[Relationship between factors examined at health examination and serum pepsinogen levels in healthy adults. Physical measurements, blood chemical tests, drinking and smoking].

OBJECTIVE: We measured serum pepsinogen (PG) levels in healthy adults and examined their physical measurements, blood chemical test values, current drinking and smoking to investigate relationships between these factors and levels of serum PG components (serum PG I, PG II and PG I/II ratio). SUBJECTS AND METHODS: A total of 452 male adults in their 40's, who were determined to be normal or to have only chronic gastritis by endoscopy or X-ray examination of the upper gastrointestinal tract, were studied. PG I and PG II levels in sera were measured, and their relationship with physical measurements and blood chemical test values, and also with current amounts of drinking and smoking, were examined. RESULTS: 1) Height, body weight, body surface area, GOT, GPT and creatinine were found to significantly differ according to serum PG I level; body surface area, GPT and ALP significantly differed according to serum PG II level. However, none of the factors examined showed any significant correlation with the PG I/II ratio. 2) When subjects were divided into positive and negative cases using the evaluation criteria of PG components for gastric cancer screening (determined as positive on the basis of serum PG I level < or = 70 ng/ml and PG I/II ratio < or = 3.0), proposed by Miki et al., none of the factors differed significantly between the two groups. CONCLUSIONS: 1) Serum PG levels were associated with stature, serum transaminase and creatinine. 2) Although serum PG levels were associated with several factors, the effect of physical measurements and blood chemical tests on the results of the evaluation criteria of PG components proposed by Miki et al, were not remarkable.

Stimulation of thrombopoiesis in mice by fibroblast growth factor 9.

Fibroblast growth factor 9 (FGF-9), a novel member of the FGF family, was found to have thrombopoietic activity in vitro and in vivo. In an in vitro megakaryocyte colony-stimulating factor assay, anti-mouse interleukin-6 (IL-6) monoclonal antibody neutralized FGF-9 activity. This suggests that the activity may be exerted via IL-6 induction. BALB/c mice that received subcutaneous FGF-9 injections of 4 to 100 micrograms/day for 2 weeks showed a dose-dependent transient increase in peripheral platelet counts 10 to 12 days after the first treatment. Histologic studies showed a marked increase in megakaryocytes in bone marrow and extramedullary hematopoiesis in the spleen and the liver. Examination of changes in the DNA content of bone marrow megakaryocytes revealed that the ploidy distribution underwent a marked shift 3 days after FGF-9 injection, with a large increase in the 2N megakaryocyte population. The major modal ploidy shifted from the normal 16N to 2N. The number of megakaryocyte progenitor cells in FGF-9-treated mice increased up to 1.5-fold in the bone marrow and 10-fold in the spleen on day 6. These results indicate that FGF-9 acts on the in vivo proliferation of megakaryocytes.

Molecular cloning of a novel cytokine cDNA encoding the ninth member of the fibroblast growth factor family, which has a unique secretion property.

Glia-activating factor (GAF) is a novel heparin-binding growth factor purified from the culture supernatant of a human glioma cell line. It shows a spectrum of activity slightly different from those of other known growth factors. We have isolated the cDNA which encodes human GAF. A homology search revealed that GAF would be the ninth member of the FGF family, and we therefore call it FGF-9. The human FGF-9 cDNA cloned by using oligonucleotide probes encoded a polypeptide consisting of 208 amino acids. Sequence similarity to other members of the FGF family was estimated to be around 30%. Two cysteine residues and other consensus sequences in family members were also well conserved in the FGF-9 sequence. FGF-9 was found to have no typical signal sequence in its N terminus like those in acidic FGF and basic FGF. Acidic FGF and basic FGF are known not to be secreted from cells in a conventional manner. However, FGF-9 was found to be secreted from cells after synthesis despite its lack of a typical signal sequence. It could be detected exclusively in the culture medium of cDNA-transfected COS cells. The amino acid sequence of proteins purified from culture supernatant of the CHO cell line, which was cDNA transfected and selected as a high producer of FGF-9, showed that no peptides were cleaved from the N terminus except the initiation methionine. The rat FGF-9 cDNA was also cloned, and the structural analysis indicated that the PGF-9 gene is highly conserved. Expression of the FGF-9 gene could be detected in the brain and kidney of the adult rat. Restricted gene expression in organs and the unique secretion nature of the protein suggest that FGF-9 plays a physiological role which differs from those of well-characterized acidic FGF and basic FGF.

Novel secretory heparin-binding factors from human glioma cells (glia-activating factors) involved in glial cell growth. Purification and biological properties.

Growth factors for rat primary glial cells were identified in conditioned medium of a human glioma-derived cell line. The factors, designated glia-activating factors (GAFs), were purified to homogeneity by a combination of heparin affinity chromatography, gel filtration, and high performance liquid chromatography on a heparin affinity column and a C4 reversed-phase column. GAFs could be resolved into three peaks by C4 column chromatography. The M(r) values of these three proteins were estimated to be 30,000, 29,000, and 25,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. These M(r) values were in good agreement with the value of 26,000 +/- 3,000 estimated from the elution volume upon gel filtration chromatography under nondenaturing conditions. These data suggested that each of the GAFs consists of a single polypeptide chain and has no subunit structures. These three purified GAFs had almost the same growth-stimulating effect on glial cells in vitro, and the half-maximal dose was around 10(-11) M. Concanavalin A staining and glycopeptide N-glycosidase treatment of GAFs indicated that an asparagine-linked oligosaccharide chain(s) was attached to these three kinds of GAFs. Microsequencing of each GAF revealed a single amino-terminal sequence with no significant homology to any known protein, and the amino-terminal sequence of the 30-kDa GAF included that of the 29-kDa GAF. GAFs also stimulated the cell growth of oligodendrocyte type 2 astrocyte progenitor cells, BALB/c3T3 fibroblasts, and PC-12 cells but not that of human umbilical vein endothelial cells.

Evidence for intramolecular disulfide bond shuffling in the folding of mutant human lysozyme.

Our previous results using the Saccharomyces cerevisiae secretion system suggest that intramolecular exchange of disulfide bonds occurs in the folding pathway of human lysozyme in vivo (Taniyama, Y., Yamamoto, Y., Kuroki, R., and Kikuchi, M. (1990) J. Biol. Chem. 265, 7570-7575). Here we report on the results of introducing an artificial disulfide bond in mutants with 2 cysteine residues substituting for Ala83 and Asp91. The mutant (C83/91) protein was not detected in the culture medium of the yeast, probably because of incorrect folding. Thereupon, 2 cysteine residues Cys77 and Cys95 were replaced with Ala in the mutant C83/91, because a native disulfide bond Cys77-Cys95 was found not necessary for correct folding in vivo (Taniyama, Y., Yamamoto, Y., Nakao, M., Kikuchi, M., and Ikehara, M. (1988) Biochem. Biophys. Res. Commun. 152, 962-967). The resultant mutant (AC83/91) was secreted as two proteins (AC83/91-a and AC83/91-b) with different specific activities. Amino acid and peptide mapping analyses showed that two glutathiones appeared to be attached to the thiol groups of the cysteine residues introduced into AC83/91-a and that four disulfide bonds including an artificial disulfide bond existed in the AC83/91-b molecule. The presence of cysteine residues modified with glutathione may indicate that the non-native disulfide bond Cys83-Cys91 is not so easily formed as a native disulfide bond. These results suggest that the introduction of Cys83 and Cys91 may act to suppress the process of native disulfide bond formation through disulfide bond interchange in the folding of human lysozyme.

Secretion in yeast of mutant human lysozymes with and without glutathione bound to cysteine 95.

A mutant human lysozyme C77A, in which Cys-77 is replaced with Ala, was secreted by Saccharomyces cerevisiae as two proteins (C77A-a and C77A-b) with different specific activities. A peptide fragment from Val93 to Ala108 was obtained from C77A-a by pepsin digestion, and examined by fast atom bombardment mass spectrometry and amino acid analysis. The results showed that glutathione was attached to the thiol group of Cys95 of the fragment through a disulfide linkage. This observation was confirmed by quantitative formation of free glutathionesulfonic acid from C77A-a by performic acid treatment. In contrast, there was no modification in the case of C77A-b. These results indicate that C77A-a contained a mixed disulfide with glutathione attached to cysteine residue 95. In C77A-b, there appears to be a free thiol of Cys95 surrounded by many side chains, which was not modified by iodoacetic acid under native conditions, suggesting that the attachment of glutathione occurs during folding. These findings further suggest that in the oxidation step of disulfide bond formation in human lysozyme secreted by yeast, mixed disulfides are formed with glutathione and that posttranslational modification with glutathione can occur even in a protein secreted by yeast.

Design and creation of a Ca2+ binding site in human lysozyme to enhance structural stability.

A Ca2+ binding site like an EF-hand motif was designed and created in human lysozyme by replacing both Gln-86 and Ala-92 with aspartic acids by site-directed mutagenesis. The mutant human lysozyme (D86/92-lysozyme) was expressed and secreted by yeast. One Ca2+ was found to bind one molecule of the purified protein with the binding constant 5.0 x 10(6) M-1. The enzymatic activity of holo-D86/92-lysozyme against glycol chitin at 40 degrees C was 2-fold higher than that of the native lysozyme. Maximal activity of the holo-D86/92-lysozyme was observed at 80 degrees C, where its relative activity normalized to the value at 40 degrees C was 6-fold and 17-fold higher than those of the native and apoenzymes, respectively. The activities of the native lysozyme and apo-D86/92-lysozyme were maximum at 65 degrees C-70 degrees C. Moreover, D86/92-lysozyme was more stable against protease digestion than the native lysozyme. These results indicate that the creation of the calcium binding site like an EF-hand motif in the human lysozyme enhances its structural stability.