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Biomedical subjects

K Morikawa

Publications and source records attributed to K Morikawa.

At least 145 records · Page 8Linked to original sources
PubMed1994

Overproduction, purification, and characterization of ferrochelatase from Escherichia coli.

To establish a system for overproduction of the ferrochelatase [EC 4.99.1.1] from Escherichia coli, a plasmid designated pFC3 was constructed. The 35-kDa protein was accumulated in E. coli DH5 alpha cells that harbored pFC3 to a level equal to approximately 9% of the total protein (roughly 50 mg/liter) upon thermal induction. This 35-kDa protein was identified as the ferrochelatase of E. coli by Western blotting and amino-terminal amino acid sequence analysis. The protein with ferrochelatase activity was purified from the cells by three simple steps with a yield of 17%. The optimum pH of the purified enzyme was around 8.0. The molecular weight of the enzyme was estimated to be 35-kDa from column chromatography on Sephacryl S-300, a value consistent with that estimated from SDS-polyacrylamide gel electrophoresis, suggesting that the enzyme is a monomer. The isoelectric point of the enzyme was approximately 4.7. Determination of the far-ultraviolet circular dichroism spectrum allowed us to calculate the alpha-helix and beta-sheet contents of the enzyme as 10 +/- 0.2 and 39 +/- 0.2%, respectively. High-level production of the ferrochelatase from E. coli will greatly facilitate detailed structural analysis of this protein.

Amino Acid Sequence↗
PubMed1994

Immunosuppressive activity of bromocriptine on human T lymphocyte function in vitro.

Bromocriptine (BRC), a dopamine type 2 agonist, prevents secretion of pituitary prolactin (PRL). BRC has been shown to impair lymphocyte responsiveness toward antigenic stimulation by decreasing serum PRL levels. Hypoprolactinaemia induced by BRC produces a similar immunosuppressive effect, as observed in hypophysectomized rats, which is restored by the administration of PRL. Therefore, the immunosuppression induced by BRC has been interpreted as the result of hypoprolactinaemia. However, the direct mechanism of BRC in immune response has never been evoked. We recently reported that BRC has an immunosuppressive activity on human B lymphocyte function in vitro. In the present study we demonstrate that BRC suppresses T cell proliferation by means of blocking IL-2 production by T cells as well as mixed lymphocyte reaction (MLR) in a dose-dependent manner. We could not detect the immunoreactive PRL activity in the conditioned medium from polyclonal T cell mitogen-stimulated T cell cultures. Then, the immunosuppressive activity of BRC on human T cell function appeared to be independent of its hypoprolactinaemic effect. Treatment with low-dose cyclosporin A (CsA) or FK506 in combination with BRC has proved more effective than either drug alone in suppression of T cell proliferation and CD25 antigen expression. Thus, the therapeutic application of BRC in combination with immunosuppressants may enhance the immunosuppressive effect, while at the same time decreasing the toxicity.

Bromocriptine↗
PubMed1994

Immunomodulatory effects of three macrolides, midecamycin acetate, josamycin, and clarithromycin, on human T-lymphocyte function in vitro.

The effect of three macrolide antibiotics, midecamycin acetate, josamycin, and clarithromycin, on human T-cell function was investigated in vitro. Midecamycin acetate and josamycin suppressed the proliferative response of peripheral blood mononuclear cells stimulated by polyclonal T-cell mitogens at concentrations between 1.6 and 8 micrograms/ml. At higher concentrations (40 to 200 micrograms/ml), all these drugs showed a marked inhibitory effect. At concentrations of 1.6 to 40 micrograms/ml, these drugs suppressed interleukin-2 (IL-2) production induced by mitogen-stimulated T cells, but not the expression of IL-2 receptor (CD25), in a dose-dependent manner. Therefore, the suppressive action on T-lymphocyte proliferation seems to be based on the ability of these drugs to inhibit IL-2 production by T cells. The drug also inhibited mixed lymphocyte reaction at the same concentrations. Combined treatment with these macrolides and the known immunosuppressants such as FK506 and cyclosporin A resulted in an increased inhibition of T-cell proliferation. The immunomodulatory properties of the antibiotics may have clinical relevance for modulation of the immune response in transplant patients and in patients with inflammatory diseases.

Adjuvants, Immunologic↗
PubMed1994

Renal handling of albumin and beta-2-microglobulin in neonates.

Urinary albumin and beta 2-microglobulin (B2M) were measured during the neonatal period. Urinary albumin decreased postnatally in term neonates, while it remained almost constant in preterm neonates. Urinary B2M showed a peak level on day 7 both in term and preterm neonates. There was some trend towards higher levels of albumin and B2M with decreasing gestation, showing that glomerular permeability increases and proximal tubular protein reabsorption decreases with increasing degrees of prematurity. In sick preterms who were depressed at birth and had respiratory failure, both parameters were elevated during the first 2 weeks, indicating the presence of glomerular and tubular damage in this period. The changes in B2M with gestation or clinical condition were more pronounced than those in albumin.

Age Factors↗
PubMed1994

Characterization of acetylcholinesterase-inhibition by itopride.

Itopride is a gastroprokinetic benzamide derivative. This agent inhibited both electric eel acetylcholinesterase (AChE) and horse serum butyrylcholinesterase (BuChE). The IC50 of itopride with AChE (2.04 +/- 0.27 microM) was, however, 100-fold less than that with BuChE, whereas in the case of neostigmine with AChE (11.3 +/- 3.4 nM), it was 10-fold less. The recovery of AChE activity inhibited by 10(-7) M neostigmine was partial, but that inhibited by up to 3 x 10(-5) M itopride was complete when the reaction mixture was subjected to ultrafiltration. Double reciprocal plots of the experimental data showed that both Km and Vmax were affected by itopride, suggesting that the inhibition is a "mixed" type, although primarily being an uncompetitive one. The inhibitory effect of itopride on cholinesterase (ChE) activity in guinea pig gastrointestine was much weaker than that on pure AChE. However, in the presence of a low dose of diisopropyl fluorophosphate, just enough to inhibit BuChE but not AChE, the IC50s of itopride against ChE activities were found to be about 0.5 microM. In conclusion, itopride exerts reversible and a "mixed" type of inhibition preferably against AChE. The IC50 of itopride for electric eel and guinea pig gastrointestinal AChE inhibition was 200 times and 50 times as large as that of neostigmine, respectively.

Acetylcholine↗
PubMed1993

Crystal structures of ribonuclease HI active site mutants from Escherichia coli.

In order to investigate the relationships between the three-dimensional structure and the enzymic activity of E. coli RNase HI, three mutant proteins, which were completely inactivated by the replacements of three functional residues, Asp10 by Asn (D10N), Glu48 by Gln (E48Q), and Asp70 by Asn (D70N), were crystallized. Their three-dimensional structures were determined by x-ray crystallography. Although the entire backbone structures of these mutants were not affected by the replacements, very localized conformational changes were observed around the Mg(2+)-binding site. The substitution of an amide group for a negatively charged carboxyl group in common induces the formation of new hydrogen bond networks, presumably due to the cancellation of repulsive forces between carboxyl side chains with negative charges. These conformational changes can account for the loss of the enzymic activity in the mutants, and suggest a possible role for Mg2+ in the hydrolysis. Since the 3 replaced acidic residues are completely conserved in sequences of reverse transcriptases from retroviruses, including human immunodeficiency virus, the concepts of the catalytic mechanism deduced from this structural analysis can also be applied to RNase H activity in reverse transcriptases.

Binding Sites↗
PubMed1993

Cooperative stabilization of Escherichia coli ribonuclease HI by insertion of Gly-80b and Gly-77-->Ala substitution.

The insertion of a Gly residue (designated as Gly-80b) between the C-cap of the alpha II-helix (Gln-80) and the N-cap of the alpha III-helix (Trp-81) in Escherichia coli ribonuclease HI enhances the protein stability by 0.4 kcal/mol in delta G (Kimura, S., Nakamura, H., Hashimoto, T., Oobatake, M., & Kanaya, S. (1992) J. Biol. Chem. 267, 21535-21542). Another mutation within the alpha II-helix, Gly-77-->Ala, reduces the stability by 0.9 kcal/mol. Simultaneous introduction of these mutations enhances the stability by 0.8 kcal/mol, indicating that the effects of these mutations are cooperative and not simply independent. We determined the crystal structures of these three mutant proteins (G80b-, A77-, and A77/G80b-RNase H) to investigate this cooperative mechanism of the protein stabilization. The structures revealed that the inserted Gly-80b assumes a left-handed helical conformation in both the G80b- and the A77/G80b-RNase H. This inserted glycine residue allows the formation of a "paperclip", which is a common motif at the C-termini of alpha-helices. Accompanying the formation of the paperclip motif, two intrahelical hydrogen bonds are formed between the backbone atoms (O78-N80b and O80b-N84). The stabilization caused by the insertion of Gly-80b can be ascribed to the formation of these hydrogen bonds. The Gly-77-->Ala substitution destabilizes the protein due to the deformed packing interactions in the hydrophobic core around Ala-77 and the stress in the wedged indole ring of Trp-81. These effects are alleviated by the insertion of Gly-80b, which relaxes the backbone structure.(ABSTRACT TRUNCATED AT 250 WORDS)

Alanine↗
PubMed1993

Stabilization of Escherichia coli ribonuclease HI by cavity-filling mutations within a hydrophobic core.

The crystal structure of Escherichia coli ribonuclease HI has a cavity near Val-74 within the protein core. In order to fill the cavity space, we constructed two mutant proteins, V74L and V74I, in which Val-74 was replaced with either Leu or Ile, respectively. The mutant proteins are stabilized, as revealed by a 2.1-3.7 degrees C increase in the Tm values, as compared to the wild-type protein at pH values of 3.0 and 5.5. The mutant protein V74A, in which Val-74 is replaced with Ala, was also constructed to analyze the reverse effect. The stability of V74A decreases by 7.6 degrees C at pH 3.0 and 12.7 degrees C at pH 5.5 in Tm as compared to those values for the wild-type protein. None of the three mutations significantly affect the enzymatic activity. The crystal structures of V74L and V74I, determined at 1.8-A resolution, are almost identical to that of the wild-type protein, except for the mutation site. In the two mutant proteins, calculation by the Voronoi procedure shows that the cavity volumes around the individual mutation sites are remarkably reduced as compared to that in the wild-type protein. These results indicate that the introduction of a methylene group into the cavity, without causing steric clash, contributes to an increase in the hydrophobic interaction within the protein core and thereby enhances protein stability. We also discuss the role of the Leu side chain, which can assume many different local conformations on a helix without sacrificing thermostability.

Amino Acids↗
PubMed1993

Folding topology and DNA binding of the N-terminal fragment of Ada protein.

Three amino terminal fragments of Escherichia coli Ada protein (39 kDa) with different molecular masses (14 kDa, 16 kDa and 20 kDa) were prepared in large quantities from an E. coli strain harboring plasmids constructed for the overproduction of the truncated proteins. The three fragments can be methylated to an extent similar to that of the intact molecule. The methylated 16 kDa fragment specifically binds to the ada box on a DNA duplex. NMR analyses revealed that the 14 kDa fragment comprises two alpha-helices and a beta-sheet with parallel and anti-parallel mixed strands. A comparison of the 15N-1H HMQC spectra of the fragments has led to the conclusion that this tertiary structure within the 14 kDa fragment is retained in the larger 16 kDa and 20 kDa fragments.

Amino Acid Sequence↗
PubMed1993

Crystal structures of ribonuclease F1 of Fusarium moniliforme in its free form and in complex with 2'GMP.

RNase F1, a guanine-specific ribonuclease from Fusarium moniliforme, was crystallized in two different forms, in the absence of an inhibitor and in the presence of 2'GMP. The crystal structure of the RNase F1 free form was solved by the molecular replacement method, using the co-ordinates of the RNase T1 complex with 2'GMP, and was refined to a final R-factor of 18.7%, using the data extended to 1.3 A resolution. For the crystal structure of the RNase F1 complex with 2'GMP, the solution of the molecular replacement method was obtained on the basis of the co-ordinates of the RNase F1 free form, and was refined to a final R-factor of 16.8%, using the data up to 2 A resolution. The two crystal structures of the RNase F1 free form and the complex with 2'GMP are very similar to each other as reflected by a small root-mean-square displacement (r.m.s.d.) value of 0.43 A for all C alpha atoms. The main differences between the two structures are associated with binding of 2'GMP in the substrate recognition site in the loop between Tyr42 and Glu46. A structural comparison between RNase F1 and RNase T1 shows a substantial similarity between all the C alpha atoms, as evidenced by a r.m.s.d. value of 1.4 A. The loop from residues 32 to 38 was strikingly different between these two enzymes, in both its conformation and its hydrogen bonding schemes. The side-chain of a catalytically active residue, His92, is shifted away from the catalytic site in RNase F1 by 1.3 A and 0.85 A with respect to the corresponding positions in the RNase T1 free form and in the RNase T1 complex with 2'GMP, respectively. In the RNase F1 complex, the guanine base of 2'GMP has a syn conformation about the glycosyl bond, and the furanose ring assumes a 3'-exo pucker, which is different from that found in the complex with RNase T1. In the catalytic site of the RNase F1 complex with 2'GMP, one water molecule was observed, which bridges the phosphate oxygen atoms of 2'GMP and the side-chains of the catalytically important residues, His92 and Arg77, through hydrogen bonds. A water molecule occupying the same position was found in the RNase F1 free form. The significance of this water molecule in the hydrolytic reaction is discussed.

Amino Acid Sequence↗
PubMed1993

Crystal structure of ribonuclease H from Thermus thermophilus HB8 refined at 2.8 A resolution.

The crystal structure of Thermus thermophilus RNase H was determined at 2.8 A resolution. The structure was solved by the molecular replacement method, based on the accurately refined structure of Escherichia coli RNase HI, which shows 52% amino acid sequence identity. Crystallographic refinement led to an R-factor of 0.205, with a 0.019 A root-mean-square deviation from ideal bond lengths and 0.048 A from ideal bond angle distances. Structural comparison shows a striking similarity in the overall folding of the thermophilic and mesophilic enzymes. The root-mean-square displacement is 0.95 A between equivalent alpha-carbon atoms from all elements of secondary structure (five alpha-helices and five beta-strands). However, some notable differences, which account for the enhanced thermostability of T. thermophilus RNase H, are observed in loop structures and side-chain conformations. The substitution of Gly for the left-handed helical residue (Lys95) in the E. coli enzyme is proposed to substantially enhance the thermostability, due to the release of steric hindrance caused by the beta-carbon atom. Furthermore, it is likely that the expansion of an aromatic cluster, arising from the replacement of Ile78 in the mesophilic enzyme by Phe, and the increased number of salt-bridges additively contribute to the stability.

Amino Acid Sequence↗
PubMed1993

Crystal structure of Escherichia coli RNase HI in complex with Mg2+ at 2.8 A resolution: proof for a single Mg(2+)-binding site.

To obtain more precise insight into the Mg(2+)-binding site essential for RNase HI catalytic activity, we have determined the crystal structure of E. coli RNase HI in complex with Mg2+. The analyzed cocrystal, which is not isomorphous with the Mg(2+)-free crystal previously refined at 1.48 A resolution, was grown at a high MgSO4 concentration more than 100 mM so that even weakly bound Mg2+ sites could be identified. The structure was solved by the molecular replacement method, using the Mg(2+)-free crystal structure as a search model, and was refined to give a final R-value of 0.190 for intensity data from 10 to 2.8 A, using the XPLOR and PROLSQ programs. The backbone structures are in their entirety very similar to each other between the Mg(2+)-bound and the metal-free crystals, except for minor regions in the enzyme interface with the DNA/RNA hybrid. The active center clearly revealed a single Mg2+ atom located at a position almost identical to that previously found by the soaking method. Although the two metal-ion mechanism had been suggested by another group (Yang, W., Hendrickson, W.A., Crouch, R.J., Satow, Y. Science 249:1398-1405, 1990) and partially supported by the crystallographic study of inactive HIV-1 RT RNase H fragment (Davies, J.F., II, Hostomska, Z., Hostomsky, Z., Jordan, S.R., Matthews, D. Science 252:88-95, 1991), the present result excludes the possibility that RNase HI requires two metal-binding sites for activity. In contrast to the features in the metal-free enzyme, the side chains of Asn-44 and Glu-48 are found to form coordinate bonds with Mg2+ in the metal-bound crystal.

Binding Sites↗
PubMed1993

Recombinant human IL-5 augments immunoglobulin generation by human B lymphocytes in the presence of IL-2.

The biological activity of interleukin-5 (IL-5) on human B lymphocytes was examined by using a newly purified recombinant human IL-5 preparation. Different densities of B cells from human tonsil samples were isolated by Percoll density gradient from nonrosetted cells. The IL-5 preparation did not act as a B cell growth factor on the large (in vivo-activated) or small (resting) B cells after activation with polyclonal B cell activators. The IL-5 augmented the immunoglobulin (Ig) generation of Staphylococcus aureus Cowan strain I (SAC)-activated B blasts in the presence of interleukin-2 (IL-2). The effect of IL-5 on Ig generation was greater in pokeweed mitogen (PWM)-driven B blasts than that in SAC-induced B blasts when assessed by enzyme-linked immunosorbent assay. The amounts of mu RNA isolated from IL-5/PWM-treated cells were also larger than those from IL-5/IL-2-treated B blasts. These results suggest that the human IL-5 can increase Ig generation of B cells in the presence of IL-2 by working at the level of mRNA coding for Ig.

B-Lymphocytes↗
PubMed1993

Clinical course and outcome of idiopathic membranous nephropathy in Japanese children.

We retrospectively studied 12 Japanese children (8 boys, 4 girls) with idiopathic membranous nephropathy (IMN), aged 2.9-15.8 (mean 7.7) years at onset. All patients were identified through either screening or a routine urinalysis; proteinuria was present in all, haematuria, which was macroscopic in 4, in 11. Three had nephrotic syndrome (NS) at or soon after onset. Stages on electron microscopy, performed in 10 patients, were I in 3, II in 5 and III in 2. Steroids alone or with cyclophosphamide were administered to 5 patients, including the 3 patients showing NS. Complete remission of proteinuria occurred in 8 patients 0.3-1.6 (mean 0.6) years after onset, and proteinuria did not recur. After a follow-up of 1.6-11.6 (mean 5.9) years, these 8 patients were in complete remission and the remaining 4 had only mild proteinuria; none had hypertension or impaired renal function. Thus, we infer that IMN in Japanese children may have a better course and outcome than IMN in non-Japanese children. Based on a comparative study of Japanese (previously reported cases added to ours) and non-Japanese (mostly Caucasian) children with IMN, this was confirmed; it is possible that steroid therapy in Japanese patients is more effective in inducing remission of NS and preserving renal function.

Adolescent↗
PubMed1993

Urinary alpha 1-microglobulin as an index of proximal tubular function in early infancy.

Urinary alpha 1-microglobulin (U-A1M) was measured in healthy term infants on days 1, 4, 7, 14, 28, 90 and 180 of life. U-A1M was high until day 14 and declined thereafter. It was significantly correlated with urinary beta 2-microglobulin (U-B2M) throughout the study, but not with serum A1M on days 1 or 7. Similar to U-B2M, U-A1M in the clinically stable term infants with intrauterine growth retardation (n = 4-7) was not elevated on days 1-7. In the sick infants who needed immediate resuscitation at birth (n = 4-8), U-A1M was well as U-B2M was high on days 1-7 and then decreased to normal levels, suggesting that U-A1M can be used as a sensitive marker of acute proximal tubular damage and its recovery. These observations indicate that U-A1M is a useful index of proximal tubular function in early infancy.

Alpha-Globulins↗