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Biomedical subjects

K Morikawa

Publications and source records attributed to K Morikawa.

At least 199 records · Page 11Linked to original sources
PubMed1991

Effect of mutagenesis at each of five histidine residues on enzymatic activity and stability of ribonuclease H from Escherichia coli.

To examine the role of histidine residues in ribonuclease H from Escherichia coli, kinetic parameters for the enzymatic activity and conformational stabilities against guanidine hydrochloride denaturation of mutant enzymes, in which each of the five histidine residues was replaced with alanine, were determined and compared with the wild-type enzyme. The mutation of His83 resulted in a marked increase in Km along with an increase in kcat. The mutation of His114 caused a large reduction in both the free energy of unfolding in water, delta GH2O, and the mid-point of the unfolding curve, [D]1/2. These results indicate that His83, which is one of the four well-exposed histidine residues in the crystal structure, is located close to a substrate-binding site, and His114, which is buried inside the protein molecule, contributes to the conformational stability, probably through the formation of a hydrogen bond with a main-chain carbonyl group. None of the histidine residues is required for activity.

Base Sequence↗
PubMed1991

Structural models of ribonuclease H domains in reverse transcriptases from retroviruses.

Tertiary models of ribonuclease H (RNase H) domains in reverse transcriptases (RTs) from Moloney murine leukemia virus (MuLV) and human immunodeficiency virus (HIV-1) were built based upon the X-ray structure of RNase H from Escherichia coli (E. coli RNase H). In two models of RT-RNase H domains, not only active site residues but also residues, which construct a hydrophobic core and hydrogen bonds, are located in the same positions as those of E. coli RNase H. The whole backbone structure and the electrostatic molecular surface of MuLV RT-RNase H model are similar to those of E. coli RNase H. On the contrary, HIV-1 RT-RNase H model lacks the third helix and the following loop, resulting no positive charge clusters around the hybrid recognition site. Referring the complex models of RTs with their substrate hybrid, the interaction between DNA-polymerase and RNase H domains in RTs was discussed.

Amino Acid Sequence↗
PubMed1991

Stabilization of Escherichia coli ribonuclease H by introduction of an artificial disulfide bond.

To examine the effect of the introduction of a disulfide bond on the stability of Escherichia coli ribonuclease H, a disulfide bond was engineered between Cys13, which is present in the wild-type enzyme, and Cys44, which is substituted for Asn44 by site-directed mutagenesis. The disulfide bond was only formed between these residues upon oxidation in vitro with redox buffer. The conformational and thermal stabilities were estimated from the guanidine hydrochloride and thermal denaturation curves, respectively. The oxidized (cross-linked) mutant enzyme showed a Tm of 62.3 degrees C, which was 11.8 degrees C higher than that observed for the wild-type enzyme. The free energy change of unfolding in the absence of denaturant, delta G[H2O], and the mid-point of the denaturation curve, [D]1/2, of the oxidized mutant enzyme were also increased by 2.1-2.8 kcal/mol and 0.36-0.48 M, respectively. Introduction of a disulfide bond thus greatly enhanced both the thermal and conformational stabilities of the enzyme. In addition, kinetic analyses for the enzymatic activities of mutant enzymes suggest that Thr43 and Asn44 are involved in the substrate-binding site of the enzyme.

Base Sequence↗
PubMed1991

Synthesis, antitumor activity, and nephrotoxicity of the optical isomers of 2-aminomethylpyrrolidine(1,1-cyclobutane-dicarboxylato)platinum(II).

The optical isomers of 2-aminomethylpyrrolidine(1,1-cyclobutane- dicarboxylato)platinum(II) (DWA2114, 1), which has potent antitumor activity against various tumors, were synthesized. They were examined for antitumor activity against Colon 26 carcinoma in a sc-iv system, and changes in urinary protein and sugar levels in drug-treated mice were used as an index of nephrotoxicity. In their effect on tumors, (+)-(S)-2-aminomethylpyrrolidine(1,1-cyclobutanedicarboxylato++ +)platinum(II) (6b) was more potent than the enantiomer 6a in that the effective dose of 6b was smaller than that of 6a; but, both drugs exhibited potent antitumor activity. On the other hand, a distinct difference between 6a and 6b was shown in their nephrotoxicity. Isomer 6b induced a great increase in urinary protein and sugar levels in mice, whereas 6a caused no increase in these levels.

Animals↗
PubMed1991

Establishment of a novel cell line with T-lineage phenotype (HPB-MLp-W) from a non-Hodgkin's lymphoma patient.

We report the characterization of a novel human T-cell line, HPB-MLp-W, which was established from blastic cells of a lymph node specimen from a patient with non-Hodgkin's lymphoma. They demonstrated the T-cell association antigens, CD2 and CD4, but no CD3, CD8, CD1, CD5, CD7 nor T-cell antigen receptor on their cell surfaces. They were also positive for Ia and Ki-1 antigen, and negative for CD25 (Tac-1). The cell line HPB-MLp-W had the same pattern of antigen expression as the patient's cells. Southern-blot analysis of DNA showed a rearrangement of the T-cell receptor-alpha and beta genes. To our knowledge, this is a novel cell line with unique T-lineage marker, to be established from a case of non-Hodgkin's lymphoma.

Antigens, CD↗
PubMed1991

Regulation of lymphocyte blastogenesis and antibody production by soluble factor released by a human B-lymphoblastoid cell line.

Yam 1B, a human B lymphoblastoid cell line, spontaneously produced an immunoregulatory factor, which suppresses blastogenesis and antibody formation by human lymphocytes. The Yam 1B cells, which were derived from the peripheral blood of an adult T-cell leukemia patient, have been established and maintained in our laboratory since 1985. This cell line expressed mature B-cell surface antigens including surface immunoglobulin M (IgM), CD23, and HLA-DR; had cytoplasmic IgM; and secreted small amounts of IgM in the culture supernatants. Yam 1B was positive for Epstein-Barr virus-associated antigen (EBNA) but negative for adult T-cell-associated antigen (ATLA). The serum-free Yam 1B culture supernatants (SN) inhibited the expression of transferrin R, but neither the expression of interleukin 2 (IL-2) R(CD25) nor the production of IL-2 in the lymphocytes stimulated with phytohemagglutin. Yam 1B SN also inhibited DNA synthesis by human T and B lymphocytes and immunoglobulin generation by normal B cells as well as by Epstein-Barr virus-transformed human B lymphoblastoid cell lines. The inhibitory activity of Yam 1B SN was inactivated at 56 degrees C and at pH 10 but was relatively stable at pH 2. It was abrogated by digestion with pronase and was partially stable by digestion with trypsin. Fractions collected from a Sephacryl S-300 gel filtration column (Pharmacia Fine Chemicals, Uppsala, Sweden) were found to have a peak of inhibitory activity of cell proliferation associated with molecules of apparent MWr of 43,000 to 67,000. The inhibitory activity of Yam 1B SN was not blocked by the anti-transforming growth factor beta antibody.(ABSTRACT TRUNCATED AT 250 WORDS)

B-Lymphocytes↗
PubMed1991

Effects of chronic bromocriptine-induced hypoprolactinemia on plasma testosterone responses to human chorionic gonadotropin stimulation in normal men.

To study the role played by normal levels of plasma prolactin (PRL) in the secretion of testosterone (T) in the testes, we induced hypoprolactinemia with a daily dose of 5 mg bromocriptine administered orally in five normal men 20 to 35 years of age for 8 weeks. The basal PRL, T, luteinizing hormone, follicle-stimulating hormone, and maximum responses of plasma T to human chorionic gonadotropin (hCG) stimulation were measured every 2 weeks. Basal levels of plasma T were reduced in the 1st 2-week-long period of hypoprolactinemia. In the 4-week-long period of hypoprolactinemia, the maximal response of plasma T to hCG stimulation was significantly reduced. The findings suggest that normal levels of plasma PRL may play an important role in the secretion of T in the human testes in vivo.

Adult↗
PubMed1991

Comparison of the antitumour effects and nephrotoxicity-inducing activities of two new platinum complexes, (-)-(R)-2-aminomethylpyrrolidine(1,1-cyclobutanedicarboxylato+ ++)-platinum (II) monohydrate, and its enantiomeric isomer.

New platinum complexes, (-)-(R)-2-aminomethylpyrrolidine(1,1- cyclobutanedicarboxylato)platinum(II) monohydrate (DWA2114R) and its enantiomeric isomer, (+)-(S)-2-aminomethylpyrrolidine(1,1- cyclobutanedicarboxylato)platinum(II) monohydrate (DWA2114S), were compared in their antitumour effects and nephrotoxicity-inducing activities. Both compounds were effective against the murine tumours L1210 and Colon 26 by i.p. injection of 20-100 mg kg-1. While DWA2114S showed marked increases in blood urea nitrogen (BUN) and urinary protein and sugar in BDF1 mice treated i.p. at the maximum tolerated dose, DWA2114R showed no increases in these parameters. To clarify the difference of nephrotoxicity between the isomers, tissue distribution was examined. Renal Pt concentration in DWA2114S-treated mice was more than 5-fold higher compared with that in DWA2114R-treated mice 2h after i.p. injection of 80 mg kg-1. However, there were no such marked differences in the lung, liver, heart, spleen and plasma. The low content of Pt in the kidneys of DWA2114R-treated mice could explain its lower nephrotoxicity. The in vitro experiments for uptake of the drugs into the cultured normal rat kidney cells and fresh splenocytes revealed that the Pt amount in the cells treated with DWA2114S, especially in the kidney cells, was much higher than DWA2114R.

Animals↗
PubMed1991

Toxicological and tumoricidal evaluations of a new platinum complex, (-)-(R)-2-aminomethylpyrrolidine(1,1-cyclobutanedicarboxylato+ ++)platinum( II) monohydrate, in rats.

The toxicities and antitumor activity of a new anticancer platinum compound, (-)-(R)-2-aminomethylpyrrolidine(1,1-cyclobutanedicarboxylato++ +) platinum(II) monohydrate (DWA2114R), were examined in rats by single intravenous injection in comparison with those of cis-diammine(1,1-cyclobutanedicarboxylato)platinum(II) (CBDCA) and cis-diamminedichloroplatinum(II) (CDDP). The lethal dose (LD) of DWA2114R (100 mg/kg) or CBDCA (80 mg/kg) caused a slight decrease in body weight (less than 10%) and no significant change in the levels of blood urea nitrogen and urinary sugar and protein. In contrast, a sub-LD level of CDDP (8 mg/kg) seriously decreased body weight (20%) and markedly elevated the levels of these nephrotoxicity parameters. Monitoring the numbers of peripheral blood cells for 3 weeks after the drug injection revealed that all three drugs showed severe thrombocytopenia, moderate leukopenia and slight anemia. However, CBDCA induced the most severe thrombocytopenia among these drugs. The number of platelets was reduced by 60% in rats injected with a half LD of CBDCA. A moderate reduction in platelet count (35-43%) was caused by an equitoxic dose of DWA2114R or CDDP, but abated about 3 days faster than that caused by CBDCA. Interestingly, only CDDP caused an irreversible anemia. Each drug showed a potent antitumor activity at weakly toxic doses against Walker 256 carcinosarcoma transplanted intramuscularly into rats. These results indicate that DWA2114R could be a promising new platinum anticancer agent with an improved toxicity profile.

Animals↗
PubMed1991

The role of CD45RA on human B-cell function: anti-CD45RA antibody (anti-2H4) inhibits the activation of resting B cells and antibody production of activated B cells independently in humans.

Anti-CD45RA antibody defined by anti-2H4 monoclonal antibody has been reported to split CD4+T cells into two distinct subpopulations. CD45RA antigen is present on the surface of virtually more than 95% B lymphocytes in the purified tonsillar B-cell preparations. We examined the role of CD45RA antigen on human B-cell function using this antibody. The addition to anti-2H4 to tonsillar B cells inhibited the proliferative response induced by Staphylococcus aureus Cowan strain I(SAC) in a dose-dependent manner. Kinetic analysis indicated that anti-2H4 exerted its inhibitory effect when added within the first 24 h of culture initiation during a 72-h culture period. Anti-2H4 inhibited the transferrin receptor expression without interfering with the expression of the IL-2 receptor on SAC-stimulated B cells in a short-term culture. Anti-2H4 blocked the progress of SAC-stimulated B cells from the G1 to S phase of the cell cycle. These events suggested that anti-CD45RA MoAb inhibited the proliferative response by directly acting on B cells in the G1 phase. In addition, anti-CD45RA antibody also had a suppressive effect on early phase of B-cell differentiation. This effect appeared to be independent of its suppressive effect on proliferation, because anti-CD45RA did not inhibit the proliferative response of preactivated B cells with lymphokines. These studies suggested that the restricted epitope recognized by anti-2H4 antibody may be directly involved in regulatory function on B cells.

Antibodies, Monoclonal↗
PubMed1991

Stimulatory effect of N-[4-[2-(dimethylamino)-ethoxy] benzyl]-3,4-dimethoxybenzamide hydrochloride (HSR-803) on normal and delayed gastrointestinal propulsion.

To estimate the effect of a new gastroprokinetic agent, N-[4-[2-(dimethylamino)ethoxy]benzyl]-3,4-dimethoxybenzamide hydrochloride (HSR-803), on non-ulcer dyspepsia, the influence of HSR-803 on gastrointestinal propulsion was assayed in dogs, rats and mice in comparison with some gastroprokinetic agents. HSR-803 (30 mg/kg, p.o.) significantly enhanced gastric emptying in dogs, and it significantly improved the delayed gastric emptying induced by dopamine (0.4 mg/kg, i.p.) and morphine (1 mg/kg, s.c.) in rats. Metoclopramide (30 mg/kg, p.o.) also significantly restored the dopamine-induced delay, but at a dose of 10 mg/kg, p.o., it enhanced the morphine-induced delay in gastric emptying in rats. HSR-803 (10-100 mg/kg, p.o.) increased small intestinal transit in mice in a dose-dependent manner, and the effect was abolished by atropine (0.3 mg/kg, i.p.). Metoclopramide also increased small intestinal transit, but domperidone and cisapride had no effect. In delayed small intestinal transit in mice, HSR-803 (10-100 mg/kg, p.o.) improved the morphine (0.3 mg/kg, s.c.)-induced delay in a dose-dependent manner. In conclusion, because of the promotion of normal and delayed gastrointestinal propulsion, HSR-803 seems to be a promising gastroprokinetic agent for the treatment of non-ulcer dyspepsia. The action of HSR-803 is likely to be exerted through cholinergic stimulation.

Acetaminophen↗
PubMed1991

The role of CD45 in the activation, proliferation and differentiation of human B lymphocytes.

We examined the role of CD45 antigen in human B cell function, using the anti-CD45 antibody, T29/33. The addition of T29/33 to B cells inhibited the proliferative response induced by various polyclonal B cell activators in a dose-dependent manner at concentrations of 0.01 to 10 micrograms/ml. Kinetic analysis indicated that T29/33 exerted its inhibitory effect when added within the first 24 h of culture initiation during a 72-h culture period, but had little effect when added at 48 h. Pre-treatment of high-density B cells with T29/33 antibody for 48 h showed a marked inhibitory effect on the proliferative response of these B cells when they were subsequently stimulated with SAC. Antibody to CD45 appeared to block the G0(G1) to S phase transition in the cell cycle analysis by propidium iodide staining. T29/33 antibody suppressed the RNA and DNA synthesis induced by SAC stimulation in B cells. These data suggested that small resting B cells were sensitive to anti-CD45-induced suppression. T29/33 antibody also suppressed immunoglobulin synthesis in B cells, independently of its suppressive effect on proliferation. These results indicated that CD45 antigen defined by T29/33 is involved in the activation, proliferation, and differentiation signals of human B lymphocytes.

Antigens, CD↗
PubMed1991

Atomic structure of a pyrimidine-dimer specific excision-repair enzyme from bacteriophage T4.

T4 endonuclease V is an enzyme responsible for the first step of a pyrimidine-dimer specific excision-repair process. The three-dimensional structure of this enzyme has been determined at 1.6A resolution by X-ray crystallography. The enzyme consists of one single compact domain classified into the all alpha structure. This single domain possesses two distinct catalytic activities, a pyrimidine-dimer glycosylase and an apurinic/apyrimidinic endonuclease. The backbone of the enzyme represents a unique folding scheme incompatible with close packing of alpha-helices. The refined structure suggests possible residues that participate in interactions with DNA.

Amino Acid Sequence↗
PubMed1991

[Clinical implications of the O-antigen serovar E and drug resistance in persistent Pseudomonas infection].

We studied bacteriological parameters in patients with persistent Pseudomonas aeruginosa (PA) infection, including distribution of the PA O-antigen serovars, incidence of serovar conversion during treatment and relationship between these serovars and susceptibility to antibiotics. PA was identified using an automated bacterial identification system (Avantage Microbiology Center, Abbot Lab., USA) and the PA O-antigen serovar was performed with a monoclonal serovar diagnostic kit (Mei-Assay Pseudomonas Aeruginosa, Meiji Seika, Tokyo). Antibiotic susceptibility was evaluated by minimum inhibitory concentration according to the NCCLS method. The PA was isolated in 14.7% of the total of 1,900 isolates in our hospital from April, 1987 to March, 1988, and was most frequently isolated from urine specimens (51.9%), followed by sputum (40.0%). The serovar E was isolated in 57 (36.3%) of 157 PA strains tested, followed by the serovar B (21.0%) and G (17.8%). The serovar E was widely distributed in the wards of hematology (55.6%), neurosurgery (54.2%) and urology (42.3%). The serovar E showed high resistance rates to many antibiotics: 62.5% to piperacillin; 58.3%, cefsulodin; 58.3%, cefoperazone; and 70.8%, gentamicin. Serovar conversion during treatment was noticed in 8 of 24 patients with persistent PA infection. Three of 5 patients in whom the serovar had converted to the serovar E died. These results suggest that PA infection with the serovar E, especially with that converting from another serovar is an unfavorable prognostic sign.

Blood Group Antigens↗
PubMed1991

Induction of non-MHC-restricted cytotoxicity in a patient with pure red cell aplasia: functional relevance to antigen-specific cytotoxic T cells.

A functional analysis of an expanded T-cell subpopulation was studied in a patient with pure red cell aplasia who had no evidence of malignancy. In the time of an aggravation of the anemia, an expanded population of large granular lymphocytes (LGL) with T-cell receptor (TCR) alpha beta +CD3+CD8+ phenotype was noted, which reverted to normal with remission. These T cells displayed a polyclonal pattern in DNA analysis. Functionally, the T cells, with suppressor/cytotoxic phenotype exhibited normal capabilities for transducing the signals of the proliferative response, and of interleukin-2R (IL-2R) expression and IL-2 production via the TCR-CD3 complex structure. While they suppressed erythroid colony formation in vitro, neither non-major histocompatibility complex-restricted cytotoxic activity, cytotoxic T-cell activity, nor suppressive activity for immunoglobulin synthesis by B cells was detected. Pretreatment by anti-CD3 or anti-T-cell receptor antibody generated cytotoxicity for FcR+ target cells in the patient cells, but no such augmentation was found with other monoclonal antibodies of FcR- target cells. These findings indicated that the expanded T cells are functionally relevant to antigen-specific cytotoxic T lymphocytes.

Adult↗
PubMed1990

Influence of organ environment on extracellular matrix degradative activity and metastasis of human colon carcinoma cells.

Orthotopic implantation of human colon carcinoma cells is useful for studying the behavior of metastatic subpopulations. We observed that the parental line and variants of human colon carcinoma KM12 cells were all tumorigenic following implantation into the subcutis or cecal wall of BALB/c nude mice. Their ability to metastasize to distant organ sites varied, however, with the site of growth. Subcutaneous (SC) tumors did not produce visceral metastases, whereas cecal tumors metastasized to the regional mesenteric lymph nodes and to the liver. To examine the influence of organ environment on the extracellular matrix-degrading activity of the tumors, we inoculated human colon carcinoma cells into the subcutis or cecal wall and after 7 weeks isolated and cultured the tumors in serum-free medium. The conditioned media of SC tumors contained very low levels of type IV collagenase (gelatinase) and heparanase (heparan sulfate-specific endo-beta-D-glucuronidase), whereas the media of the cecal wall tumors contained high levels of both. Zymograms of the media revealed that the intracecal human colon carcinomas secreted more than three times the amount of latent and active forms of 92-kd type IV collagenase than did the SC tumors. Moreover, only the conditioned media of intracecal tumors contained latent and active forms of 64-kd type IV collagenase. Histochemical analysis using rabbit antiserum raised against the synthetic peptides of 72-kd procollagenase type IV showed type IV collagenase in the intracecal tumors; human colon carcinoma growing SC, however, were not stained significantly. These results suggest that factors in the organ environment may affect production and secretion of tumor extracellular matrix-degrading enzymes, and these factors may modify the metastatic behavior of human colon carcinoma cells in nude mice.

Animals↗
PubMed1990

Three-dimensional structure of ribonuclease H from E. coli.

The three-dimensional structure of RNase H from Escherichia coli was determined at 1.8 A resolution by X-ray crystallography. The enzyme was found to belong to the alpha + beta class of structures, consisting of two distinct domains. The structure implies a possible region interacting with a DNA-RNA hybrid. The Mg2(+)-binding site essential for activity is located near a cluster of four acidic amino acids--one glutamic and three aspartic acid residues. These residues are completely conserved in the homology alignment of sequences of RNase H and reverse transcriptases from retroviruses and retrovirus-like entities. The structural motif of beta strands around the Mg2(+)-binding site has similarities to that in DNase I.

Amino Acid Sequence↗