Biomedical subjects
Publications and source records attributed to S Hirono.
Structures of two enzyme-inhibitor complexes of human immunodeficiency virus-1 protease with allophenylnorstatine derivatives were obtained from molecular dynamics simulation in aqueous solution. The stronger inhibitor gave considerably smaller fluctuation at P3 site, which formed hydrogen bonding with the enzyme flap region.
We examined hepatocyte growth factor-producing organs in normal and carbon tetrachloride-treated Japanese monkeys, a species more related to human beings than rats, by measuring hepatocyte growth factor protein and hepatocyte growth factor mRNA in various tissues. Hepatocyte growth factor protein and hepatocyte growth factor mRNA levels were determined by use of an enzyme-linked immunosorbent assay for human hepatocyte growth factor and Northern-blot analysis using human hepatocyte growth factor cDNA as a probe, respectively. Although very slight amounts of hepatocyte growth factor protein were found in various organs of normal monkeys, the hepatocyte growth factor protein levels were remarkably increased only in the livers and spleens of carbon tetrachloride-treated monkeys. Hepatocyte growth factor mRNA was distinctly detected in the livers, spleens, lungs, intestinal tracts, adrenal glands and aortas of normal monkeys. After the administration of carbon tetrachloride, the hepatocyte growth factor mRNA levels in the liver, spleen and gastrointestinal tract were markedly increased; the level in the lung was unchanged, differing from the findings in rats; and the levels in adrenal gland and aorta were unaltered. These results indicate that the liver is an important hepatocyte growth factor-producing organ in monkeys during liver regeneration and that hepatocyte growth factor induced in the liver may stimulate hepatocyte proliferation by way of a paracrine mode.
We performed molecular dynamics (MD)/free energy perturbation (FEP) calculations to reproduce the experimental free energy difference of denaturation for staphylococcal nuclease mutant Met32-->Ala (M32A) and to predict the stability of the mutant Met32-->Leu (M32L). The calculated free energy difference of denaturation for the M32A of -1.9 kcal/mol was in good agreement with the experimental value. In the M32A, a small hydrophobic core formed by three aromatic rings (Tyr-27, Phe-34, Phe-76) in a wild-type crumbled as a result of exposure to water. The van der Waals interactions in the native state of the M32A were weaker than those of the wild-type, which strongly suggests that the Met-32 is important for the stability of the enzyme. The M32L has not been available yet, but is expected to retain the small hydrophobic core. The free energy difference of denaturation for the M32L was calculated to be 1.6 kcal/mol. The MD/FEP simulation showed that the native state structure of the M32L was only slightly changed when compared with that of the wild-type. It was suggested that the M32L is more stable than the wild-type because the electrostatic interactions in the denatured state are more disadvantageous than those in the native state.
The stability of two mutants G88V (Gly-88-->Val) and A69T (Ala-69-->Thr) of staphylococcal nuclease was analyzed by molecular dynamics simulations. The calculated free energy differences of denaturation for G88V and A69T were -1.1 and -2.8 kcal/mol, respectively. These values are in good agreement with the experimental values. The free energy differences divided into electrostatic and van der Waals components were analyzed. These two mutants are mainly destabilized due to van der Waals interactions. There is little difference between the electrostatic contribution to the free energy change in the native state and that in the denatured state. In each mutant structure, a small cavity appears in the vicinity of the perturbed residue. It is suggested that intramolecular van der Waals interactions of the mutants are weaker than those of the wild-type. Furthermore, analyses of the contributions of each residue near the perturbed residue and of water to the free energy difference of denaturation suggest that the interaction between water and the perturbed residue plays a very important role in the stability of staphylococcal nuclease, and that a small hydrophobic core consisting of the three aromatic rings (Tyr-27, Phe-34, Phe-76) and the side chain of Met-32 is also important for the stability.
1,4-Dihydropyridine derivatives having two nitrooxyalkyl moieties as esters at the 3 and 5 positions possess antihypertensive activity. To understand how substituents affect the biological activity, the quantitative structure-activity relationship (QSAR) of 27 compounds was analyzed using the Fuzzy adaptive least-squares (FALS 91) method. The QSAR models suggested that the hydrophobicity and electronic effect at the 4 position of the 1,4-dihydropyridine along with the special structures of the nitrooxyalkylester components are important for antihypertensive activity.
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To evaluate the role of protein phosphorylation reactions in signal transduction of human hepatocyte growth factor (hHGF), now known to be the same protein as the scatter factor and tumor cytotoxic factor, we examined the effects of various inhibitors of protein kinases on the mitogenic activity of hHGF on rat hepatocytes in primary culture. Genistein, a specific inhibitor of tyrosine kinase, dose-dependently inhibited the effect of hHGF in stimulating DNA synthesis of hepatocytes. By contrast, 1-(5-isoquinolinesulfonyl)-2- methylpiperazine (H7), a specific inhibitor of protein kinase C, potentiated the stimulatory effect of hHGF on DNA synthesis of hepatocytes. H7 was effective at over 2 micrograms/ml and potentiated the effect of hHGF over 2-fold at 20 micrograms/ml. On the other hand, an inhibitor of Ca++/calmodulin-dependent protein kinase inhibited both the basal and hHGF-stimulated DNA synthesis in the cells, whereas an inhibitor of cyclic nucleotide-dependent protein kinases had little effect on the action of hHGF. These results suggest that tyrosine phosphorylation is required for stimulation of hepatocyte DNA synthesis by hHGF and that the action of hHGF is negatively regulated by protein kinase C activation.
To characterize the receptor(s) for human hepatocyte growth factor (hHGF), a physiological hepatotrophic factor involved in liver regeneration following hepatic injury, recombinant hHGF (rhHGF) was radioiodinated. The labeled rhHGF retained its full biological activity on adult rat hepatocytes in primary culture. The specific binding of [125I]iodo-rhHGF to hepatocytes reached a plateau within 240 min at 4 degrees C. Scatchard plot analysis of the binding data suggested the presence of two classes of high affinity binding sites for [125I]iodo-rhHGF. One of the sites had a dissociation constant (Kd) of about 4.6 pM with 300 sites/cell and the other has a Kd of about 275 pM with 15,160 sites/cell. Unlabeled rhHGF displaced cell surface-bound [125I]iodo-rhHGF in a dose-dependent manner as did native hHGF purified from plasma of patients with fulminant hepatic failure. However, other growth factors to rat hepatocytes in primary culture such as insulin and human epidermal growth factor, and proteins which have high amino acid sequence-homology to hHGF such as plasminogen and prothrombin, did not compete with [125I]iodo-rhHGF in the binding, which suggests the binding was specific to hHGF. Covalent cross-linking experiment of [125I]iodo-rhHGF to cell surface receptor(s) on hepatocytes showed there were two macromolecular species with apparent molecular weights of 330,000 and 230,000. Unlabeled rhHGF and native hHGF competed for the binding of [125I]iodo-rhHGF to the two macromolecular species, but insulin, human epidermal growth factor, plasminogen, and prothrombin did not. Based upon our estimated molecular weight of rhHGF = 84,000, these results suggest that hHGF specifically binds to two polypeptides of 246,000 and 146,000 daltons which are likely to represent the hHGF receptors of primary cultured rat hepatocytes.
An asymptomatic 49-year-old woman with a diffuse abnormal shadow detected on a routine chest rentogenogram was referred for evaluation. Ultrasonography, computed tomography (CT) and magnetic resonance imaging (MRI) revealed space-occupying lesions in the liver and spleen. Lymphadenopathy was observed in the mediastinum and abdomen on CT, and multiple lesions of low signal intensity were observed in the spine on MRI. Furthermore, the presence of multiple tumorous nodules in the liver suggesting metastatic hepatic cancer was demonstrated on laparoscopy. Biopsy of the liver tumors revealed sarcoidosis; transbronchial biopsy specimens showed identical histological features. After administration of prednisolone for one month, the space-occupying lesions in the liver, spleen and spine improved. This case of sarcoidosis exhibited hepatic tumorous nodules and space-occupying lesions in the spleen and the spine which mimicked the presence of metastatic hepatic cancer.
3-Benzoylpropionic acid derivatives possess an immunomodulative activity and suppress adjuvant arthritis. To understand how substituents affect the biological activity, the quantitative structure-activity relationships of 30 compounds were analyzed by the adaptive least-squares method. For the suppressing activity in rats, the electronic effects and the structural feature of the substituent on benzene ring were suggested to be important. To reinforce and confirm the correlation, 4 additional compounds of phenoxybutyric acid derivatives were synthesized and tested with the rat adjuvant-induced arthritis. These compounds were found to have potent suppressing activity.
A monoclonal antibody was raised against a mineralized tissue-specific sialoprotein containing no phosphorus using partially purified noncollagenous bone matrix proteins from rats as antigen. Then the sialoprotein was purified by high performance liquid chromatography from rat mandibulae using the monoclonal antibody as a marker. The sialoprotein (59-kDa bone sialoprotein (BSP)) with a molecular weight of 59,000 contained 1.4% sialic acid but no detectable phosphorus. Immunohistochemical studies with the antibody showed that the protein was specific to mineralized tissues such as bone and dentin, and present in osteoblasts, osteocytes, and bone matrix. No other soft tissues, such as the cartilage, liver, kidney, and periosteum, were stained. However, Western blot analysis showed that plasma contained immunoreactive 59-kDa BSP. The quantitative amino acid composition of 59-kDa BSP resembled that of human alpha 2-HS glycoprotein (alpha 2-HSG) (Lee, C.-C., Bowman, B.H., and Yang, F. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 4403-4407; Kellermann, J., Haupt, H., Auerswald, E.-A., and Muller-Esterl, W. (1989) J. Biol. Chem. 264, 14121-14128) and rat 64-kDa protein (Franzén, A., and Heinegård, D. (1985) in The Chemistry and Biology of Mineralized Tissues (Butler, W.T., ed), p. 132, EBSCO Media, Birmingham, AL). Amino acid sequence analyses of the amino-terminal region and four peptide fragments of 59-kDa BSP revealed that about 50% of the amino acids were homologous with those of human alpha 2-HSG, which is known to be synthesized by the liver, transported in the bloodstream, and incorporated into calcified tissues. But when newborn rat calvaria, primary cultures of osteoblast-rich cells, and adult rat hepatocytes were incubated with radioactive leucine, immunoreactive 59-kDa BSP was detected in their conditioned medium by fluorography. Several characteristics, including the amino acid sequence, suggest that 59-kDa BSP may be the rat counterpart of human alpha 2-HSG. However, rat 59-kDa BSP is a single peptide and synthesized by both osteoblasts and hepatocytes, whereas human alpha 2-HSG is known to be a heterodimer and to be synthesized by the liver.
We present the results of free energy perturbation calculations on binding and catalysis of a tetrapeptide substrate, acetyl-Phe-Ala-Ala-Phe-NMe, by native subtilisin BPN' and a subtilisin BPN' mutant (Thr220----Ala220). The calculated difference in the free energy of binding was 0.70 +/- 0.72 kcal/mol. The calculated difference in the free energy of catalysis was 1.48 +/- 0.89 kcal/mol. These calculated values compare well with the experimental values in which another substrate, succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, was used. These findings suggest that Thr220 is more important for catalysis than substrate binding.
The N-terminal amino acid of the heavy chain of native (purified from human plasma) and recombinant human hepatocyte growth factor (hHGF) was determined by analyses of amino acid composition and sequence of peptide fragments derived by enzymatic cleavage, peptide mapping, and fast atom bombardment mass spectrometry. Our results indicate that the N-terminal amino acid of the heavy chain of hHGF, both native and recombinant, is pyroglutamate, derived from glutamine at the 32nd residue from the initiation methionine.
The levels of human hepatocyte growth factor (hHGF) in sera obtained from patients with various liver diseases were determined using adult rat hepatocytes maintained in primary culture. The mean hHGF activity for 22 patients with fulminant hepatic failure was about nine times greater than that found in normal human serum. The increase in serum hHGF activity seen in two patients with "acute-on-chronic" hepatitis was similar to that found in patients with fulminant hepatic failure. The serum level of hHGF from patients with acute hepatitis is related to the stage of their illness. The average value for 31 patients was about three times that of normal human serum. In some patients, the time course for the increase in serum hHGF activity was similar to that demonstrated for alpha-fetoprotein. The mean hHGF activity in serum for the 33 patients with chronic hepatitis and from 25 patients with liver cirrhosis was increased also compared with that of normal human serum. In addition, serum hHGF activity in three of seven patients studied after partial hepatectomy for a space-occupying lesion of the liver was increased. These data suggest that the increase in serum hHGF activity present in patients with various liver diseases reflects a self-defense mechanism that is involved in the process of liver cell regeneration.
Human hepatocyte growth factor (hHGF) was purified from the plasma of six patients with fulminant hepatic failure due to hepatitis B in two and non-A, non-B hepatitis in four. The purified hHGF from each patient contained two major protein bands having molecular weights of 79,000 and 86,000 and several minor bands having molecular weights between 76,000 and 92,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis performed under nonreduced conditions. After reduction with 2-mercaptoethanol, three major bands having molecular weights of 58,000, 34,500, and 31,500 were evident. In addition, a band having a molecular weight of 21,000 was detected. hHGF activity was destroyed by its reduction. The hHGF purified from patients demonstrated a dose response in terms of an increase in DNA synthesis using cultured hepatocytes. The hHGF concentration in the plasma of the patients with grade III-IV hepatic coma was calculated to be in the range of 1.8-3.0 nM. Finally the heavy chain of hHGF was not recognized by an anti-human albumin antibody, indicating that hHGF is not biliprotein, an albumin-bilirubin complex, that has been reported to be a putative liver growth factor.
We present free energy perturbation calculations on the complexes of Glu46----Ala46 (E46A) and Glu46----Gln46 (E46Q) mutants of ribonuclease T1 (RNaseT1) with inhibitors 2'-guanosine monophosphate (GMP) and 2'-adenosine monophosphate (AMP) by a thermodynamic perturbation method implemented with molecular dynamics (MD). Using the available crystal structure of the RNaseT1-GMP complex, the structures of E46A-GMP and E46Q-GMP were model built and equilibrated with MD simulations. The structures of E46A-AMP and E46Q-AMP were obtained as a final structure of the GMP----AMP perturbation calculation respectively. The calculated difference in the free energy of binding (delta delta Gbind) was 0.31 kcal/mol for the E46A system and -1.04 kcal/mol for the E46Q system. The resultant free energies are much smaller than the experimental and calculated value of approximately 3 kcal/mol for the native RNaseT1, which suggests that both mutants have greater relative adenine affinities than native RNaseT1. Especially E46Q is calculated to have a larger affinity for adenine than guanine, as we suggested previously from the calculation on the native RNaseT1. Thus, the molecular dynamics/free energy perturbation method may be helpful in protein engineering, directed toward increasing or changing the substrate specificity of enzymes.
We have found a hepatotrophic factor in plasma or sera of patients with fulminant hepatic failure and have purified human hepatocyte growth factor from plasma of these patients. In this study we developed an enzyme-linked immunosorbent assay with high specificity and sensitivity for human hepatocyte growth factor in human serum. This assay for serum human hepatocyte growth factor is a sandwich method consisting of three steps. The standard curve for human hepatocyte growth factor appeared to be linear in the range of 0.20 to 12.50 ng purified human hepatocyte growth factor/ml (2.35 to 147 pmol/L). The assay took about 4 hr. Serum human hepatocyte growth factor values in patients with fulminant hepatic failure measured by enzyme-linked immunosorbent assay showed a strong positive correlation with that by bioassay using rat hepatocytes in primary culture. The mean value of serum human hepatocyte growth factor for 30 normal subjects was 0.24 +/- 0.12 (S.D.) ng/ml; that for 23 patients with fulminant hepatic failure was 8.06 +/- 1.76 (S.E.M.) ng/ml- greater than 30 times greater than the mean value for normal subjects. Serum human hepatocyte growth factor levels in patients with acute hepatitis, chronic hepatitis and cirrhosis were found to be slightly higher than those in normal subjects, but only the increase in serum human hepatocyte growth factor of acute hepatitis patients was statistically significant. The enzyme-linked immunosorbent assay for serum human hepatocyte growth factor should prove useful for serum human hepatocyte growth factor level measurement in patients with various liver diseases.
We present a calculation of the relative changes in binding free energy between the complex of ribonuclease T1 (RNase Tr) with its inhibitor 2'-guanosine monophosphate (2'GMP) and that of RNase T1-2'-adenosine monophosphate (2'AMP) by means of a thermodynamic perturbation method implemented with molecular dynamics. Using the available crystal structure of the RNase T1-2'GMP complex, the structure of the RNase T1-2'AMP complex was obtained as a final structure of the perturbation calculation. The calculated difference in the free energy of binding (delta delta Gbind) was 2.76 kcal/mol. This compares well with the experimental value of 3.07 kcal/mol. The encouraging agreement in delta delta Gbind suggests that the interactions of inhibitors with the enzyme are reasonably represented. Energy component analyses of the two complexes reveal that the active site of RNase T1 electrostatically stabilizes the binding of 2'GMP more than that of 2'AMP by 44 kcal/mol, while the van der Waals' interactions are similar in the two complexes. The analyses suggest that the mutation from Glu46 to Gln may lead to a preference of RNase T1 for adenine in contrast to the guanine preference of the wild-type enzyme. Although the molecular dynamics equilibration moves the atoms of the RNase T1-2'GMP system about 0.9 A from their X-ray positions and the mutation of the G to A in the active site increases the deviation from the X-ray structure, the mutation of the A back to G reduces the deviation. This and the agreement found for delta delta Gbind suggest that the molecular dynamics/free energy perturbation method will be useful for both energetic and structural analysis of protein-ligand interactions.