Prediction of outcome in fulminant hepatic failure by serum human hepatocyte growth factor.
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Growth Substances↗
Biomedical subjects
S Hirono
Publications and source records attributed to S Hirono.
Effects of protein kinase inhibitors on the mitogenic activity of human hepatocyte growth factor on rat hepatocytes in primary culture.
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.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
Identification and partial characterization of two classes of receptors for human hepatocyte growth factor on adult rat hepatocytes in primary culture.
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.
Animals↗
Sarcoidosis with tumorous hepatic and bone lesions mimicking disseminated malignancy: a case report.
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.
Diagnosis, Differential↗
Structure-activity studies of 3-benzoylpropionic acid derivatives suppressing adjuvant arthritis.
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.
Animals↗
Purification, characterization, and studies on biosynthesis of a 59-kDa bone sialic acid-containing protein (BSP) from rat mandible using a monoclonal antibody. Evidence that 59-kDa BSP may be the rat counterpart of human alpha 2-HS glycoprotein and is synthesized by both hepatocytes and osteoblasts.
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.
Amino Acid Sequence↗
Free energy perturbation calculations on binding and catalysis after mutating threonine 220 in subtilisin.
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.
Amino Acid Sequence↗
Identification of the N-terminal residue of the heavy chain of both native and recombinant human hepatocyte growth factor.
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.
Amino Acid Sequence↗
Human hepatocyte growth factor in blood of patients with fulminant hepatic failure. I. Clinical aspects.
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.
Growth Substances↗
Human hepatocyte growth factor in blood of patients with fulminant hepatic failure. Basic aspects.
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.
Animals↗
Relative binding free energy calculations of inhibitors to two mutants (Glu46----Ala/Gln) of ribonuclease T1 using molecular dynamics/free energy perturbation approaches.
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.
Adenosine Monophosphate↗
Levels of the human hepatocyte growth factor in serum of patients with various liver diseases determined by an enzyme-linked immunosorbent assay.
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.
Enzyme-Linked Immunosorbent Assay↗
Calculation of the relative binding free energy of 2'GMP and 2'AMP to ribonuclease T1 using molecular dynamics/free energy perturbation approaches.
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.
Adenosine Monophosphate↗
Neoplastic angioendotheliosis: demonstration of immunoglobulin gene rearrangements by the Southern blot hybridization technique.
Using the Southern blot hybridization technique, two cases of 'neoplastic angioendotheliosis' (NAE) were examined for immunoglobulin gene rearrangements. For DNA analysis, frozen kidney (Case 1) and lung (Case 2) tissue, in which intravascular neoplastic cells were abundant, were used. In each case rearrangements were observed in both heavy and light chain genes. On examination of the heavy chain gene, the JH DNA probe detected one rearranged band in Case 1 and two in Case 2 after EcoRI digestion. On examination of the light chain genes, the C kappa DNA probe revealed one rearranged band in Case 1 and two in Case 2 after BamHI digestion, while the C lambda 2 DNA probe revealed only germline configurations after EcoRI digestion in each case. Thus, B-lymphocytic differentiation at the gene level was demonstrated in neoplastic cells in both cases. Since no more than two rearrangements were detected in each immunoglobulin gene, the intravascular neoplastic cells were considered to be monoclonal in nature.
Aged↗
Quantitative structure-activity relationships for calmodulin inhibitors.
Using the discriminant analysis method, we completely distinguished 24 calmodulin inhibitors in three groups, as classified by Zimmer et al. The resultant discriminant functions distinguished the three groups in terms of positive potential surface area on the side chain, as well as the total and neutral surface areas on the ring in the inhibitor molecules. Group assignment of additional calmodulin inhibitors from other sources was then estimated according to the discriminant functions. The relationship between structure and inhibitory potency on calmodulin-activated phosphodiesterase for group I inhibitors, together with those estimated, was studied using the adaptive least squares method with several parameters dependent on molecular conformations. A "best conformer" was selected for each inhibitor on the basis of quantitative structure-activity relationship (QSAR). The results of QSAR analysis of group I inhibitors showed that hydrophobicity was important for the ring moiety but not for the side chain. The negative potential surface area of the side chain is necessary for activity. It is desirable for the nitrogen atom in the side chain, which is considered the center of the negative potential area, to be located far from the ring moiety. Thus, the ring moiety and side chain may possibly play different roles in interactions with the receptor system.
Calmodulin↗
Fuzzy adaptive least squares and its use in quantitative structure-activity relationships.
Fuzzy adaptive least squares (FALS), a pattern recognition method designed to correlate molecular structure with activity rating, has been developed. A novel feature of FALS is that the degree to which each sample belongs to an activity class is given using a membership function. The algorithm involves an iterative modification of forcing factors to maximize the sum of the membership function values over all samples. This paper first describes the method and calculation procedure of FALS89 (1989 version of FALS), and then shows its application to the correlation of structure with a potency rating of anticarcinogenic mitomycin derivatives and arginine-vasopressin antagonists. FALS89 applied to these samples showed considerably high reliability in both recognition and leave-one-out prediction.
Antibiotics, Antineoplastic↗
Molecular cloning and sequence analysis of cDNA for human hepatocyte growth factor.
Amino acid sequences of four peptide fragments of human hepatocyte growth factor purified from the plasma of patients with fulminant hepatic failure were determined. Based on the amino acid sequence of one of the fragments, two oligodeoxyribonucleotide mixtures were synthesized and used to screen a human placenta cDNA library. On the screening, two overlapping cDNA clones for human hepatocyte growth factor were isolated and the nucleotide sequence of the cDNA was determined. The entire primary structure of the protein was deduced from the sequence. The protein consists of 728 amino acid residues, including a possible signal peptide at the N-terminus. The sequence revealed that the heavy and light chains which comprise the protein are encoded by the same mRNA and are produced from a common translation product by proteolytic processing.
Amino Acid Sequence↗
Clinical significance of human hepatocyte growth factor in blood from patients with fulminant hepatic failure.
We have recently found the presence of human hepatocyte growth factor in sera of patients with fulminant hepatic failure and have purified human hepatocyte growth factor from plasma of a patient with fulminant hepatic failure. In this paper, we report the clinical significance of human hepatocyte growth factor in blood from patients with fulminant hepatic failure. The effect of sera or plasma from 17 patients with fulminant hepatic failure on liver cell growth was examined by use of adult rat hepatocytes in primary cultures. Sera or plasma from 16 of the 17 patients with fulminant hepatic failure stimulated DNA synthesis in hepatocytes more effectively than normal human serum. The mean growth-promoting activity for the 17 patients with fulminant hepatic failure was about 16 times higher than that obtained for normal human serum. This growth-promoting activity of the patients' blood was not related to sex, age, clinical outcome of the patients or type of fulminant hepatic failure, but was intimately related to the clinical grade of hepatic coma. Sera or plasma with Grade III and IV coma showed stimulatory activity on DNA synthesis more markedly than sera or plasma from patients with coma of less than Grade II. In the surviving group, this activity decreased as the hepatic coma of patients improved. In fact, this activity of sera from patients at the recovery stage showed no significant increase compared with that of normal human serum. In the group of terminal patients, this activity increased as the coma developed.(ABSTRACT TRUNCATED AT 250 WORDS)
Adult↗