Mode of inhibitory action of cholecystokinin in amylase release from isolated rat pancreatic acini--inhibition of secretory process post to protein kinase C-calcium ion systems

The incubation of isolated rat pancreatic acini with low doses (1 x 10(-11)-1 x 10(-10) M) of cholecystokinin-octapeptide (CCK8) induced amylase release. This CCK8-induced amylase release has been shown to be mediated through the protein kinase C activation and the Ca2+ mobilization which are linked to the phospholipase C-mediated hydrolysis of phosphoinositides. However, the incubation of the acini with high doses (1 x 10(-9)-1 x 10(-7) M) of CCK8 reduced amylase release to the level less than that induced by the maximally effective dose (1 x 10(-10) M) of this secretagogue. Under the same conditions, the high doses of this secretagogue did not inhibit the phospholipase C-mediated hydrolysis of phosphoinositides. The stimulatory action of the maximally effective dose of CCK8 in amylase release was mimicked by the simultaneous addition of protein kinase C-activating 12-O-tetradecanoylphorbol-13-acetate (TPA) and Ca2+ ionophore A23187. A high dose (1 x 10(-7) M) of CCK8 reduced the amylase release induced by the combination of TPA and A23187. These results suggest that the high doses of CCK8 inhibit the secretory process post to the protein kinase C-Ca2+ systems and thereby reduce the amylase release induced by the maximally effective dose of CCK8 in rat pancreatic acini.     

Primary structure of non-histone protein HMG1 revealed by the nucleotide sequence

The isolation and sequencing of a cDNA clone coding for the entire sequence of pig thymus non-histone protein HMG1 are described. The sequence analysis reveals a complete 2192-nucleotide sequence with a 5'-terminal untranslated region of 11 nucleotides, 642 nucleotides of an open reading frame that encoded 214 amino acids, and a 3'-terminal untranslated region of 1539 nucleotides. The HMG1 protein, deduced from the nucleotide sequence, has a molecular weight of 24,785 and a C-terminal of a continuous run of 30 acidic amino acids, encoded by a simple repeating sequence of (GAN)30. The predicted amino acid sequence is homologous to HMG1, HMG2, and HMG-T sequences from several sources, suggesting that the protein conformation is under evolutionary constraints. Northern blot analysis reveals that another hybridizable RNA species of smaller size is present. Southern blot analyses suggest that pig genome contains several HMG1 gene equivalents.     

5C6-F4, a novel 100,000-dalton rat lymphocyte activation antigen defined by monoclonal antibody

Con A-activated rat thymocytes were used to immunize mice, and immune spleen cells were fused with NS/1 myeloma cells. One clone, designated 5C6-F4, reacted strongly with Con A-activated rat thymocytes and some LPS-activated rat spleen cells but not with normal thymocytes, spleen cells, or bone marrow cells of rat origin. The 5C6-F4 did not react with Con A-activated thymocytes of mouse origin. Immunoprecipitation of 5C6-F4 antigen from surface-iodinated Con A-activated rat thymocytes or LPS-activated rat spleen cells revealed its m.w. to be approximately 100,000. The kinetic studies of the expression of 5C6-F4 antigen revealed that 5C6-F4 antigen was detectable at 6 hr after Con A stimulation of rat spleen cells, whereas IL 2 receptor (IL 2R) was detectable at 12 hr. The appearance of 5C6-F4 antigen and IL 2R precede the onset of DNA synthesis of Con A-activated spleen cells. Thus, 5C6-F4 antigen is classified as early activation antigen. The 5C6-F4 inhibits the lymphocyte proliferation induced by mitogen and the IL 2-driven rat T cell proliferation. Sequential immunoprecipitation study as well as binding inhibition study indicated that the 5C6-F4 antigen is distinct from IL 2R molecule. The 5C6-F4 antigen appears to be a novel rat lymphocyte activation antigen that exhibits immunoregulatory function and also may serve as a useful marker of T cell activation.     

Generation of cytotoxic T lymphocytes from thymocyte precursors to trinitrophenyl-modified self antigens. II. Establishment of a T cell hybrid clone constitutively producing killer-helper factor(s) (KHF) and functional analysis of released KHF

T cell hybridoma lines were constructed by fusion of Mycobacterium tuberculosis-primed and boosted BALB/c T cells with the AKR-derived T lymphoma cell line BW5147. Certain of the hybridomas prepared in this manner secreted constitutively into their culture supernatants biologically active molecules that displayed precursors of cytotoxic T cell activating properties characteristic of killer-helper factor (KHF). Cell surface analysis revealed that the hybridomas were indeed somatic cell hybrids between the two respective partner cells used for fusion. KHF properties of these hybridoma supernatants were verified by their capacity to stimulate peanut agglutinin-binding (PNA+) C3H/He thymocytes to respond in vitro to 2,4,6-trinitrophenyl(TNP)-modified syngeneic stimulator cells in conjunction with suboptimal doses (10 U/ml) of interleukin 2 (IL 2) for the generation of H-2-restricted, TNP-reactive cytotoxic T cells. The biologically active molecules secreted by a T cell hybrid clone (2Y4) were, like conventional KHF, distinct from IL 1, IL 2, or immune interferon (IFN-gamma). The partially purified KHF derived from 2Y4 cells shows activity at apparent m.w. range of 34,000 to 60,000 on gel permeation, and is relatively homogeneous with respect to isoelectric point, which was approximately 4.5 to 4.7. The partially purified 2Y4-KHF is able to augment proliferation of as well as the expression of IL 2 receptors on PNA+ thymocytes in conjunction with IL 2. Finally, addition of 2Y4-KHF on day 0, followed by the addition of IL 2 on day 2 for 7 days of culture was effective in generating potent CTL responses, whereas addition of IL 2 on day 0, followed by the addition of 2Y4-KHF on day 2 to the culture was ineffective.     

Stimulation of Amino Acid Uptake and Na+, K+-ATPase Activity by Norepinephrine in Superior Cervical Sympathetic Ganglia Excised from Adult Rats

Active uptake of a labelled nonmetabolizable amino acid, alpha-aminoisobutyric acid (AIB), into isolated superior cervical sympathetic ganglia (SCG) excised from adult rats was considerably stimulated by the addition of either norepinephrine (NE, 50 microM) or 3,4-dihydroxyphenylethylamine (dopamine, DA, 100 microM) to the medium during aerobic incubation for 2 h at 37 degrees C. The NE-induced increase in AIB uptake was significantly antagonized by the addition of an alpha 1-adrenoceptor antagonist (prazosin, 10 microM) in SCG axotomized 1 week prior to the examination, in which most of the ganglionic neurons had degenerated and reactive proliferation of the satellite glial components was in progress. The addition of neither acetylcholine (ACh, 1 mM) plus eserine (0.1 mM) nor cyclic nucleotides (1 mM) changed the AIB uptake by the SCG. In the axotomized SCG, the NE-evoked increase in AIB uptake was much more pronounced than that of intact or denervated SCG. A kinetic study of the active AIB uptake in the SCG showed that NE produced a decrease of the Km value and an increase in the Vmax, especially in the axotomized SCG. Ganglionic Na+, K+-ATPase activity was greatly stimulated in the presence of NE, but not by ACh. These results strongly suggest that the NE-induced enhancement of active AIB uptake in the isolated SCG is occurring in glial cells rather than in neuronal cells, with a possible alteration of membrane properties for amino acid uptake and with an apparent regulation by the stimulated transport enzyme Na+, K+-ATPase.     

Enhanced formation of mouse hybridomas without hat treatment in a serum-free medium

Summary A newly developed, serum-free medium (NYSF-404) selects for antibody-producing hybridomas after fusion of antigen-sensitized mouse spleen cells with myeloma cell lines P3-X63-Ag8-U1 (P3-U1), P3-X63-Ag8-6.5.3 (Ag8.653), or P3-NSI/1-Ag4-1 (NS-1). Without the need for hypoxanthine-aminopterinthymidine (HAT) selection of hybrid cells, frequency of hybridoma formation in medium NYSF-404 is higher (twice) than that in serum- and HAT-containing medium. Colonies developed upon limiting dilution in the presence of the mortal parent myeloma cells in medium NYSF-404 and pure culture of antibody-secreting cells could be subsequently established. The results suggest that fusions can be done in serum-free medium and that the clonal growth of hybridomas is dependent on factors produced by parent myeloma cells under serum-free culture conditions. Such factors seem deficient in serum- and HAT-containing medium or are masked by serum.     

Release of endogenous prostaglandins by mild hyperosmotic saline inhibits tetragastrin-stimulated gastric acid secretion in rats

Filling of the gastric lumen of rats with 1.0 M NaCl solution (5 ml) for 10 min under urethane anesthesia caused an increase in the gastric fluid concentrations of prostaglandin (PG) E₂, 13, 14-dihydro-15-keto-PGE₂ and 6-keto-PGF_1α as determined by radioimmunoassay. PGE₂ was the major PG generated. The levels of PGE₂ in the gastric fluid were increased dose-dependently after filling the lumen with 0.3, 0.5, 0.7 or 1.0 M NaCl solutions. The pH of the gastric fluid increased similarly after 0.5 to 1.0 M NaCl solutions. Indomethacin (10 mg/kg, i.p.) suppressed the PGE₂ increase caused by 1.0 M NaCl solution, but did not prevent the increase of the pH of the gastric fluid induced by intragastric 1.0 M NaCl. Infusion of tetragastrin (62.5 μg/kg/hr, i.v., for 10 min) caused a marked increase of acid secretion without modifying intragastic concentration of PGE₂. The acid secretion due to tetragastrin was completely inhibited after intragastric administration of 1.0 M NaCl solution, while indomethacin restored the tetragastrin-induced acid secretion, with prevention of a rise of intragastric PGE₂ levels. These observations suggest that 1.0 M NaCl solutions suppress basal intragastric acid through a mechanism which is independent of prostaglandins. In contrast, the suppression of tetragastrin-induced acid secretion by intragastric 1.0 M NaCl solution appears to be mediated through a release of prostaglandins     

Chromosomal localization of the Epstein-Barr virus (EBV) genome in Bloom's syndrome B-lymphoblastoid cell lines transformed with EBV

The localization of the Epstein-Barr virus (EBV) genome in chromosomes of human B-lymphoblastoid cell lines (LCLs) transformed with EBV, and the effect of EBV DNA on the level of sister chromatid exchange (SCE) in Bloom's syndrome (BS) B-LCLs, were examined with chromosomal in situ hybridization techniques using a ³H-EBV DNA probe. EBV DNA was detected in chromosomes 1–5 and 13–15 at specific G band regions in BS as well as in normal B-LCLs, regardless of SCE. Several chromosomal sites (1p31, 1q31, 4q22–24, 5q21, 13q21, 14q21) carrying EBV DNA seemed to be very characteristic in normal as well as in BS B-LCLs. There was no statistically significant difference in silver grain counts due to EBV DNA and their distribution in different chromosomes or groups among normal and BS B-LCLs with normal and high SCE. These findings strongly indicate that EBV infection did not introduce a correcting factor for BS SCE.     

Amidase-like activity of calpain I and calpain II on substance P and its related peptides

Porcine calpains (Ca2+-dependent cysteine proteinases) I and II, which had been purified each to a homogeneous state, were found to hydrolyze specifically carboxyl-terminal amide of substance P and several other biologically active peptidyl amides. This amidase-like activity was demonstrated both by determining released ammonia and by separating products on high-performance liquid chromatography followed by amino acid analysis. The calpain-catalyzed deamidation of substance P occurred exclusively at the carboxyl-terminal amide, leaving the side-chain glutamine intact. Enkepharinamide and MSH-release inhibiting factor were scarcely deamidated. Calpains I and II showed similar specificities for these amide substances and similar profiles of inhibitions by various protease inhibitors, but distinctly different Ca2+ requirements. The specificity constants, kcat/Km, for substance P were found to be three to four orders of magnitude higher than those for the synthetic substrates.     

Antileukemic effect of coral-prostanoids clavulones from the stolonifer Clavularia viridis on human myeloid leukemia (HL-60) cells

To elucidate the biological activities of coral-prostanoids, clavulones, discovered from the Japanese stolonifer Clavularia viridis, we examined the effect of clavulone on the cell growth of human cancer (human promyelocytic leukemia (HL-60) cells and HeLa cells) and normal (Chang liver cells and lung fibroblasts) cells in vitro. Clavulone showed strong antiproliferative and cytotoxic activities in the human cells and it had some selectivity to leukemic (HL-60) cells over other HeLa cells or normal cells on the basis of the IC50 values and cytotoxic effect of the cells. The IC50 value of clavulone in the HL-60 cells was about 0.4 microM (0.2 micrograms/ml). Over 1.0 microM (0.5 micrograms/ml), clavulone showed a significant cytotoxic activity on the HL-60 cells. The data on DNA synthesis and flow cytometric analysis revealed that clavulone arrests the cells in the G1-phase and inhibits the cell growth of HL-60 cells by inhibiting S-phase DNA synthesis. These results suggest that clavulone has a potent antileukemic effect on HL-60 cells.