Biomedical subjects
Publications and source records attributed to M Kubota.
The gene encoding trehalose synthase (catalyzing the conversion of maltose into alpha, alpha-trehalose by intramolecular transglucosylation) was cloned from Pimelobacter sp. R48. Sequence analysis revealed a 1719-bp synthase gene and a 573-residue amino-acid sequence. The 220 N-terminal residues were homologous to those of maltases from Saccharomyces carlsbergensis and Aedes aegypti.
A 5.1-kbp genomic DNA fragment was cloned from trehalose-producing bacterium Arthrobacter sp. strain Q36. Sequence analysis of the DNA fragment revealed two open reading frames of 2325 and 1794 bp, encoding maltooligosyltrehalose synthase (TreY) and maltooligosyltrehalose trehalohydrolase (TreZ), respectively. The 3' end of treY overlaps with the 5' end of treZ by one nucleotide, and it is suggested that treYZ constitutes and operon. The deduced amino acid sequences of both enzymes have several regions common to amylolytic enzymes belonging to an 'alpha-amylase family'.
Explore the source record for details and available documents.
The number of reported cases of malignancy developing in growth hormone (GH) users worldwide has increased to more than 40. However, the causal relationship between GH administration and the occurrence of malignancies is still uncertain. We investigated somatic cell mutation frequencies (Mfs) or variant frequency (Vf) at three gene loci in patients with pituitary dwarfism receiving GH therapy to clarify the genetic effect of GH. Eighty-eight patients receiving GH therapy for at least 3 months and 42 age-matched healthy controls were studied. Mfs at hypoxanthineguanine phosphoribosyltransferase (HPRT) and T-cell receptor (TCR) loci in GH users were not significantly higher than in the controls. Although a few patients seemed to have a slightly increased Vf at the glycophorin A (GPA) locus, the difference was not statistically significant. In addition, there was no tendency for the Mfs (Vf) at these loci to increase with the duration of the GH therapy. These data seem to exclude the possibility that GH induces genetic instability in patients with pituitary dwarfism who are receiving GH therapy.
The effect of mutational loss of thymidine kinase (TK) on the sensitivity to alkylating agents was investigated in promyelocytic, HL-60, and T-lymphoblastoid, Molt-3, human leukemia cell lines. Although both cell lines exhibited approx. 1% residual TK activity, only HL-60 TK deficient cells had a decreased intracellular TTP pool, i.e., 20% of that of the wild-type. When treated with N-methyl-N'-nitronitrosoguanidine or ethyl methanesulfonate, HL-60 TK deficient cells showed significantly increased killing and mutation frequencies at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus relative than did wild-type. Pretreatment of cells with O6-benzylguanine, an inhibitor of O6-alkylguanine-DNA alkyltransferase, partially abolished those differences. Molt-3 wild-type and TK deficient cells had similar cell survivals and HGPRT mutation frequencies following treatment with alkylating agents. These results indicate that TK deficiency, only when a concomitant decrease of TTP pool is detected, plays a pivotal role in the sensitivity to the cytotoxic and mutagenic effects of alkylating agents.
Treatment of circulating human neutrophils with recombinant human granulocyte colony-stimulating factor (rhG-CSF) for 30 min augmented superoxide generation and chemotaxis induced by N-formylmethionyl-leucyl-phenylalanine (fMLP) in a dose dependent manner. When neutrophils were treated with 1 microM of methotrexate (MTX) for 60 min after incubation with rhG-CSF (10 ng/ml), the effects of rhG-CSF on superoxide generation and chemotaxis were inhibited by approximately 49 and 29%, respectively. Although inhibitory effects of MTX were also seen in neutrophils not pretreated with rhG-CSF, the degree of inhibition was much less. The addition of either hypoxanthine or guanosine at a concentration of 100 microM to the culture medium significantly attenuated the effects of MTX. However, in neutrophils obtained from a patient with Lesch-Nyhan syndrome, which lacked hypoxanthine-guanine phosphoribosyl transferase activity neither hypoxanthine nor guanosine had any rescue effect. These results suggest that MTX inhibits superoxide generation and chemotaxis in rhG-CSF-activated neutrophils, at least in part, by disturbing purine nucleotide biosynthesis.
A method is described for the determination of Tl in soils by FAAS, involving extraction of Tl from 5 g of soil by digestion with HClO(4)/HNO(3) followed by separation of the extracted Tl into 5 mL of diisopropylether from HBr solution, including Ce(SO(4))(2). Tl in the organic phase is determined by direct aspiration into the spectrophotometer. The percentage relative standard deviation (% RSD) for 5 replicate samples is about 1%. The detection limits (S/N = 3) of this method are 0.001 mg/L for aqueous solution and 0.02 mg/kg DW for soil, when 50 mL of soil solution corresponding to 2.5 g soil are used. The Tl concentration even of unpolluted soils can be determined. The method was shown to be unaffected by the presence of various ions in soil and was able to recover nearly 100% Tl added to soils. The arithmetic mean (range) of 18 Japanese unpolluted surface soils was 0.33 (0.10-0.56)mgTl/kg DW.
Substance P is known to elicit diverse actions via activating multiple subtypes of tachykinin receptors, and these actions appear to be involved not only in synaptic transmission but also in synaptic plasticity during development of the mammalian central nervous system. The availability of sensitive quantitation of individual tachykinin receptor subtypes is crucial for elucidating the physiological function specifically mediated by activation of a particular receptor subtype. We thus attempted to develop an assay to determine the level of messenger RNA molecule encoding the neurokinin-1-type tachykinin receptor and apply it for assessment of developmental changes in the neurokinin-1 receptor gene expression in the rat brain to explore the role of tachykinin receptors during ontogeny. The assay was designed to use a competitive reverse transcription-polymerase chain reaction co-amplifying endogenous neurokinin-1 receptor messenger RNA and internal standard, which enabled specific quantification of the number of neurokinin-1 receptor transcripts, ranging from 3.1 x 10(3) to 1.3 x 10(5) molecules/microgram total RNA. The levels of neurokinin-1 receptor gene expression were examined in three different brain regions of the rat aged 0-56 days after birth. The order of neurokinin-1 receptor messenger RNA expression was hippocampus > cerebral cortex > > cerebellum at all ages examined except postnatal day 0, where its expression was more abundant in the cerebral cortex than in the hippocampus. From postnatal day 3 onward, the hippocampus contained 140-160% of the cortical levels. Although the tachykinin receptor expression in the cerebellum was too low to be accurately assessed by conventional techniques, our assay enabled us to determine the amount of cerebellar neurokinin-1 receptor messenger RNA that changed in the range 7-23% of the cortical level during postnatal development. A prominent feature revealed by this assay is that the neurokinin-1 receptor gene expression in the rat brain is developmentally regulated. The hippocampus displayed a transient peak of neurokinin-1 receptor messenger RNA at postnatal day 3 and a subsequent gradual decrease. In the cerebral cortex, the amount of the message was highest at birth, and was followed by a moderate decrease during postnatal development. At 56 days after birth, the expression levels in both brain regions were down-regulated to approximately 50% of their maximal levels. The transitory pattern of gene expression was also observed in the cerebellum. The results of this study demonstrate that the reverse transcription-polymerase chain reaction-based assay is useful to quantitate precisely the neurokinin-1 tachykinin receptor message in limited tissue samples derived from discrete brain regions. Together with previous findings, the increased level of neurokinin-1 receptor messenger RNA expression in immature rat brain shown by the present analysis suggests that the neurokinin-1-type tachykinin receptor may play a role in the synaptic plasticity associated with morphological and functional development of the mammalian CNS.
It is still controversial as to whether physiological portal insulin delivery has metabolic advantages over peripheral insulin delivery. To clarify this issue, glycemic regulation during intravenous (IVGTT) and oral (OGTT) glucose tolerance tests and hyperglycemic clamp studies with either peripheral or portal glucose infusion was investigated in left-segmentally pancreatectomized dogs with portal ([PPx] n = 7) or systemic ([Tx] n = 7) venous drainage of the remaining pancreas. In Tx dogs, systemic diversion of pancreatic venous effluent was accomplished by gastroduodenal-caval shunt. Data obtained were compared with those in normal control dogs ([NC] n = 7). The loss of pancreatic beta-cell mass in PPx dogs decreased insulin responses to peripheral and portal glucose loads. In contrast, Tx dogs showed insulin responses comparable to those of NC dogs to glucose loads via both routes. Against peripheral glucose loads (IVGTT and hyperglycemic clamp with peripheral glucose infusion), PPx and Tx dogs showed deteriorated glucose handling. Against portal glucose loads (OGTT and hyperglycemic clamp with portal glucose infusion), deteriorated glucose handling was observed in Tx dogs, but not in PPx dogs. Deterioration in glycemic regulation against portal glucose loads in left-segmentally pancreatectomized dogs with peripheral insulin delivery but not in pancreatectomized dogs with portal delivery indicates that intraportal hyperglycemia and hyperinsulinemia are essential for promoting hepatic glucose handling.
Nail regeneration was studied in 48 digits replanted after amputation through the distal phalanx. Twenty-seven were amputated through Tamai's zone I, at the nailbed level, and 21 digits were amputated through zone II, proximal to the nail. The nails of 9 digits in zone I and 14 in zone II showed almost normal nail regeneration. We observed that replantation after amputations distal to the lunula will show near normal nail regeneration if there is minimal postoperative circulatory disturbance. Amputations proximal to the lunula, however, have a greater chance of causing damage to the germinal matrix, which results in more problems with nail growth.
We investigated serum levels of interferon alpha, interferon gamma, tumour necrosis factor alpha, interleukin-2 (IL-2) and interleukin-6 (IL-6) in patients with necrotizing lymphadenitis (Kikuchi's disease) (NL). Four male patients, diagnosed as having NL following biopsy of the affected lymph nodes and by the clinical course, were included in this study. All four patients had higher than normal serum interferon gamma and IL-6 levels during the acute phase, which returned to normal levels during the convalescent phase. Interferon alpha, tumour necrosis factor alpha and IL-2 were, however, within the normal ranges. Our findings indicate the possibility that interferon gamma and IL-6 may play an important role in the pathogenesis of NL.
No effective therapy has yet developed for liver fibrosis by directory inhibiting the accumulation of extracellular matrix. The effect of a newly synthesized prolyl4-hydroxylase (PH) inhibitor, HOE 077 (pyridine-2, 4-di-carboxylic-di(2-methoxyethyl)amide), was examined using the model of choline-deficient L-amino acid (CDAA) defined diet-induced liver fibrosis in 16-week-old male Wistar rats. HOE 077 at doses up to 200 ppm prevented fibrosis in a dose-dependent manner, as indicated by reduced hydroxyproline content in liver as well as inhibition of increased serum fibrotic markers (PIIIP, 7S, hyaluronic acid). HOE 077 at 200 ppm reduced expression of type III procollagen alpha 1, messenger RNA (mRNA) in the liver, with a good correlation with serum PIIIP and hydroxyproline content of the liver. Histologically, HOE 077 at 200 ppm also reduced proliferation of myofibroblastlike cells (activated Ito cells). These results indicate that a PH inhibitor can prevent fibrosis by inhibiting not only the hydroxylation of proline but also the activation of Ito cells, which are considered the main collagen-producing cells, resulting in reduced expression of procollagen mRNA.
To better define the sphingolipid metabolism during focal brain ischemia, levels of ceramide, sphingomyelin, cerebroside and gangliosides were determined in rat cerebral cortex during focal ischemia produced by occlusion of the middle cerebral artery. Sphingomyelin began to decrease at 2 hours of ischemia and continued to decrease for 96 hours. In contrast, ceramide increased at 6 hours and increased to 4.2-fold at 96 hours after ischemia, and the fatty acid composition of ceramide was solely nonhydroxylated fatty acid similar to sphingomyelin. Hydroxylated fatty acid-linked cerebroside decreased at 6 hours of ischemia, whereas any significant decrease of nonhydroxylated fatty acid-linked cerebroside didn't occur for 96 hours of ischemia. There were no measurable changes in the levels of gangliosides. These results suggested that ceramide was produced in the cerebral cortex by the breakdown of sphingomyelin during early ischemia.
Intolerable malodor emanating from ulcerated tumors as a result of anaerobic infection is a serious problem in the management of advanced and recurrent breast cancer. Metronidazole can control this malodor, but its oral use may cause adverse reactions. We therefore formulated a metronidazole gel, since no equivalent preparation is commercially available in Japan, and used it in five female patients (four with advanced cancer and one with recurrent cancer) admitted to our hospital between March 1994 and July 1995. The patients were aged between 47 and 71 (median: 59) years, and the duration of morbidity in the four patients with advanced cancer ranged from 10 months to four years. In three patients, the tumors were larger than 10 cm x 10 cm. Metronidazole gel was applied to the surface of ulcerated tumors once or twice daily. Independent assessments by the patient, doctor and nurse were unanimous, and revealed that the malodor was alleviated in one patient after three days, and removed in four patients after two to five (median: four) days of metronidazole gel treatment. Culture of swabs showed a decrease or disappearance of anaerobic colonies. Adverse reactions characteristic of metronidazole did not occur. The topical use of metronidazole in a gel form will improve the quality of life for patients with malodorous ulcerated tumors and facilitate intensive treatment of the underlying disease.
A case of atypical proliferative glomerulonephritis (PGN) without mesangial immunoglobulin (Ig) A deposition (so-called non-IgA PGN) showing exacerbation of heavy proteinuria under long-term observation is reported. Examinations of first renal biopsy specimens revealed membranoproliferative glomerulonephritis (MPGN)-like findings. Urinary protein excretion completely disappeared after treatment with prednisolone (PSL) and an antiplatelet drug, i.e., dipyridamole. Negative reaction for urinary protein continued for more than 10 years. Fourteen and a half years after the first biopsy, the patient had heavy proteinuria again. Results of the second renal biopsy showed marked proliferation of glomerular mesangial cells. Under electron microscopy, lobulation and double contours of the glomerular capillary walls were also observed segmentally. Depositions of IgG, IgM, IgA, and C3 were observed mainly in the glomerular capillary walls, but not in the mesangial areas; however, these findings were not compatible with IgA nephropathy or MPGN. No hypocomplementemia was observed during the clinical course. The patient was treated with 30 mg of PSL and 75 mg of dipyridamole daily and showed a good response to such treatment. It appears that this patient had a rare case of atypical non-IgA PGN.
Explore the source record for details and available documents.
We here report a case of periventricular leukomalacia (PVL) associated with hypocarbia which remained even after extubation. The patient had no risk factors affecting PVL development other than hypocarbia. We consider that the irregular tachypnea, which remained after extubation might be attributable to overdriving of ventilation of central neurogenic origin. Our patient's clinical course suggests that sodium bicarbonate drip infusion is a very effective way to alter the set point of respiratory neuronal drive of the patient with central neurogenic hyperventilation.
PURPOSE: To evaluate the usefulness of a radiographic system with monochromatic synchrotron radiation to depict small vessels and peripheral secretory ducts. MATERIALS AND METHODS: Radiography of various organs was tested in 14 anesthetized dogs and pancreatography was performed in an excised human pancreas by using the following system: monochromatic synchrotron radiation with an energy level just above the k absorption edge of iodine as an x-ray source and a high-definition TV system with a high-light-sensitivity image pick-up tube camera coupled with a fluorescent screen as a detector. RESULTS: This system allowed depiction of small vessels (diameter < 50-100 microns) of the heart (penetrating transmural artery), brain (perforating arteries that arise directly in the circle of Willis), and intestinal organs (vasa recta and their submucosal communications) and of small branches (down to the fifth order) of the pancreatic duct. CONCLUSION: The synchrotron radiation system may be useful for evaluating microcirculatory disorders and early-stage malignant tumors in various human organs.