PubMed Health⌕ Search

Biomedical subjects

S Ohkubo

Publications and source records attributed to S Ohkubo.

At least 55 records · Page 3Linked to original sources
PubMed1995

Effects of norfloxacin on the retina in rabbits.

BACKGROUND: Fluoroquinolones have a strong affinity with melanin, and their ocular effects have been reevaluated. Norfloxacin, one of the fluoroquinolones, has broad-spectrum activity against aerobic gram-positive and gram-negative bacteria. We examined the retinal toxicity and intraocular pharmacokinetics of intravitreal norfloxacin in rabbits. METHODS: Twenty-three albino and 23 pigmented rabbits were divided into three groups to evaluate retinal toxicity and two groups to investigate the intraocular pharmacokinetics. Each of these five groups was further divided into two subgroups (albino rabbits and pigmented rabbits). RESULTS: With 500 micrograms norfloxacin, the oscillatory potential of the electroretinogram was transiently and selectively deteriorated in albino and pigmented rabbits, whereas the electroretinogram remained unchanged with 50 micrograms in pigmented rabbits. No changes were observed in the visual evoked potential or on histology of the retina 7 days after an intravitreal injection of 50 or 500 micrograms norfloxacin. The electroretinogram and the retinal histology became abnormal 7 days after four intravitreal injections of 500 micrograms norfloxacin at 7-day intervals. As regards the intraocular pharmacokinetics after an intravitreal injection, the norfloxacin concentration in the chorioretina was as high as that in the vitreous 3 h after injection and was much higher than that in the vitreous 7 days after injection. Similar results were obtained after multiple injections. CONCLUSIONS: These results indicate a high concentration of norfloxacin in the melanin-containing ocular tissues.

Animals↗
PubMed1995

Dissociation between the mitogenic effect and antitumor activity of seed extract from Aeginetia indica L.

The extract from seeds of Aeginetia indica L. (AIL), a parasitic plant, induces potent antitumor immunity against Meth A fibrosarcomas in BALB/c mice. AIL also possesses a thymocyte co-stimulatory effect in vitro with suboptimal dose of Con A, a B cell mitogenic effect, and stimulates AIL-primed CD4+ T cells to produce Th1-type cytokines. In this study, we investigated the relationship between mitogenicity and antitumor activity with AIL. When AIL was analyzed by SDS-PAGE, there was strong and diffuse staining in the region between 14 kDa and the bottom of polyacrylamide gel and it was unaffected when AIL was digested with proteinase K (PK) before SDS-PAGE. Some bands with different molecular mass were also found in silver-stained gel and they disappeared completely by incubating AIL with PK before SDS-PAGE. The in vitro thymocyte co-stimulatory and B cell mitogenic effects were not influenced by digesting AIL with PK but were completely suppressed by the oxidation of AIL with sodium periodate before culture. In contrast, the in vivo antitumor activity was completely abolished by PK, but it was not affected by periodate oxidation. We generated mAbs specific for AIL and investigated the influence on the antitumor activity of AIL in vivo. Around 60-80% of tumor-bearing mice failed to recover from a challenge tumor when they were treated with supernatants isolated from mAb-induced precipitation reactions. Immunoblotting (Western blotting) revealed that all the mAbs reacted exclusively with a 50-60 kDa protein and that this reactivity was not influenced after oxidizing the blots with sodium periodate. We demonstrated that AIL contains polysaccharides and proteins. The polysaccharides induced B cell mitogenic and thymocyte co-stimulatory effects in vitro, while the proteins, especially a 50-60 kDa protein containing non-carbohydrate epitopes recognized by the mAbs, mediated antitumor activity in vivo.

Animals↗
PubMed1995

Characterization of murine PACAP mRNA.

A murine PACAP precursor cDNA was isolated by screening a brain cDNA library. The amino acid sequence of the precursor was highly similar (from 81% to 93% similarity) to its rat, human, and ovine counterparts. The primary structure of murine PACAP is identical with those from sheep, humans, and rats, indicating that the mature PACAP is well conserved among mammals. Northern blot analysis revealed that the approximately 2.4 kb transcript for the PACAP precursor is expressed in murine brain. The verification that murine PACAP is identical to its human counterpart provides a rationale for physiological and pathophysiological studies of PACAP in mice.

Amino Acid Sequence↗
PubMed1995

Two enhancer elements for DNA replication of pSC101, par and a palindromic binding sequence of the Rep protein.

The minimal replication origin (ori) of the plasmid pSC101 has been previously defined as an approximately 220-bp region by using plasmids defective in the par region, which is a cis-acting determinant of plasmid stability. This ori region contains the DnaA binding sequence, three repeated sequences (iterons), and an inverted repeat (IR) element (IR-1), one of the binding sites of an initiator protein, Rep (or RepA). In the present study, we show that plasmids containing par can replicate at a nearly normal copy number in the absence of IR-1 but still require a region (the downstream region) between the third iteron and IR-1. Because par is dispensable in plasmids retaining IR-1, par and IR-1 can compensate each other for efficient replication. The region from the DnaA box to the downstream region can support DNA replication at a reduced frequency, and it is designated "core-ori." Addition of either IR-1 or par to core-ori increases the copy number of the plasmid up to a nearly normal level. However, the IR-1 element must be located downstream of the third iteron (or upstream of the rep gene) to enhance replication of the plasmid, while the par region, to which DNA gyrase can bind, functions optimally regardless of its location. Furthermore, the enhancer activity of IR-1 is dependent on the helical phase of the DNA double helix, suggesting that the Rep protein bound to IR-1 stimulates the activation of ori via its interaction with another factor or factors capable of binding to individual loci within ori.

Bacterial Proteins↗
PubMed1995

Gq/11 communicates with thromboxane A2 receptors in human astrocytoma cells, rabbit astrocytes and human platelets.

Western blot analysis was performed to clarify the presence of trimeric G protein subfamily in membranes derived from human astrocytoma cells (1321N1), cultured rabbit astrocytes and human platelets, using G protein antisera GS alpha, Gi alpha, Gq/11 alpha, and G beta were found to exist in membranes derived from human astrocytoma cells and rabbit astrocytes as well as human platelets. However, only small amount of G(o) alpha was detected in any membranes. Gq/11 alpha was expressed much more in human platelets than in human astrocytoma cells or rabbit astrocytes. 9,11-Epithio-11,12-methanothromboxane A2 (STA2), a stable analogue of thromboxane A2 (TXA2), activated GTPase in membranes derived from human astrocytoma cells, rabbit astrocytes and human platelets with different potencies. STA2-induced GTPase activation in human platelet membranes was partly inhibited by treatment with QL antibody at 0 degrees C for 90 min. Furthermore, STA2-induced GTPase activation in membranes derived from human astrocytoma cells and rabbit astrocytes were potently inhibited by treatment with QL antibody. The results obtained indicate that TXA2 receptors in human astrocytoma cells and rabbit astrocytes communicate with Gq/11 as well as in human platelets.

Animals↗
PubMed1994

Biosynthesis of human endothelins in transformants expressing cDNAs for human prepro-endothelins.

To understand the biosynthesis of the human ET family, three kinds of cloned cDNA for human prepro-endothelin-1 (prepro-ET-1), prepro-ET-2, and prepro-ET-3 were stably expressed in Chinese hamster ovary cells (CHO-K1). Immunoreactive (ir-) ET polypeptides in the culture media of the transformants were purified by reverse-phase high-performance liquid chromatography (HPLC) coupled with sandwich-type enzyme immunoassay (EIA). Amino acid sequencing and FAB mass spectrometry of the purified ir-ET polypeptides revealed the presence of human big ET-1 (1-38), big ET-2 (1-38), and big ET-3 (1-41)NH2 as intermediate forms. These results directly revealed the biosynthetic pathways of three human ETs at the peptide level.

Amino Acid Sequence↗
PubMed1994

[Intraocular penetration of oral levofloxacin in rabbits].

The intraocular penetration of levofloxacin was studied in albino and pigmented rabbits after oral administration of 14C-levofloxacin at a dose of 20 mg/kg. The radioactivity of the eyeball of albino rabbits in the autoradiogram was much higher at 1 hr than at 2 or 6 hr after administration. On the other hand, the distribution of radioactivity in pigmented rabbits was similar at 1, 2, and 6 hr after administration. The radioactivity levels in melanin-containing tissues such as the iris-ciliary body and the choroid-retina were significantly higher in pigmented rabbits than in albino rabbits 2 hr after oral administration. The radioactivity levels in the ocular tissues without melanin except for the cornea were similar in albino and pigmented rabbits. These results indicate that the intraocular pharmacokinetics of levofloxacin are markedly affected by its affinity for melanin.

Administration, Oral↗
PubMed1994

[Endophthalmitis caused by Paecilomyces lilacinus after cataract surgery: a case report].

A case of fungal endophthalmitis caused by Paecilomyces lilacinus after cataract surgery is reported. An 84-year-old woman had been suffering from diabetes mellitus and hypertension. She underwent an uncomplicated extracapsular cataract extraction with implantation of a posterior chamber intraocular lens in the right eye. Approximately one month after the surgery iritis was noted, and treated systemically and topically with corticosteroids and antibiotics. The inflammation was improved by these therapies at first, but worsened when the dose of oral corticosteroid was reduced. White fluffy exudates were present on the iris near the chamber angle with the hypopyon. Removal of the intraocular lens, vitrectomy and medicinal therapy were undertaken. The eye was salvaged but the final visual acuity was counting fingers. Paecilomyces lilacinus was identified from the intraocular specimen taken at the time of surgery. The clinical effects and the results of in-vitro sensitivity test showed that fluconazole and amphotericin B were ineffective, and that econazole, thimerosal and miconazole were effective.

Aged↗
PubMed1993

A phase II study of combination therapy with 5'-deoxy-5-fluorouridine and cisplatin in the treatment of advanced gastric cancer with primary foci.

BACKGROUND: 5'-Deoxy-5-fluorouridine (5'-DFUR, doxifluridine) is a recently developed prodrug of oral 5-fluorouracil (5-FU), which is used clinically in Japan for the treatment of gastric, colorectal, and breast cancer. 5-FU has been reported to act synergistically with cisplatin (CDDP) in experimental and clinical studies. The authors conducted a multicenter Phase II study of combination therapy with 5'-DFUR and CDDP to evaluate the therapeutic usefulness of this regimen in the treatment of unresectable and advanced gastric cancers with primary foci. None of the patients had previously undergone chemotherapy. Their ages ranged from 27 to 75 years and performance status was grade 0 to 3. METHODS: 5'-DFUR (1400 mg/m2/d) was administered orally on days 1 through 4 and 15 through 18, and CDDP (80 mg/m2/d) was injected intravenously on day 5. This treatment cycle was repeated every 4 weeks. An independent panel of specialists evaluated the clinical response. RESULTS: Fifty-one patients were studied. Clinical evaluation of response was possible in 43 patients who met the protocol requirements. The overall response rate was 50.0% (14 of 28, 95% confidence limits, 30.7%-69.4%) for patients with measurable lesions. The median duration of response was 5.2 months (156 days). The overall median survival time was 8.9 months (268 days) for evaluated patients. Therapeutic toxicity of World Health Organization (WHO) grade > or = 3 was manifested as anorexia and nausea or vomiting in 20.9% and 18.6% of the patients, respectively. However myelotoxicity and nephrotoxicity of WHO grades 3 and 4 occurred in less than 10% of the patient group. No drug-related mortality occurred. CONCLUSIONS: Combined therapy with 5'-DFUR and CDDP is a safe and effective treatment regimen for advanced gastric cancers with primary foci which stresses the patient's quality of life, especially when used in an outpatient setting.

Adenocarcinoma↗
PubMed1993

Expression in baculovirus vector system of the nucleocapsid protein gene of rinderpest virus.

The rinderpest (RV) nucleocapsid (NP) gene segment was inserted into the genome of Autographa californica nuclear polyhedrosis virus (AcNPV) adjacent to the polyhedrin promoter. The expression of NP protein in Sf9 cells was confirmed by indirect immunofluorescence and by Western blotting analysis with monoclonal antibodies. Recombinant RV-NP protein was purified by ultracentrifugation on a sucrose density gradient, and used as an antigen for an enzyme linked immunosorbent assay to detect anti RV-NP antibody. Both IgM and IgG antibodies against RV-NP were detected in the sera of rabbits infected with the L strain of RV. The pattern of development of IgG anti RV-NP antibody closely correlated with that of virus neutralizing antibody. In rabbits inoculated with recombinant vaccinia virus expressing RV-H gene (RRV-H), anti RV-NP was not detected. The results indicated that the baculovirus vector system can be used for the preparation of the diagnostic antigen of rinderpest as well as to distinguish between natural infection and vaccination with RRV-H.

Animals↗
PubMed1993

Production of immunoreactive pituitary adenylate cyclase activating polypeptide (PACAP) by human neuroblastoma cells, IMR-32: detection and characterization with monoclonal and polyclonal antibodies against different epitopes of PACAP.

Sensitive and specific two-side enzyme immunoassays (two-site EIAs) for pituitary adenylate cyclase activating polypeptides, PACAP38, and PACAP27, have been established using six monoclonal antibodies against PACAP38, and a rabbit antibody against a C-terminal portion of PACAP27. In extracts of rat hypothalamus, these EIAs detected not only PACAP38 and PACAP27 but also an immunoreactive (ir-) PACAP lacking an epitope of a monoclonal antibody, PA-1C, which recognizes the C-terminal portion of PACAP38. By the use of these EIAs, it was found that one of the human neuroblastoma cell lines, IMR-32, produced ir-PACAP. In reverse-phase (RP-)HPLC, intracellular and extracellular ir-PACAPs were separated into two peaks, of which one was eluted at a position close to that of PACAP38 and the other in rather hydrophobic fractions. Those ir-PACAPs also lacked PA-1C epitope of PACAP38. SDS-PAGE and immunoblot analysis of the two peaks of the RP-HPLC indicated that they consisted of several components including those with apparent molecular weights of 6.5 k and 10 k for the first peak ir-PACAP, and 14 k and 20 k for the second peak ir-PACAP. These results indicate that IMR-32 produces a precursor of PACAP and related peptides generated in various processing steps. Although the significance of the modification in the C-terminus of PACAP38 is unknown, IMR-32 may be a cell line useful for studying the regulation of the biosynthesis of PACAP.

Animals↗
PubMed1993

Minimal essential origin of plasmid pSC101 replication: requirement of a region downstream of iterons.

The minimal replication origin (ori) of the plasmid pSC101 was defined as an about 220-bp region under the condition that the Rep (or RepA) protein, a plasmid-encoded initiator protein, was supplied in trans. The DnaA box is located at one end of ori, as in other plasmids, like mini-F and P1. The other border is a strong binding site (IR-1) of Rep which is palindromic sequence and lies in an about 50-bp region beyond the repeated sequences (iterons) in ori. This IR-1 is located just upstream of another strong Rep binding site (IR-2), the operator site of the structure gene of Rep (rep), but its function has not been determined. The present study shows that the IR-1 sequence capable of binding to Rep is essential for plasmid replication with a nearly normal copy number. Furthermore, a region between the third iteron and IR-1 is also required in a sequence-specific fashion, since some one-base substitution in this region inactivate the origin function. It is likely that the region also is a recognition site of an unknown protein. Three copy number mutations of rep can suppress any one-base substitution mutation. On the other hand, the sequence of a spacer region between the second and the third iterons, which is similar to that of the downstream region of the third iteron, can be changed without loss of the origin function. The requirement of the region downstream of iterons in pSC101 seems to be unique among iteron-driven plasmid replicons.

Bacterial Proteins↗
PubMed1993

Elevated serum fucose levels in idiopathic nephrotic syndrome.

We measured serum fucose concentrations in 34 children with idiopathic nephrotic syndrome (INS), 23 with chance proteinuria and/or hematuria (CPH), and 20 healthy children as controls. The serum fucose levels in the patients with INS were significantly elevated at onset (14.0 +/- 3.7 mg/dl, n = 4, p < 0.02), in relapses (19.1 +/- 3.7 mg/dl, n = 10, p < 0.001), and in remission (13.9 +/- 7.0 mg/dl, n = 30, p < 0.01) as compared with CPH patients (10.6 +/- 3.2 mg/dl, n = 23) and controls (9.1 +/- 3.1 mg/dl, n = 20). Those in remission were further divided into 2 groups and the mean fucose concentrations were significantly different in the two remission groups: 20.1 +/- 4.6 mg/dl in 14 patients whose blood samples were taken within one week of remission and 9.0 +/- 2.9 mg/dl in the other 16 patients whose samples were taken at 1 to 6 months of remission. The mean value in the latter remission group was significantly lower in comparison with the former group, but not different from the controls. Gel-chromatography of serum samples from patients with INS revealed a single peak of fucose in the high molecular fraction, and this was also found in the same fractions of serum inhibitor of lymphocyte blastogenesis in INS. We concluded that serum fucose concentrations are elevated in INS patients and that, because of the large molecular weight, the fucose is probably in a form bounded to some glycoproteins in the serum. Considering various reports on fucose, serum fucose may be associated with immunodepression in INS patients.

Child↗
PubMed1992

Expression of human pituitary adenylate cyclase activating polypeptide (PACAP) cDNA in CHO cells and characterization of the products.

cDNA encoding human PACAP precursor was expressed in non-neuroendocrine Chinese hamster ovary cells, CHO-K1, The cells were transfected with expression vector (pTS705) containing the human PACAP cDNA by electroporation. A cell line which produced more than 80 ng/ml of immunoreactive PACAP (ir-PACAP) into the conditioned medium was established. RP-HPLC analysis of culture medium of this established cell line exhibited the presence of two types of PACAP, i.e. PACAP38 and PACAP27. At the same time, it was also revealed that immunoreactive PACAP-related peptide (ir-PRP) was secreted into the cultured medium. The ir-PACAPs were confirmed to ahve biological activities such as induction of cAMP and neurite outgrowth in rat pheochromocytoma PC12h cells.

Adenylyl Cyclases↗
PubMed1992

Structure of the human pituitary adenylate cyclase activating polypeptide (PACAP) gene.

The human gene encoding pituitary adenylate cyclase activating polypeptide (PACAP) was isolated and its nucleotide sequence was determined. By comparison with a human PACAP cDNA, the exon/intron organization of PACAP gene was determined. The last exon encoded the longer form of PACAP, PACAP38 and 3'-untranslated sequences, suggesting that the shorter form of PACAP, PACAP27 is not generated by alternative splicing mechanisms. The 5'-flanking region of the PACAP gene contains several sequence motifs homologous to CRE, TRE, and GHF-1. On the basis of DNA isolated from mouse A9 microcell hybrid clone containing a single human chromosome, the PACAP gene was assigned to human chromosome 18. Furthermore, we determined the locus of the gene to be 18p11 by the chromosomal in situ hybridization technique.

Amino Acid Sequence↗