Biomedical subjects

J Hata

Publications and source records attributed to J Hata.

At least 181 records · Page 10Linked to original sources

PubMed1991

Purification and characterization of a Coffea canephora alpha-D-galactosidase isozyme.

Exoglycosidases modify carbohydrate epitopes on glycoproteins and glycolipids. The alpha-D-galactosidase from Coffea canephora is an important exoglycosidase which degrades the human blood group B epitope. Although multiple isozymes have been described, they have never been demonstrably purified and thoroughly characterized. We have developed a technique to purify an isozyme to homogeneity. The isolated enzyme has a molecular weight of 36.7 kDa by SDS PAGE and 34.0 kDa by gel filtration. The isozyme is highly selective for alpha-D-galactosides and inactive against other low molecular weight substrates. It hydrolyzes the the terminal alpha-D-galactosyl residue from the blood group B epitope. Protease activity is below detectable limits. The isozyme has a broad pH optima at 6.3, a pl of 7.03, is unaffected by ionic strength, and is stable at 4 degrees C.

Amino Acids↗

PubMed1991

Correlation of chromosome abnormalities with histological and clinical features in Wilms' and other childhood renal tumors.

Chromosomes and histology were successfully studied in 33 childhood renal tumors. Thirty-one tumors were classified as one of four subtypes of Wilms' tumor. Of 24 typical Wilms' tumors, 12 had hyperdiploidy with nonrandom trisomies, mostly including +6 and/or +12. Three typical Wilms' tumors with an 11p13 deletion or a pericentric inversion with a break in 11p13 were not associated with aniridia. Two other typical Wilms' tumors with the 11p13 deletion and one fetal rhabdomyomatous nephroblastoma with an 11p13 translocation were associated with aniridia. Two cystic partially differentiated nephroblastomas showed hyperdiploidy with +12. Of four clear cell sarcomas of the kidney, three had normal diploidy and the other had a 2;22 translocation. Two congenital mesoblastic nephromas had hyperdiploid karyotype with trisomy 11, which was never seen in the 31 Wilms' tumors. Our findings and a review of data on 102 reported Wilms' tumors revealed 11p13 abnormalities in 24 tumors, 11p15 abnormalities in five tumors, and partial deletions of 1p, 7p, 11q, 12q, 16q, or 17p or monosomy of No. 21 or No. 22 each in four or more tumors. These findings suggest that increased copy number of genes on the nonrandom trisomic chromosomes might contribute to the genesis of many Wilm's tumors and that deletion of various tumor suppressor genes other than a Wilms' tumor gene, WT1 in 11p13, might also play a critical role in the development of some tumors.

Child↗

PubMed1991

Close similarity between cultured human omental mesothelial cells and endothelial cells in cytochemical markers and plasminogen activator production.

The mesothelial cells obtained from human omental adipose tissue showed a typical cobblestone monolayer and reacted strongly with keratin, but did not have Von Willebrand factor. Ultrastructurally these cells revealed the existence of desmosome-like cell junctions as well as intracellular canaliculi, tubular structures surrounded by microvilli, and tonofilament-like filaments. The mesothelial cells grew much faster in the medium containing epidermal growth factor, actively took up acetylated-low density lipoprotein into their cytoplasm, and released angiotensin-converting enzyme. They also released urokinase-type plasminogen activator, but only half as much as do human umbilical vein endothelial cells; release of tissue-type plasminogen activator was not observed. Inasmuch as the mesothelial cells also released plasminogen activator inhibitor-1, as do human umbilical vein endothelial cells, we could not detect u-PA activity in culture medium. u-Pa may play a role in the protection against adhesion among visceral organs. These observations indicate that cultured human mesothelial cells have characteristics closely related to those found in human endothelial cells.

Adipose Tissue↗

PubMed1991

Evidence for heterogeneous groups of neuronal differentiation of Ewing's sarcoma.

We have investigated the capability of differentiation of Ewing's sarcoma (ES) towards a neuronal direction through the establishment of four extraosseous ES cell lines and by in vitro stimulation with dibutyryladenosine cyclic monophosphate (db-cAMP) of eight ES lines. All except one of the lines expressed the molecule defined by 5C11, the antibody specifically reactive with ES. Two ES lines expressed a 200 kilodalton (kD) neurofilament protein (NFP) although their original tumours were negative for NFP. Elongation of cytoplasmic processes and increased NFP expression were observed after db-cAMP treatment of these lines and microtubules in the cytoplasmic processes were ultrastructurally demonstrated. Six lines were NFP negative, but three lines changed their morphology after induction of 200 kD NFP expression by db-cAMP treatment. The other three showed no definitive differentiation after db-cAMP treatment. Chromosomal analysis of the new ES lines showed the typical t(11;22) in one line and a +der(22) in two lines. No correlation was observed between the chromosomal abnormality and the differentiation capability. We conclude that ES is a heterogeneous group of tumours with respect to capability of differentiation into the neuronal lineage, but it is clearly distinguished from peripheral primitive neuroectodermal tumours by its 5C11 reactivity.

Adolescent↗

PubMed1991

Monoclonal antibody against bone marrow stromal cells. Its production and characterization.

Bone marrow stromal cells play an essential role in the proliferation and differentiation of hematopoietic stem cells (1, 2). As a means of analyzing of the bone marrow microenvironment immunohistochemically, we attempted to produce a rat monoclonal antibody against the murine preadipocyte line H-1 derived from long-term bone marrow culture (LTBMC) of C57BL/6 mice (3, 4). A newly established monoclonal antibody, designated R4-A9, was obtained from a hybridoma prepared by fusion of Y.B2/3.0Ag20(YO) rat myeloma cells with spleen cells of LEW rats immunized with H-1 cells. The immunofluorescence of live H-1 cells showed that the antigen reacting with this antibody was strongly expressed on the cell surface. The specificity of R4-A9 was assessed immunohistochemically on frozen sections of various tissues from normal adult mice. R4-A9 demonstrated specificity for hematopoietic stroma in bone marrow and spleen. No staining was observed in thymus, lymph nodes or other tissues examined, with the exception of Leydig cells in the testis and the endothelium of small arteries in several organs. Detailed immunohistochemical observations at both the light microscopy and electron microscopy level showed that R4-A9 selectively reacted with the sinusoidal endothelium, perisinusoidal adventitial cells (5) (adventitial reticular cells (6] and intersinusoidal reticular cells (5) and the reticular cells of the splenic red pulp. These findings indicate that reticular cells and the endothelium of the bone marrow possess the common cell surface molecules recognized by R4-A9. SDS-PAGE analysis showed that R4-A9-immunoprecipitated proteins had a molecular mass of 100 kDa under reducing conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Characterization of a radioimmunoassay to determine plasma total renin.

The determination of plasma total renin is useful not only as a tool to investigate the physiology of hypertension but also as a marker for Wilms' tumor. A radioimmunoassay (RIA) system to determine plasma total renin was newly developed using monoclonal antibodies specific for both inactive and active renin (inactive + active = total); in this study, an effort was made to confirm that this RIA system truly determines plasma total renin concentrations. First, it was found that this monoclonal antibody stains only the juxtaglomerular apparatus of the kidney. Second, the data determined by the RIA were compared with those obtained by the conventional enzymatic method: samples were activated, and renin activity was assayed by measuring angiotensin I. The coefficient of the data obtained by this RIA system and by the conventional method was 0.921 (p less than 0.01) based on all 89 samples, and 0.809 (p less than 0.01) based on 86 of the 89 samples whose values were less than 600 pg/ml by RIA. As a result of these studies, it was concluded that the newly developed RIA system does determine total renin levels in patients' plasma.

Antibodies, Monoclonal↗

PubMed1991

Murine cytomegalovirus infection model in Balb/c mice. 3. Immunoglobulin production during infection.

In mice infected with a lethal dose of murine cytomegalovirus (MCMV) the serum immunoglobulin (Ig) levels and the Ig-bearing cells in the spleen dropped to barely detectable levels 2 days after infection. In mice with acute but non-lethal MCMV infection, the serum IgM was twice and the IgG 32 times that of the uninfected controls by Day 8 of infection; the numbers of spleen cells bearing IgM and the IgG subclasses (IgG1, IgG2a, IgG2b, IgG3) were also greatly increased. In the asymptomatically infected group, serum IgM remained unchanged but the IgG increased to 16 times that of uninfected controls by Day 11 of infection; the numbers of spleen cells bearing IgM and IgG subclasses were also increased, although to a lesser extent than in the acute, non-lethally infected mice. In the latter two groups, serum IgA and IgA-bearing cells in the spleen did not alter significantly. Complement-requiring neutralizing antibodies to MCMV were detected 8 days post infection.

Animals↗

PubMed1990

Immunological quantitation and immunohistochemical localization of leukotriene A4 hydrolase in guinea pig tissues.

We prepared a highly specific polyclonal antibody against leukotriene (LT) A4 hydrolase using a recombinant human enzyme. Using this antibody, we quantified LTA4 hydrolase protein content in the cytosols of guinea pig tissues. The enzyme protein content correlated well with the enzyme activity with a correlation coefficient of 0.87. However, the enzyme activity per mg of the enzyme in the cytosols was low, particularly in the liver and adrenal gland, compared with the specific activity of the purified enzyme. These observations suggest the presence of inhibitory substances and/or inactive enzymes in the cytosols of these tissues. To determine the cellular localization of LTA4 hydrolase in tissues other than blood cells, we carried out immunohistochemical examinations of guinea pig tissues. We identified epithelial cells in the tracheobronchial system and gastrointestinal tract, smooth muscle cells in the bronchi and aorta, vascular endothelial cells, and the intestinal plexus as novel cellular sources of the enzyme in the parenchyme of the tissue. Thus, LTA4 hydrolase was widely distributed in various types of parenchymal cells in the tissues, and this observation warrants further investigations on the biological activities of LTB4 in these cells and tissues.

Animals↗

PubMed1990

Spontaneous transformation and immortalization of human endothelial cells.

A new cell line from the human umbilical vein has been established and maintained for more than 5 yr (180 generations; 900 population doublings). This strain, designated ECV304, is characterized by a cobblestone monolayer growth pattern, high proliferative potential without any specific growth factor requirement, and anchorage dependency with contact inhibition. Karyotype analysis of this cell line reveals it to be of human chromosomal constitution with a high trisomic karyotype (mode 80). Ultrastructurally, endothelium-specific Weibel-Palade bodies were identified. Although one of the endothelial cell markers, Factor VIII-related antigen (VIIIR:Ag) was negative in this cell line, immunocytochemical staining for the lectin Ulex europaeus I (UEA-I), and PHM5 (anti-human endothelium as well as glomerular epithelium monoclonal antibody) was positive, and angiotensin-converting enzyme (ACE) activity was also demonstrated. In addition, ECV304 displayed negativity for alkaline and acid phosphatase and for the epithelial marker keratin. All of these findings suggest that ECV304 cells originated from umbilical vein endothelial cells by spontaneous transformation. Ultrastructurally, no viruslike particles have been detected intracellularly. Nude mouse tumorigenicity and rabbit cornea tests were both positive. This is a report on a novel case of phenotypic alteration of normal venous endothelial cells of human origin in vitro, and generation of a transformant with indefinite life spans. This line may be useful in studies of some physiologically active factors available for medical use.

Cell Division↗

PubMed1990

Determination of plasma total renin level by RIA with a monoclonal antibody: value as a marker for nephroblastoma.

Plasma total renin is a new, useful marker for nephroblastoma, but the conventional method for determining its level is sophisticated and requires large blood samples. To develop a simpler technique, a monoclonal antibody specific for both inactive and active renin (inactive + active = total) was raised, and a radioimmunoassay (RIA) system was established. This monoclonal antibody stains only the juxtaglomerular apparatus; values determined by this RIA did not change before and after activation. So far, the RIA system has been applied to 136 samples from 92 patients. Plasma total renin levels were also determined with the conventional method: samples were activated, then renin activity was assayed by measuring angiotensin I. The coefficient of the data obtained by these two different techniques was 0.921 (P less than .01). Plasma total renin levels in patients with nephroblastoma were significantly increased (546.5 +/- 297.8 pg/mL) over those in patients with neuroblastoma (218.6 +/- 46.5 pg/mL) and in controls (165.8 +/- 67.5 pg/mL, P less than .01). After removal of Wilms' tumors, the levels decreased to normal when sampled every 2 weeks. It was concluded that a newly developed RIA system is more useful in determining plasma total renin levels in patients with nephroblastoma.

Antibodies, Monoclonal↗

PubMed1990

Electroporation as a new technique for producing transgenic fish.

A recombinant plasmid, pMV-GH, containing rainbow trout growth hormone cDNA fused to mouse metallothionein I promoter, was introduced into medaka (Oryzias latipes) by electroporation. Of 3109 fertilized eggs treated with electric pulses (750 V/cm, 50 microseconds, 5 times), 783 (25%) hatched out. Four percent of the hatchlings were transgenic. To obtain transgenic lines, 180 hatchlings were maintained and 35 of them grew into adult fish. Two of these fish were transgenic. When one transgenic fish was mated with a normal female, the transgene was found in all the F1 offspring assayed. In F2 offspring obtained by mating transgenic F1 fish, 88% were transgenic.

Animals↗

PubMed1990

Generation of transgenic mice with elevated blood pressure by introduction of the rat renin and angiotensinogen genes.

The role of the renin-angiotensin system in blood pressure control and in the development of hypertension was investigated by generating transgenic mice carrying the rat renin or angiotensinogen gene or both genes under the control of the mouse metallothionein I promoter. The systolic blood pressure was significantly elevated in transgenic mice carrying both transgenes but was maintained normally in those bearing either of the transgenes. The transgene was effectively and properly transcribed to form the mature mRNA in the transgenic mice. The production of rat renin and angiotensinogen in the transgenic mice carrying the corresponding transgene was also verified by immunoanalyses of these proteins. Furthermore, the specific angiotensin-converting enzyme inhibitor captopril was effective in reducing the elevated blood pressure of the hypertensive transgenic mice. These results indicate that the combined action of the exogenous rat renin and angiotensinogen is responsible and necessary for elevation of blood pressure in the hypertensive transgenic mice.

Angiotensinogen↗

PubMed1990

Disseminated adenovirus (type 19) infection in a neonate. Rapid detection of the infection by immunofluorescence.

A case of fatal disseminated adenovirus infection in a neonate who suffered from severe keratoconjunctivitis and pneumonitis is reported. The diagnosis was made seven days after the onset of illness based on the detection of adenovirus antigen in the smears of the tracheal suction and conjunctival swab by immunofluorescence. Viral antigen was detected in the frozen or formalin-fixed autopsy specimens of the lungs, kidneys, spleen, liver and lymph nodes. Typical crystal arrangement of adenovirus virions was observed in the alveolar epithelial cells by electron microscopy. The isolated virus was identified to be of type 19 by a neutralization test. The IF examination using adenovirus group specific immune reagents on the smears of clinical specimens appears to be useful for rapid diagnosis of viral infections.

Adenoviridae Infections↗

PubMed1990

Establishment of specific monoclonal antibodies against recombinant human granulocyte colony-stimulating factor (hG-CSF) and their application for immunoperoxidase staining of paraffin-embedded sections.

Detection of granulocyte colony-stimulating factor (G-CSF), one of the substances responsible for proliferation and differentiation of granulocytes, has been performed up to the present by use of the granulocyte colony-formation assay, because of the lack of a specific anti-G-CSF antibody. This has prevented the advancement of biological investigations of cell dynamics linked to G-CSF, e.g., cell localization of G-CSF and its pathophysiological changes. In the present work, two monoclonal antibodies (MAb), 1E7 and 4A6, against recombinant human G-CSF (rhG-CSF) were developed by cell hybridization between NS-1 myeloma cells and splenocytes from a mouse immunized with rhG-CSF. 1E7 and 4A6 were shown to be reactive with hG-CSF but not with other CSF (hGM-CSF, hIL-3, and mouse GM-CSF) by Western blot analysis. An immunoperoxidase staining method using these MAb was then established. This method was applicable to frozen sections, paraffin-embedded sections, and cells fixed with 4% paraformaldehyde. Positive staining for G-CSF was observed in tumor cells secreting G-CSF and also in Chinese hamster ovary (CHO) cells transfected with hG-CSF cDNA. However, no staining was seen in tumor cells secreting no G-CSF, untransfected CHO cells, lung fibroblasts, or bone marrow stromal cells after short periods of culture. These results confirmed the immunospecificity of MAb 1E7 and 4A6 and the validity of their application to immunohistochemistry using paraffin-embedded sections.

Animals↗

PubMed1990

[Clinical evaluation on differential quantitation of human chorionic gonadotropin in testicular cancer].

In 113 patients with testicular germ cell tumor, the authors determined human chorionic gonadotropin (hCG) and the free beta subunit levels in sera by differential quantitation using homologous hCG-beta radioimmunoassay (RIA) and hCG enzyme immunoassay (EIA), respectively. In 59 patients with seminoma, intact hCG was positive in 6 patients (10.1%), whereas free hCG-beta was positive in 23 patients (39.0%). Syncytiotrophoblastic giant cells were detected in 4 cases out of 6 patients with positive intact hCG seminoma. Seventeen cases (28.8%) revealed positive free hCG-beta only without elevation of intact hCG. In 54 patients with nonseminoma, both intact hCG and free hCG-beta were positive in 36 cases (66%). In some cases, hCG-beta/hCG ratio increased up to 275% when recurrence of tumor developed. Molecular heterogeneity of hCG is closely related to proliferation and differentiation of hCG producing cells. The differential quantitation of intact hCG and hCG-beta using these two assays is valuable for clinical detection of molecular heterogeneity of hCG in testicular tumor.

Adolescent↗

PubMed1990

Concurrent murine cytomegalovirus and Klebsiella pneumoniae infections in germfree mice.

The effects of concurrent murine cytomegalovirus (MCMV) and Klebsiella pneumoniae infections were studied in germfree (GF) mice. The mice received sublethal doses, 5 x 10(5) pfu, of MCMV. K. pneumoniae was injected in doses of 40 to 100 cfu, which by itself killed 0-33% of GF mice. When K. pneumoniae was given to GF mice infected with MCMV, the mortality increased up to 100%, with distinct enhancement persisting until day 10 of the MCMV infection. The virus titer in various organs did not change after superinfection with K. pneumoniae, while the viable counts of K. pneumoniae in organs remained remarkably high until death, suggesting the cause of death to be severe generalized infection by the bacteria. When compared to specific pathogen-free (SPF) mice, GF mice were more susceptible to both MCMV and K. pneumoniae infection, had higher titers of the virus for longer periods in various organs, and showed extension in the duration of enhanced mortality by the bacteria. Histopathologically, the spleen and liver were found to be the most severely affected tissues, more so in GF than in SPF mice, with recovery from the changes being slower in the GF animals.

Animals↗

PubMed1990

Molecular diversity of precursor B acute lymphoblastic leukemias identified by the immunoglobulin heavy chain gene organization.

Acute leukemias with morphological, cytochemical, and immunological characteristics correlating to precursor B lymphocyte and with germ line configuration of immunoglobulin heavy (IgH) chain loci were studied for the organization of antigen receptor genes including C mu, Ig light (IgL) chain, T cell receptor (TCR) beta and gamma. Three of the five lymphoblast samples retained the germ line configuration of both Ig JH and C mu region. The other two samples showed deletion of the entire JH region resulting in the rearrangement of the C mu region. None of these five cases had a Ig L chain gene rearrangement. The three cases with germ line IgJH and C mu loci were revealed to belong to stage I (HLA DR+), stage II (HLADR+, CD19+), and stage III (HLADR+, CD19+, CD10+) B precursor ALLs, respectively. The two cases with deletion of IgJH region also belonged to stage II and III B precursor ALL. Thus immunologically classified stage II and III B precursor ALLs include those with germ line IgH region, representing ALLs at a very early stage of B cell development. A subgroup of B precursor ALL with deleted IgJH region, which is abortive at the molecular genetic level, was also identified. Karyotype abnormalities involving chromosomal region 11q23 in the leukemias with germ line IgH region are also discussed.

B-Lymphocytes↗

PubMed1989

Human yolk-sac tumor antigen 2G10: biochemical characterization and significance as a serum antigen.

The biochemical characterization of human yolk-sac tumor (YST) antigen 2G10, detected by monoclonal antibody (MAb) M912-2G10, was studied. Previous results indicated that glycolipids having a non-reducing terminal N-acetyllactosamine structure were the epitope of 2G10 on human erythrocytes. In this study, the glycoprotein nature of 2G10 on the infantile embryonal carcinoma line, MTE, was investigated. 2G10 activity, measured by a new enzyme-linked immunosorbent assay (2G10-ELISA), was recovered in residual fractions of MTE from which glycolipids were removed. Chromatographically, 3H-galactose-labelled 2G10 on MTE had a molecular weight (mw) of about 580 kDa, which decreased after pronase or alkaline-borohydride treatment. Our results indicate the glycoprotein nature of 2G10 on MTE. Furthermore, 2G10, both on erythrocytes and on MTE, was sensitive to galactosidase but not to neuraminidase and fucosidase, suggesting that terminal galactose is involved in the antigenic structure. It was also found by 2G10-ELISA that 2G10 sheds from tumor cells. Shedding occurs in nude mice transplanted with MTE as well as in patients with germ-cell tumors (GCTs). The serum level of 2G10 in non-tumor patients was low, but high levels were detected in patients with YSTs and with GCTs having YST components. Immunohistochemically, the presence of 2G10-positive YST components was shown in patients who had high serum levels of 2G10. Sera from other urogenital and childhood solid tumors did not have elevated 2G10. The mw of shed 2G10 was lower than that of 2G10 on the cell surfaces. Our results clearly indicate the usefulness of serum 2G10 as a tumor marker for GCTs having YST components.

Animals↗