% (36/61) developed to normal piglets. Heat-stress conditions of 42 degrees C for 180 min, 43 degrees C for 30 min, 44 degrees C for 10 min, and 45 degrees C for 10 min were determined as critical with respect to the in vitro and in vivo survival of porcine embryos. Porcine day 6 embryos constitutively synthesized hsp70 even without heat stress, while hsp90 was detected only at trace level. Neither hsp70 nor hsp90 levels increased in the embryos subjected to heat stresses. In conclusion, porcine day 6 embryos could continue to develop in vivo or during in vitro culture after exposure to acute and temporary rise in temperature. However, no increase of hsp70 and hsp90 was observed in the heat-stressed porcine embryos, while hsp70 was detected in the nonheat-stressed porcine embryos. The precise mechanism of the thermotolerance was unclear.

Animals↗

PubMed1996

Characterization of CD4-CD8- T cell receptor alpha beta + T cells appearing in the subarachnoid space of rats with autoimmune encephalomyelitis.

Inflammation of the central nervous system (CNS) in experimental autoimmune encephalomyelitis (EAE) starts in the subarachnoid space (SAS) and spreads later to the adjacent CNS parenchyma. To characterize the nature of lesion-forming T cells in situ in more detail, T cells were isolated from the SAS and their surface phenotype and the nucleotide sequence of the junctional region of the T cell receptor (TCR) was determined and compared with those of the lymph node (LN) and spinal cord (SC) T cells. Characteristically, more than 70% of SAS TCR alpha beta + T cells isolated at the early stage of EAE lacked both CD4 and CD8 molecules, whereas those from LN and SC were either CD4+ or CD8+. Analysis of nucleotide sequences of the junctional region of TCR revealed that T cells bearing a sequence identical to that for encephalitogenic T cell clones were found in both SAS and SC. Furthermore, purified CD4-CD8- T cells expressed CD4 molecules after culture. At the same time, these T cells acquired reactivity to myelin basic protein and induced passive EAE in naive animals after adoptive transfer. Our results suggest that CD4-CD8- T cells in the SAS are precursors of lesion-forming T cells in the SC and that phenotype switching takes place during the process of T cell infiltration into the CNS parenchyma. The double-negative nature of these T cells may explain an escape of encephalitogenic T cells from negative selection in T cell differentiation.

Amino Acid Sequence↗

PubMed1996

Characterization, molecular cloning and expression of megakaryocyte potentiating factor.

We examined whether the conditioned media of 64 kinds of cell lines, which have been maintained by a protein-free culture system, could produce megakaryocyte potentiating (Meg-POT) activity. In these cell lines, HPC-Y5, established from human pancreatic cancer, was shown to have the highest level of activity. The megakaryocyte potentiating factor (MPF) was purified from its conditioned medium by a combination of ion-exchange chromatography, gel filtration and reversed-phase HPLC. The purified MPF showed Meg-POT activity almost equal to human (Hu) interleukin 6 (IL-6) in the presence of murine IL-3 in a colony-forming assay with mouse bone marrow cells. The molecular weight of MPF was estimated to be 33 kDa by SDS-PAGE. Glycopeptidase F digestion and amino sugar analysis of the factor demonstrated that MPF is a glycoprotein carrying at least one N-linked sugar chain. The N-terminal amino acid sequence of MPF was determined to be Leu-Ala-Gly-Glu-Thr-Gly-Gln-Glu-Ala-Ala-Pro-Leu-Asp-Gly-Val-Leu-Ala-Asn. The same or homologous amino acid sequence has not been found in known proteins, demonstrating that MPF may be a novel cytokine which has Meg-POT activity. Then, we isolated HuMPF cDNA from an HPC-Y5 cDNA library using polymerase chain reaction and plaque hybridization methods. The HuMPF cDNA encodes a polypeptide consisting of 622 amino acids, including a signal peptide of 33 amino acids, and with a deduced molecular weight of 68 kDa, although HPC-Y5 cells secrete a 33 kDa form of HuMPF. HuMPF cDNA does not show any significant homology with other known sequences. The cDNA was expressed in COS-7 and Chinese hamster ovary (CHO) cells, and Meg-POT activity was detected in their culture supernatant. The COS-7 cells secreted only a 33 kDa recombinant (r)HuMPF, however, an additional 30 kDa form was detected in the culture medium of CHO cells. The 33 kDa rHuMPF from CHO cells showed Meg-POT activity, but not the purified 30 kDa rHuMPF. The difference in structure and activity between the 33 and 30 kDa forms of HuMPF was ascribed to the existence in the 33 kDa form of the C-terminal 25 amino acid residues. The expression of MPF mRNA was examined by Northern blot analysis using labeled MPF cDNA as a probe. MPF mRNA was detected in HPC-Y5 cells, with an approximate molecular size of 2.4 kb. We also examined the expression of the MPF gene in various human tissues, and the 2.4 kb band was detected only in lung. Then, the immunohistocytochemical analysis and in situ hybridization revealed that MPF-producing cells were identified as lung macrophages. MPF may exhibit other biological activities such as regeneration of the lung tissues.

Amidohydrolases↗

PubMed1996

Subculture of proliferating adult rat hepatocytes in medium supplemented with nicotinamide and EGF.

To establish parenchymal hepatocyte cell lines, we tried to subculture the primary hepatocytes isolated from adult rats. The hepatocytes were cultured in serum-free modified Dulbecco's modified Eagle's medium supplemented with 10 mM nicotinamide and 10 ng/ml epidermal growth factor. When 6 x 10(5) cells were plated on 35-mm dishes coated with rat tail collagen, the cells proliferated and reached confluence at Day 6 to Day 8. The first subculture was carried out at Day 8 using 0.005% collagenase and gentle pipettings. Most cells were recovered and plated on the new dishes coated with the collagen (first passage). The attached cells could proliferate and reached near confluence when the cells occupied more than two-thirds of the dish surface. About a week after the first subculture, the second one was conducted. Although the number of the recovered cells was smaller than at the first passage, the cells could attach and proliferate to a certain extent. Thereafter, they were maintained for more than 2 mo, but they never overgrew. Albumin secretion into the culture medium was confirmed in the subcultured cells. Ultrastructurally, these subcultured cells possessed hepatic characteristics such as peroxisomes with a crystalline nucleiod and bile-canaliculus structures. When 10% fetal bovine serum and ascorbic acid 2-phosphate were added to the cells of the second passage, they began to proliferate very slowly. These proliferating cells were mainly mononucleate and had a small cytoplasm. In addition, some of them could differentitate into typical mature hepatocytes by forming a three-dimensional structure interacting with nonparenchymal cells. In this experiment, we showed the successful subculturing of parenchymal hepatocytes isolated from adult rats and provided evidence that the subcultured cells still have the potential to proliferate and to differentiate.

Animals↗

PubMed1996

Analysis for heterozygosity of protein S mRNA: application to genetic screening and family studies in hereditary protein S deficiency.

We genetically screened patients with hereditary protein S deficiency for heterozygosity of protein S mRNA using PCR-RFLP for Pro626 polymorphism. All patients who showed hemizygous state of protein S mRNA, characterized by markedly decreased levels of one allele, had a phenotype of type I protein S deficiency. A putative mutation, such as a nonsense or splice site mutation, in the silent alleles may have affected the mutated mRNA metabolism and reduced the mutated mRNA accumulation, and consequently resulted in type I protein S deficiency in these patients. We also applied this mRNA-based analysis to family studies in hereditary protein S deficiency. In a family with type I protein S deficiency, all affected individuals showed a loss of one allele at the mRNA level and the silent allele cosegregated with the disease phenotype. Detection of hemizygous expression of protein S mRNA provided direct evidence for type I protein S deficiency without further precise genetic analysis. Our findings indicate that this mRNA-based analysis can be a useful strategy for genetic screening and family studies in hereditary protein S deficiency.

Codon↗

PubMed1996

Antibodies to E1 and E2/Protein X components of pyruvate dehydrogenase complex in sera of patients with primary biliary cirrhosis.

AIMS/METHODS: Using purified E1 component of pyruvate dehydrogenase complex (PDC) from bovine heart, we measured the levels of anti-E1 antibodies in PBC sera using ELISA and determined the degree of inhibition that these antibodies exerted on E1 enzyme activity. We also estimated levels of anti-E2/Protein X (Pro-X) antibodies in PBC sera using purified E2 and Pro-X of PDC which were copurified with E1. RESULTS/CONCLUSIONS: Anti-E1 antibodies were detected in 87.5% (35/40) of PBC sera. Some of these sera inhibited E1 enzyme activity but inhibition did not correlate with levels of anti-E1 antibodies. A high positive correlation (r = 0.918) was found between levels of anti-E1 and anti-E2/Pro-X antibodies, suggesting that anti-PDC antibody production was stimulated by PDC itself. Levels of IgG class anti-E2/Pro-X antibodies were significantly higher in sera of symptomatic PBC patients than in those of asymptomatic PBC patients. It was also found that patients who were positive for only IgM class anti-E2/Pro-X antibodies had early-stage PBC.

Animals↗

PubMed1996

Subungual dermatofibroma of the thumb.

We report a rare case of subungual dermatofibroma in a 38-year-old man who was noted to have bulging and thinning of the thumb nail plate. Growth of the nail plate to normal thickness and shape was achieved after surgical removal of the tumour and careful repair of the nail matrix.

Adult↗

PubMed1996

Influence of OH group and sugar bonded to flavonoids on flavonoid-mediated suppression of tumor growth in vitro.

Many flavonoids extracted from higher plants, synthesized or purified flavonoids, and their aglycones showed anti-tumor activity. Using purified or synthesized flavonoids, we studied the effect of the chemical structure of 2-benzoic flavone on the growth of human tumor cells (HCT-15 in vitro. The type of sugar combined with the A phenol of flavonoids played an important role in the tumor suppression; i.e., glucose attachment at the A phenol caused suppression of tumor cell growth, but other sugars such as rhamnose and lutinoside at that position did not suppress the growth of the cells. OH groups bonded to the B phenol also had a great effect on the growth. Flavonoids with OH groups conjugated to the 3', 4', and 5' of B phenol were stronger in anti-tumor effect than those with the OH groups attached at the 3' and 4' or 4' only, although anthocyanins were generally more effective than the other flavonoids.

Antineoplastic Agents, Phytogenic↗

PubMed1996

Antitumor effect of hydrolyzed anthocyanin from grape rinds and red rice.

When Balb/C mice that were fed with red glutinous rice, white ordinary rice, or commercially available standard food were inoculated with syngeneic Meth/A lymphoma cells i.p., the group fed the red rice survived longer than the other two groups. In order to determine if the anthocyanins contained in red-coloured seeds and fruit rinds were responsible for the tumor suppressive effect, we added anthocyanins extracted from grape rinds and glutinous red rice to petri dishes that had been seeded with HCT-15 cells. After 4 days of culture, cell counts were made. These anthocyanins were not effective in suppressing the tumor growth. However, anthocyanidins, which were generated by keeping anthocyanins in 5 to 6% HC1 methanol for 5 to 6 hours, were effective in the suppression of tumor growth. Flowcytometric histograms were made after 2 days of culture with these bioflavonoids. The histogram of cells treated with crude anthocyanin was similar to that of the control. Hydrolyzed anthocyanidins gave the elevation of S phase, suggesting a block in the step from S-phase to G2-phase. It seemed that the anthocyanidins contained in the grape rinds and red rice were effective on the suppression of cell growth as observed previously for anthocyanins extracted from the petals of higher plants.

Animals↗

PubMed1996

Effects of oxygen radical scavengers on connexins 32 and 26 expression in primary cultures of adult rat hepatocytes.

Although we recently reported our success in inducing and maintaining the gap junction proteins connexin 26 (Cx26) and connexin 32 (Cx32) in adult rat hepatocytes cultured in serum-free L-15 medium supplemented with epidermal growth factor, dimethylsulfoxide (DMSO) and glucagon, the mechanisms by which DMSO induces gap junctions are still not clear. It is known that DMSO is not only a differentiation reagent for various cells but also a powerful scavenger of oxygen radicals. In the present study, by using this culture system and the measurement of oxidative stress by the nitro blue tetrazolium (NBT) formazan assay, we have examined the effect of oxygen radical scavengers such as DMSO, dimethylthiourea (DMTU) and alpha-tocopherol on the expression of both Cxs and on gap junctional intercellular communication (GJIC), as compared to another differentiation reagent, hexamethylene-bis-acetamide (HMBA). DMSO and DMTU clearly inhibited the oxidative stress of the cultured hepatocytes, while alpha-tocopherol and HMBA did not. The expression of Cx26 and Cx32 in the cultured hepatocytes was markedly induced by DMSO and DMTU. Furthermore, extensive GJIC was also observed with DMSO and DMTU. These results suggest that the expression of gap junctions in the hepatocytes may be closely related to oxidative stress and that oxygen radical scavengers may be important substances in inducing this expression.

Acetamides↗

PubMed1996

Structural organization and promoter activity of the human ryudocan gene.

To better understand the regulation of ryudocan (syndecan-4) expression, we have determined the structural organization of the human ryudocan gene. The human ryudocan gene extends approximately 24 kilobases and is divided into five exons, which appear to be conserved in syndecan family members. Exon I encodes the signal peptide; exons II-IV, the extracellular domain; and exon V, the transmembrane and cytoplasmic domains, which are highly homologous among syndecan family members. Primer extension analysis showed that human ryudocan gene had a single transcription initiation site, located 3 bases upstream from the described cDNA [Kojima et al. (1993) BBRC 190, 814-822]. The 5'-flanking sequences of human ryudocan gene contain a TATA-like sequence as well as a variety of other potential binding sites for transcription factors, including Sp1, Ap-2, NF-kB, E-alpha H box, H4TF-2, and LBP-1, and were capable of functioning as a promoter. The determination of the human ryudocan gene structure will allow elucidation of constitutive, cell-specific, tissue-specific, and developmentally regulated expression.

Animals↗

PubMed1996

Recovery of mRNA expression of tryptophan 2,3-dioxygenase and serine dehydratase in long-term cultures of primary rat hepatocytes.

Expression of tryptophan 2,3-dioxygenase (TO) and serine dehydratase (SDH) has not previously been maintained or re-induced in long-term cultured hepatocytes. In the present study, we succeeded in inducing expression of TO and SDH mRNAs in adult rat hepatocytes cultured in serum-free L-15 medium supplemented with epidermal growth factor and 2% dimethyl sulfoxide (DMSO). After the start of culture, the expression of TO mRNA rapidly disappeared and at 96 h it was less than 10% of that at isolation. However, after the addition of 2% DMSO from 96 h, the transcript level gradually increased and reached about 40% of that of the isolated cells at day 14. In addition, the expression of TO mRNA was enhanced in cells treated with both 10(-5) M dexamethasone and 10(-7) M glucagon. In contrast, the expression of SDH mRNA decreased very rapidly and we could not detect it after 24 h of culture. Furthermore, 2% DMSO failed to induce it. However, when both 10(-5) M dexamethasone and 10(-7) M glucagon were added to the culture medium at day 9, we observed dramatic induction of SDH mRNA 24 h later. Primary hepatocytes cultured by this method could express and maintain highly differentiated hepatic functions for a long time. Thus, this in vitro system is suitable for the investigation of hepatic functions.

Animals↗

PubMed1996

Calcium-dependent phospholipid binding to the C2A domain of a ubiquitous form of double C2 protein (Doc2 beta).

Rabphilin 3A and Doc2 alpha are synaptic vesicle-associated proteins, and are thought to function as Ca2+ sensors in neurotransmitter release. If either rabphilin 3A or Doc2 alpha plays a role in membrane trafficking, like the synaptotagmins, then non-neural forms should be present. Here we describe the isolation of a mouse cDNA which encodes a novel Doc2 homologue (Doc2 beta) that is present in all tissues. The encoded protein, which is highly homologous to human Doc2 alpha (70% identity), is composed of 412 amino acids with a calculated relative molecular mass (M(r)) of 45,837. The sequence identity is especially high in two C2 domains (74% in C2A and 84% in C2B). Northern and Western blot analyses have shown that Doc2 beta is expressed in all cell lines and tissues tested. Ca(2+)-dependent phospholipid binding assaying of recombinant fusion proteins revealed that the single C2A domain, but not the C2B domain, of Doc2 beta binds phosphatidycholine and phosphatidylserine (2.5:1, w/w) liposomes. The binding is Ca(2+)-dependent, with an EC50 value of approximately 1 microM and a Hill coefficient of approximately 3, which are comparable to those of synaptotagmins, rabphilin 3A and Doc2 alpha. Our results suggest that Doc2 beta is involved in constitutive membrane trafficking.

Adaptor Proteins, Signal Transducing↗

PubMed1996

Expression of p21(waf-1/cip-1) is significantly induced in the livers of LEC rats with chronic liver injury.

It is reported that hepatocytes isolated from LEC rats with chronic liver injury show reduced growth activity in primary culture. To elucidate the molecular basis of this phenomenon, we examined expression of p21(waf-1/ciP-1) and p27, cyclin-dependent kinase inhibitors, by northern blot analysis. The expression of p21(waf-1/cip-1 ) in the LEC rat liver was 3-fold higher than that of age-matched SD rat liver, while there was no significant difference in p27 expression level. Western blot analysis also revealed a significant increase in p21(waf-1/cip-1) in the nuclear matrix fraction of the LEC rat liver. Immunohistochemically, p21(waf-1/cip-1) was detected in the nuclei of normal LEC rat hepatocytes, but not in those of hepatocellular carcinoma cells, suggesting selective growth of neoplastic hepatocytes.

Animals↗

PubMed1996

Eosinophilia in premature infants: correlation with chronic lung disease.

We attempted to clarify the possible pathophysiological significance of eosinophilia in bronchopulmonary dysplasia (BPD). The subjects studied were 17 premature infants, i.e. seven with respiratory distress syndrome (RDS) followed by bronchopulmonary dysplasia (the BPD group: four with stage IV and three with stage III BPD) and 10 infants without BPD (the non-BPD group), who comprised seven with RDS, two with meconium aspiration syndrome and one with transient tachypnea of the newborn. Peripheral eosinophil counts, the number of nuclei of eosinophils and serum eosinophilic cationic protein (ECP) levels, and ECP and polymorphonuclear leukocyte (PMN) elastase levels of intratracheal aspirates (TA) were determined once a week during the first 4 weeks of life. Peripheral eosinophil counts were higher in infants with BPD than those in the non-BPD group. Hypersegmented nuclei of peripheral eosinophils with more than four nuclei were more frequently present in the infants with BPD. A good correlation was observed between peripheral eosinophil counts and serum ECP levels. ECP levels of the TA in the infants with BPD were significantly elevated. There was a good correlation between ECP and PMN elastase levels of the TA. Lung tissue specimens of two infants of the BPD group, both of whom had patent ductus arteriosus (PDA), were obtained from the lower portion of the left lung when they underwent an operative procedure for PDA at 24 and 25 days of life, respectively. Immunohistochemical staining of eosinophil-derived granular major basic protein (MBP) was performed on the lung tissue specimens. Infiltration of a few MBP-staining eosinophils was observed on the specimens from both infants. Our results suggest that peripheral eosinophils in sick premature infants may be activated and appear to be correlated with the severity of BPD. Further studies will be needed to more clarify the physiological role of eosinophils in premature infants.

Blood Proteins↗

PubMed1996

Alterations in the DNA topoisomerase IV grlA gene responsible for quinolone resistance in Staphylococcus aureus.

A 4.2-kb DNA fragment conferring quinolone resistance was cloned from a quinolone-resistant clinical isolate of Staphylococcus aureus and was shown to possess a part of the grlB gene and a mutated grlA gene. S-80-->F and E-84-->K mutations in the grlA gene product were responsible for the quinolone resistance. The mutated grlA genes responsible for quinolone resistance were dominant over the wild-type allele, irrespective of gene dosage in a transformation experiment with the grlA gene alone. However, dominance by mutated grlA genes depended on gene dosage when bacteria were transformed with the grlA and grlB genes in combination. Quinolone-resistant gyrA mutants were easily isolated from a strain, S. aureus RN4220, carrying a plasmid with the mutated grlA gene, though this was not the case for other S. aureus strains lacking the plasmid. The elimination of this plasmid from such quinolone-resistant gyrA mutants resulted in marked increases in quinolone susceptibility. These results suggest that both DNA gyrase and DNA topoisomerase IV may be targets of quinolones and that the quinolone susceptibility of organisms may be determined by which of these enzymes is most quinolone sensitive.

Amino Acid Sequence↗