Tomoyoshi Nishino

Purification and subunit structure of hepatocyte growth factor from rat platelets

A hepatocyte growth factor (HGF) that stimulates DNA synthesis of adult rat hepatocytes in primary culture was purified as a homogeneous material from platelets of 1000 rats by a four‐step procedure: stimulation of its release from platelets by thrombin, cation‐exchanger fast protein liquid chromatography (FPLC) on a Mono S column, heparin‐Sepharose CL‐6B chromatography, and reverse‐phase HPLC on a C4 column. The purified HGF stimulated DNA synthesis of adult rat hepatocytes in primary culture at 1 ng/ml and was maximally effective at 5 ng/ml, being about twice as potent as EGF at this concentration. HGF did not stimulate DNA synthesis of Swiss 3T3 cells. It was found to be a beat‐ and acid‐labile protein that was inactivated by reduction with dithiothreitol. The purified HGF had a molecular mass of 82 kDa, as estimated by SDS‐PAGE, and was found to be a heterodimer which dissociated into a large subunit of 69 kDa and a small one of 34 kDa by SDS‐PAGE under reducing conditions. These biological and chemical properties showed that HGF was not identical with any known growth factors, including platelet‐derived growth factor (PDGF).

Production of rabbit antibody specific for amino-terminal residues of cholecystokinin octapeptide (CCK-8) by selective suppression of cross-reactive antibody response

Antibody specific for the amino-terminal region of cholecystokinin octapeptide (CCK-8) was generated in a highly reproducible way in New Zealand white rabbits by a novel immunization procedure which involves immunization with CCK-8 peptide conjugate coupled with keyhole limpet hemocyanin (KLH) and inhibiting cross-reacting antibody formation by treatment of the animals with a potent tolerogenic conjugate of beta-alanyl-tetragastrin and a copolymer of D-glutamic acid and D-lysine (D-GL). The antisera thus produced specifically react with an amino-terminal region of CCK-8 but not with the non-sulfate form of CCK-8, nor with the carboxy-terminal region which shares a cross-reactive determinant among gastrin and cholecystokinin-related peptides (caerulein, CCK-4, CCK-8, CCK-33 and CCK-39). The antisera produced by this method allowed us to measure specifically CCK in extracts from tissue such as duodenum containing gastrin and CCK at comparable levels.

Promyelocytic leukemia cell line, HL-60, produces human hepatocyte growth factor

The human promyelocytic leukemia cell line, HL-60, stimulated with PMA, produced human HGF-like immunoreactivity (HL-60 HGF), which was detected with human HGF-specific ELISA. The purified HL-60 HGF was indistinguishable from human HGF in the plasma of patients with fulminant hepatic failure by studies of subunit constitution and amino acid composition. The HL-60 HGF mRNA corresponded to 6 kb, which was consistent with previous reported data in rat and human HGF mRNA, was detected in stimulated HL-60, by northern hybridization analysis using human HGF cDNA probe. These findings indicated that HL-60 HGF was identical to, or closely resembled, human plasma HGF. The HL-60 cell is an attractive model for studies of HGF-producing mechanisms, the manner of secretion and the nature of induction signals.

Radioimmunoreactive plasma bradykinin levels and histological changes during the course of cerulein-induced pancreatitis in rats

The plasma bradykinin (BK) and serum amylase levels and histological changes in rats during the course of acute pancreatitis induced by a large dose of cerulein were examined. Animals were given four intraperitoneal injections of 20 μg/kg body wt of cerulein at hourly intervals. The plasma concentration of BK-like immunoreactivity (BK-LI), measured by a highly sensitive and specific radioimmunoassay established in this study, was found to reach a peak 6 h after the first injection of cerulein and then to remain elevated. On the other hand, the serum amylase and the histological alterations (i.e., interstitial edema, vacuolization, and inflammatory infiltration) were maximal 9 h after the first injection and returned to nearly normal after 24 h. These observations suggest that the BK generation is indicative of the participation of the kallikrein-kinin system in the pathophysiological change and that the plasma BK-LI level is a good marker of cellular damage and inflammation within the pancreas during the course of acute pancreatitis.

A large molecular form of glucagon-like peptide-1(GLP-1) immunoreactivity is co-released with glucagon from pancreas by arginine in normal subjects

Plasma immunoreactivities of glucagon-like peptide-1 (GLP-1IR) in normal subjects were measured with a specific radioimmunoassay during the arginine tolerance test. Plasma GLP-1IR after arginine infusion showed a 3-fold increase in parallel to plasma glucagon immunoreactivity and plasma glucagon-like immunoreactivity, measured with a glucagon C-terminal specific antiserum (OAL 123) and an N-terminal and/or central region specific glucagon antiserum (OAL 196), respectively. This finding suggested that the increased immunoreactivities of GLP-1 as well as that of glucagon were of pancreatic origin. Upon gel chromatography, plasma at the basal state showed three GLP-1 immunoreactive peaks, eluted in the position of void volume, synthetic GLP-1(72-108), and a smaller molecular fraction. Gel chromatography of plasma after an arginine load showed an additional peak (Mr 13,000-15,000) with little change in other GLP-1 immunoreactive peaks. This large molecular form of GLP-1IR was also shown to exist in the human pancreatic extract. Moreover, the free GLP-1 concentrations in plasma before and after an arginine load were shown to be about equal by reverse phase HPLC. These data suggested that in normal subjects arginine stimulation co-releases GLP-1IR, predominantly large molecular form, with glucogen from the pancreas.

Effect of captopril on renal vascular resistance, renin, prostaglandins and kinin in the isolated perfused kidney

Vasodilatory and natriuretic effects of captopril were studied in the isolated hog kidney perfused with modified Krebs-Ringer solution. Renal arterial infusion of captopril caused increases in releases of renin, prostaglandins (PGE2, 6-keto-PGF1 alpha and PGF2 alpha) and kinin, and was accompanied by a decrease in the renal vascular resistance and an increase in urinary sodium excretion. Indomethacin administered with captopril diminished the saluretic effect of captopril and evoked an increase in kinin, but was associated with a marked decrease in prostaglandin and renin releases, while renal vascular resistance remained decreased. Indomethacin alone did not alter vascular resistance and kinin; however, renin and prostaglandin releases were decreased. Aprotinin administered with captopril showed a decrease in releases of prostaglandins, renin and kinin without any change in vascular resistance. These results suggest that increased release of kinin induced by captopril contributes to a reduction in renal vascular resistance. Increased prostaglandin release after captopril administration may be caused by an increase in kinin without direct involvement of captopril in prostaglandin synthesis. Renal prostaglandins may enhance sodium excretion and mediate renin secretion in captopril perfusion.

Effects of K-76COOH (MX-1) on immune response: Induction of suppressor T-cells by MX-1

K-76COOH (MX-1), isolated from the cultured supernatant of a species of fungi imperfecti, Stachybotrys complement nov. sp. K-76, is an inhibitor of the complement component, C5. The effects of MX-1 on various immune responses were investigated. MX-1 enhanced the response of spleen cells to PHA and LPS: MX-1 at 0.01-250 micrograms/ml for PHA and at 10-250 micrograms/ml for LPS. In contrast, it inhibited the response to Con A: MX-1 at 0.01-500 micrograms/ml for spleen cells and at 100-500 micrograms/ml for thymocytes. MX-1 and IL-1 synergistically acted to enhance the Con A response of spleen and thymus cells from which accessory cells and Ia-positive cells had been removed by passing through Sephadex G-10 columns and treating with anti-Ia monoclonal antibody plus complement. T-cells pretreated with MX-1, IL-1 and Con A for 3 days suppressed not only the response of B-cells to LPS but also the production of anti-SRBC antibodies. In addition, MX-1 was found to increase CD8+ T-cells. These results suggest that MX-1 acts on T-cells to induce suppressor T-cells.

Monoclonal Antibody to Pseudouridine Used to Develop a Radioimmunoassay

Pseudouridine, a component of tRNA, was modified to yield the derivatives: succinyl, palmitoyl pseudouridine, and protein conjugates. These derivatives were used in preparation of monoclonal antibodies specifically directed to pseudouridine. MAbs from three hybridomas (OAL 881, 812 and 814) were established and shown to be directed to pseudouridine, uridine and uracil. OAL 881 was equally reactive with the three substrates, OAL 812 showed the same reactivity for pseudouridine and uracil, while OAL 814 showed a reactivity mainly for pseudouridine. MAb OAL 814 was used in a radioimmunoassay (RIA) system unique for pseudouridine. A good dose-response curve was observed in the range between 31.3 and 2000 nmol/ml. Intra- and inter-assay CV values were below 4.1% and 7.4% respectively, and good results were obtained from recovery of added material and dilution tests. The ratio of pseudouridine/creatinin in urine was significantly higher in patients with cancer than in normal subjects.

Synergic effects of kallikrein-kinin and prostaglandins on renin release on infusion of isolated hog kidney with aldosterone

The effects of infusion of a large amount of aldosterone into the renal artery of isolated perfused hog kidney on the release of renin, prostaglandins (PG) and kinin and the excretion of urinary kallikrein were investigated. Infusion of aldosterone at a rate of 100 ng/min (100 to 800 ng/ml of perfusate) resulted in significant releases of renin, PG (PGE2 , 6-0-PGF1 alpha), and kinin and increase in urinary kallikrein. Infusion of aldosterone and an inhibitor of kallikrein, aprotinin, decreased the releases of renin, PG and kinin and infusion of aldosterone with indomethacin decreased the release of PG but increased that of kinin and urinary kallikrein without significant change in renin releases. These findings suggest that the release of renin by aldosterone may result from synergic effects of renal PG and the kallikrein -kinin system.