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Biomedical subjects

H Kodama

Publications and source records attributed to H Kodama.

At least 307 records · Page 17Linked to original sources
PubMed1994

Generation of lymphohematopoietic cells from embryonic stem cells in culture.

An efficient system was developed that induced the differentiation of embryonic stem (ES) cells into blood cells of erythroid, myeloid, and B cell lineages by coculture with the stromal cell line OP9. This cell line does not express functional macrophage colony-stimulating factor (M-CSF). The presence of M-CSF had inhibitory effects on the differentiation of ES cells to blood cells other than macrophages. Embryoid body formation or addition of exogenous growth factors was not required, and differentiation was highly reproducible even after the selection of ES cells with the antibiotic G418. Combined with the ability to genetically manipulate ES cells, this system will facilitate the study of molecular mechanisms involved in development and differentiation of hematopoietic cells.

Animals↗
PubMed1994

Adhesion of NFS-60 myeloid leukemia cells to MC3T3-G2/PA6 stromal cells induces granulocyte colony-stimulating factor production.

To examine the interaction between immature myeloid cells and stromal cells in the induction of granulocyte colony-stimulating factor (G-CSF) production, stromal cells of the MC3T3-G2/PA6 (PA6) murine cell line, which has preadipocyte characteristics and can support hematopoiesis, were cocultured with various myeloid cell lines and G-CSF mRNA expression was examined by Northern and reverse transcriptase-polymerase chain reaction analyses. A significant amount of G-CSF mRNA was induced by the culture of an interleukin-3/G-CSF-dependent murine myeloid leukemia cell line, NFS-60, on PA6 stromal cells for 16 hours. Using a G-CSF-dependent subline of DA-1 (DA-1N), the biologic activity of G-CSF was also detected in PA6/NFS-60 coculture supernatants, but not in the culture supernatant of PA6 or NFS-60 alone. Direct contact of NFS-60 cells with the PA6 stromal layer was essential for the induction of G-CSF mRNA, as indicated by the following observations: (1) NFS-60 cells efficiently adhered to PA6 cells; (2) medium conditioned by NFS-60 cells did not contain the activity to induce G-CSF mRNA in PA6 cells; and (3) induction of G-CSF mRNA was not observed when NFS-60 cells were separated from PA6 cells by a microporous membrane (0.45-microns pore size). Several other myeloid cell lines, including FDC-P2, 32Dcl3, WEHI-3, and DA-1, did not induce G-CSF mRNA expression after the coculture with PA6 cells, although significant numbers of these cells adhered to PA6 cells. Therefore, NFS-60 cells may express or overexpress a molecule that is involved in adhesion-mediated induction of G-CSF production.

Animals↗
PubMed1994

Liquid chromatographic-mass spectrometric analysis of N-acetylamino acids in human urine.

Liquid chromatography-atmospheric pressure chemical-ionization mass spectrometry (LC-APCI-MS) was used for the analysis of N-acetylamino acids that could not be determined with an amino acid analyzer. LC-APCI-MS could directly detect the protonated molecular ions of various synthetic N-acetylamino acids, distinguishing N-acetylserine from O-acetylserine and N(alpha)-acetyllysine from N(epsilon)- acetyllysine. Furthermore, N-acetylasparagine, N-acetylaspartic acid, N-acetylglutamine and N-acetylglutamic acid were identified in normal human urine. The assay data for N-acetylaspartic acid agree with a previous report using gas chromatography-mass spectrometry. These results demonstrate the usefulness of the apparatus described above for the analysis of N-acetylamino acids in biological samples.

Acetylation↗
PubMed1994

Expression of a heterodimeric (placental-intestinal) hybrid alkaline phosphatase in KB cells.

A hybrid heterodimeric alkaline phosphatase expressed in KB cells, consisting of placental and intestinal (fetal) subunits, was purified by use of two different immunoaffinity columns using the monoclonal antibodies 2HIMS-1 and HPMS-1. The closely related subunits were found to yield a dimeric active enzyme glycosylated as the mature heterodimeric forms. This enzyme displays intermediate properties to the placental and intestinal (fetal) isozymes with regard to heat stability, inhibition patterns with amino acids and amino acid derivatives, as well as reactivity with monoclonal antibodies specific for human alkaline phosphatase isozymes. Peptide fragments obtained from the hybrid enzyme after cyanogen bromide cleavage belong to either the placental or intestinal (meconial) isozyme as evaluated by SDS polyacrylamid gel electrophoresis, and the N-terminal amino acid sequences, corresponding to the placental and intestinal subunits, can be identified in the peptide fragments. By N-glycanase digestion or tunicamycin treatment, the molecular mass of the subunits was reduced to 62 kDa compared to 69 kDa for the native ones. The results confirm that some cell lines can synthesize hybrid alkaline phosphatases.

Alkaline Phosphatase↗
PubMed1994

Expression of Mac-2 antigen in the preosteoclast and osteoclast identified in the op/op mouse injected with macrophage colony-stimulating factor.

Osteoclast deficiency in op/op mice is cured by a single injection of 5 micrograms recombinant human macrophage colony-stimulating factor (rhM-CSF). In this study, we found that mouse osteoclasts are positive for Mac-2 antigen, but not for F4/80, MOMA-2, Mac-1, or BM8 antigen. By using F4/80 and MOMA-2 monoclonal antibodies, we confirmed the absence of mature macrophages in the femora of op/op mice and found that multiple injections of rhM-CSF are required for the recruitment of macrophages in the bones. After a single rhM-CSF injection, we found Mac-2 positive mononuclear cells in the femora of op/op mice. The time course of the appearance of Mac-2-positive cells was very similar to that of tartrate-resistant acid phosphatase (TRAP)-positive cells. In bone sections prepared from the mutant mice that received rhM-CSF 3 days earlier, 91% of the TRAP-positive mononuclear cells were also positive for Mac-2 antigen. These results demonstrate the expression of Mac-2 antigen in preosteoclasts. The antigen was detected on the plasma membrane of preosteoclasts, as well as in their cytoplasm and nucleus, and in the extracellular matrix in the space between the cells and bone. Since Mac-2 is a galactose-specific lectin, a potential role of the lectin in cell-cell and cell-matrix adhesion during osteoclast differentiation is suggested.

Animals↗
PubMed1994

Development of hyperglycaemia and insulin resistance in conscious genetically diabetic (C57BL/KsJ-db/db) mice.

A bolus injection of insulin dose-dependently reduced plasma glucose levels in genetically diabetic (db/db) mice and their normoglycaemic litter-mates (+/+ mice) aged 5, 8 and 12 weeks. Compared between the groups, the dose-response curves showed that insulin resistance was already present in the 5-week-old db/db mice when they were still normoglycaemic. The minimum effective dose of insulin was lower in the +/+ (32 micrograms/kg) than in the db/db (100 micrograms/kg) mice and the maximum response which was obtained at 320-1000 micrograms/kg of the hormone was higher in the former (about 80%) than in the latter (about 55%). Although the basal plasma glucose levels in the db/db mice were significantly increased with age as compared with those in the +/+ mice, the insulin response curves were identical in the db/db mice from 5 to 12 weeks of age. The number of insulin binding sites were significantly decreased by 22-50% (5-12-week-old) in the liver plasma membrane from the db/db mice compared with that from the +/+ mice, while its affinity was not significantly changed between the groups. Streptozotocin (100 mg/kg, i.p.) treatment increased the number of insulin receptors in the db/db mice to a number comparable with those in the +/+ mice. Coinciding with the change, the hypoglycaemic action of insulin was slightly enhanced in the streptozotocin-treated db/db mice compared with that in non-treated db/db mice, but was still considerably depressed when compared with that in +/+ mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
PubMed1994

Effect of chlamydial antibodies on the outcome of in vitro fertilization (IVF) treatment.

OBJECTIVE: Our objective was to study the relationship between the presence of Chlamydia trachomatis antibodies and the success of IVF treatment. DESIGN: We evaluated 118 in vitro fertilization and embryo transfer (IVF-ET) treatment cycles from 51 couples with a history of infertility lasting for at least 2 years. All women starting a treatment cycle had their serum chlamydial antibody titers measured by indirect immunofluorescent technique. All patients received similar ovarian stimulation regimens and the oocytes collected were inseminated with similar concentrations of motile sperm. Clinical data from couples where the female partner had C. trachomatis ab titers > or = 40 have been compared with the equivalent data from couples where the female partner had C. trachomatis ab titers < 40. RESULTS: There was no statistically significant difference between the two groups concerning age, infertility period, oocytes collected, oocytes fertilized, and fertilization rate, and the pregnancy rates were comparable. CONCLUSION: Previous exposure to C. trachomatis did not alter the success rate of IVF-ET.

Adult↗
PubMed1994

Stromal cell-dependent bone marrow culture with a nearly protein-free defined medium.

We examined the ability of a defined medium mSFO2 to support stromal cell-dependent B lymphopoiesis and myelopoiesis. Both ST2 and PA6 stromal cell lines were able to form monolayers in the presence of mSFO2 alone. While the ST2 monolayer could last for long period of time, either bFGF or EGF were required for the maintenance of the PA6 monolayer with mSFO2. mSFO2 did support the generation of distinct stages of B precursors on the ST2 layer and this culture condition was efficient enough to be used for limiting dilution assay of in vitro clonable B progenitors. While ST2 cell line failed to support long-term myelopoiesis with mSFO2, PA6 in combination of bFGF was able to support sustained generation of various hematopoietic progenitors. Indeed, 20 times increase of IL-3 reactive colony-forming cells was induced upon culturing the normal bone marrow cells on the PA6 layer for 8 days with mSFO2. Because total protein concentration of mSFO2 is only 10 micrograms/ml consisting of transferrin and insulin, the present result that mSFO2 is able to support the proliferation of normal hematopoietic progenitor cells would make an important step towards the standardization of bone marrow culture.

Animals↗
PubMed1994

Immunohistochemical demonstration of intestinal-type alkaline phosphatase in stomach tumors induced by N-methyl-N'-nitro-N-nitrosoguanidine in rats.

A polyclonal antibody against rat intestinal-type alkaline phosphatase (I-ALP) was generated and proven to be applicable immunohistochemically to paraffin-embedded sections. Expression of I-ALP in normal tissues, intestinal metaplasia and stomach tumors induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was then investigated in five different strains of rats. Male SD (Crj:CD), Lewis (LEW/Crj), WKY (WKY/NCrj), Wistar (Crj:Wistar) and F344 (F344/DuCrj) animals were given drinking water containing 100 micrograms/ml of MNNG for 30 weeks and were killed at week 50. Among the 5 strains, stomach adenocarcinomas were found most frequently in the SD case. The susceptibility of rats to induction of stomach carcinoma did not correlate with the development of intestinal metaplasias in each strain. Histochemical staining for mucin demonstrated all stomach tumors (adenomatous hyperplasias and well-differentiated adenocarcinomas) to consist mainly of gastric type cells (pyloric gland cell and surface mucous cell types), with intestinal-type tumor cells (goblet cell and intestinal absorptive cell types) being only occasional findings. Immunohistochemically, I-ALP was strongly positive on the striated cell borders of small intestinal absorptive cells of the villus and on brush borders of epithelial cells of kidney proximal tubules. I-ALP was also detected in the normal stomach, limited to the striated cell borders of absorptive cells of the upper one-fourth of intestinal metaplastic glands. I-ALP may thus be a useful marker for stomach tumor cells of intestinal absorptive cell type, indicative of maturation and differentiation. No stomach tumors consisting mainly of intestinal-type cells were found, and therefore there was no suggestion of any derivation from intestinal metaplasias.

Adenocarcinoma↗
PubMed1994

Purification and characterization of a novel enzyme, alpha-neoagarooligosaccharide hydrolase (alpha-NAOS hydrolase), from a marine bacterium, Vibrio sp. strain JT0107.

A novel enzyme, alpha-neoagarooligosaccharide hydrolase (EC 3.2.1.-), which hydrolyzes the alpha-1,3 linkage of neoagarooligosaccharides to yield agaropentaose (O-beta-D-galactopyranosyl(1-->4)-O-3,6-anhydro-alpha-L-galactopyranosyl (1-->3)-D-galactose], agarotriose [O-beta-D-galactopyranosyl(1-->4)-O-3,6-anhydro- alpha-L-galactopyranosyl (1-->3)-D-galactose], agarobiose [O-beta-D-galactopyranosyl(1-->4)-3,6-anhydro-L-galactose], 3,6-anhydro-L-galactose, and D-galactose was isolated from the marine bacterium Vibrio sp. strain JT0107 and characterized. This enzyme was purified 383-fold from cultured cells by using a combination of ammonium sulfate precipitation, successive anion-exchange column chromatography, gel filtration, and hydroxyapatite chromatography, gel filtration, and hydroxyapatite chromatography. The purified protein gave a single band (M(r), 42,000) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Estimation of the M(r) by the gel filtration method gave a value of 84,000, indicating that the enzyme is dimeric. Amino acid sequence analysis revealed it to have a single N-terminal sequence that has no sequence homology to any other known agarases. The optimum temperature and pH were 30 degrees C and 7.7, respectively. The Km and maximum rate of metabolism for neoagarobiose were 5.37 mM and 92 U/mg of protein, respectively.

Amino Acid Sequence↗
PubMed1994

The possible role of age-related increase in the plasma glucagon/insulin ratio in the enhanced hepatic gluconeogenesis and hyperglycemia in genetically diabetic (C57BL/KsJ-db/db) mice.

Genetically diabetic db/db mice and their normoglycemic littermates (+/+ mice) were studied to determine plasma levels of glucose, glucagon and insulin and hepatic gluconeogenic enzyme activities. Plasma glucose levels did not differ significantly between the 5-week-old db/db and +/+ mice, but increased with age in the former until the animals were 16-week-old. Similar age-associated changes were observed in the activities of the gluconeogenic enzymes, glucose-6-phosphatase (G-6-Pase) and fructose-1,6-diphosphatase (F-1,6-DPase). While the plasma levels of insulin and glucagon that peaked at 7 weeks of age did not parallel the hyperglycemia, the plasma glucagon/insulin (G/I) ratio roughly paralleled the hyperglycemia. Analysis of individual values for the db/db mice revealed statistically significant (P < 0.001) correlations between plasma glucose levels and hepatic G-6-Pase (r = 0.78) or F-1,6-DPase (r = 0.74) activity. There were also significant correlations between the G/I ratio and plasma glucose levels (P < 0.001, r = 0.66), hepatic G-6-Pase (P < 0.01, r = 0.48) or F-1,6-DPase (P < 0.01, r = 0.57) activity. It is thus concluded that the relative predominance of glucagon over insulin action plays an important role in the age-associated development of hyperglycemia in db/db mice. Glucagon presumably activates the hepatic gluconeogenic enzymes to enhance hepatic glucose output.

Age Factors↗
PubMed1994

Differential hypoglycemic effect of 2,5-anhydro-D-mannitol, a putative gluconeogenesis inhibitor, in genetically diabetic (db/db) and streptozotocin-induced diabetic mice.

2,5-Anhydro-D-mannitol (AM), a putative gluconeogenesis inhibitor, completely reversed the hyperglycemia in genetically diabetic (db/db) mice that exhibited hyperinsulinemia and enhanced hepatic gluconeogenic enzyme (glucose-6-phosphatase (G-6-Pase) and fructose-1,6-diphosphatase (F-1,6-DPase)) activities compared with the control +/+ mice. In contrast, AM only partially reversed the hyperglycemia of streptozotocin (STZ)-treated +/+ mice in which the hepatic gluconeogenic enzyme activities were enhanced to the same degree as in the db/db mice, whereas the blood insulin level was depressed. In the db/db mice, the STZ-treatment attenuated the hyperinsulinemia and exaggerated the hyperglycemia as well as the hepatic gluconeogenic enzyme activities, and it greatly reduced the hypoglycemic action of AM. Not only the dose-response curve of AM but also the time-course of the blood glucose level (expressed as % of pre-treatment value) following 320 mg/kg of AM were almost identical between +/+, STZ-treated +/+ and STZ-treated db/db mice. In the STZ-treated +/+ mice, a combination treatment of insulin (320 micrograms/kg) with AM (320 mg/kg) caused hypoglycemia that was greater than that induced by AM or insulin alone. On the other hand, in vitro studies with purified F-1,6-DPase revealed that phosphorylated AM (AM-1,6-diphosphate) but not AM itself inhibited the gluconeogenic enzyme activities. These results suggest that inhibition of gluconeogenesis is responsible, at least in part, for the hypoglycemic activity of AM. AM appears to inhibit hepatic gluconeogenic enzyme activities after being phosphorylated by an insulin-dependent mechanism.

Animals↗
PubMed1994

Isolation and characterization of rainbow trout (Oncorhynchus mykiss) serum amyloid P component (SAP)

We purified a Ca(2+)-dependent agarose-binding protein from rainbow trout (Oncorhynchus mykiss) sera. SDS-PAGE analysis showed the possibility that the purified protein was a polymer with a molecular weight of over 100,000 composed of covalently-bounded 32-kDa subunits. N-terminal twenty amino acid sequence of the 32-kDa protein showed partial homology with other known serum amyloid P components (SAPs) including plaice (Pleuronectes platessa) (indicated 40% homology), human (55%), hamster (45%), rat and mouse (40%) SAPs. In electron micrographs the 32-kDa protein was observed as pentameric disc-like structure. On the basis of the results, the 32-kDa agarose-binding protein of rainbow trout was concluded to belong to pentraxin family and to be a SAP homologue.

Amino Acid Sequence↗
PubMed1994

Measurement of iminodipeptides in the serum of patients with prolidase deficiency using liquid chromatography-mass spectrometry.

Iminodipeptides containing C-terminal proline or hydroxyproline were determined in sera from patients with prolidase deficiency, in their mother's serum, and in the sera of unrelated controls, using liquid chromatography-mass spectrometry with an atmospheric pressure ionization interface system. The separation was carried out on a reversed phase column using 1 g/l aqueous trifluoroacetic acid-methanol (75 + 25, by vol.). The quasi-molecular ions ([M + H])+ of various iminodipeptides containing C-terminal proline and hydroxyproline were observed in the sera of patients with prolidase deficiency, using selected ion monitoring. The quasi-molecular ions ([M + H])+ of iminodipeptides containing C-terminal proline were not observed in the sera of normal subjects or the patients' mother, but the latter did contain various iminodipeptides with C-terminal hydroxyproline. This method proved useful for the determination of iminodipeptides in the sera of patients with prolidase deficiency.

Chromatography, Liquid↗
PubMed1994

Characteristics of partially purified prolidase and prolinase from the human prostate.

Both prolidase and prolinase from the human prostate were separated into two peaks by TSK DEAE-5PW chromatography. These peaks of prolidase isozymes I and II differed from each other in their responses to preincubation with Mn2+, their substrate specificity, optimal pH, and heat stability. The molecular weights of prolidases I and II were estimated to be 110,000 and 165,000, respectively, by gel filtration. Substrate specificity of prolinase peaks I and II was almost the same, but they differed in optimal pH and heat stability. The molecular weights of prolinases I and II were about 85,000 and 63,000, respectively. These results indicate that two isozymes of prolidase and of prolinase, which differ in various characteristics, are present in the human prostate.

Aged↗
PubMed1994

Involvement of the c-kit receptor in the adhesion of hematopoietic stem cells to stromal cells.

The proto-oncogene c-kit, encoding a receptor-type tyrosine kinase, is allelic with the W locus of the mouse. The stromal cell line OP9, capable of supporting long-term hematopoiesis, was newly established from a newborn B6C3F1-op/op mouse calvaria. When bone marrow cells of WBB6F1-W/Wv mice were cocultured with the OP9 cells in liquid medium, hematopoiesis declined to a level one-thousandth of that in the cocultures of bone marrow cells of WBB6F1-+/+ mice and stromal cells by day 21. In contrast, when bone marrow cells of W/Wv mice were cocultured with OP9 cells in semisolid medium, at least 61% of the number of colonies were detected until the end of our observation period of 42 days when compared with that in control cocultures, although colonies formed by hematopoietic stem cells of W/Wv mice were significantly smaller than those of normal stem cells. After a 24-hour incubation with OP9 cells, fewer stem cells of W/Wv mice than normal ones adhered to the stromal cells. Adhesion of normal stem cells to stromal cells was inhibited by the addition of an antagonists anti-c-kit monoclonal antibody, ACK2. These results demonstrate that the c-kit receptor plays an important role not only in the proliferative response of hematopoietic stem cells but also in their adhesion to stromal cells.

Animals↗
PubMed1994

N-terminal amino acid sequence of a 28 kDa major serum high density lipoprotein of the rainbow trout Oncorhynchus mykiss.

As there are few data on primary structures of fish apolipoproteins (apo), we determined the N-terminal sequence of a 28 kDa major serum protein of rainbow trout, a possible homologue of apoAI protein in higher vertebrates. Rainbow trout serum was separated by two-dimensional electrophoresis. A spot of the 28 kDa protein (pl 5.45) was cut out and directly applied to a protein sequencer. We succeeded in sequencing 30 amino acids from the N-terminal. The sequence of the 28 kDa protein shared a high similarity with that of apoAI of Atlantic salmon (Salmo salar). These results showed that the 28 kDa protein is apoAI of rainbow trout.

Amino Acid Sequence↗