Effects of corticotropin releasing hormone on appetitive behaviors.

Corticotropin releasing hormone (CRH) is a 41-residue hypothalamic neuropeptide that has been shown to have potent behavioral effects in animals and has been implicated in clinical disorders in man. This review focuses on those aspects of the behavioral effects of CRH related to food-associated behaviors. The effects of CRH on food intake are compared with its effects on performances maintained by food presentation, and contrasted with the effects of CRH on performances maintained by other events. The effects of CRH antagonists and drugs that interact with the behavioral effects of CRH are also reviewed, particularly with respect to their direct effects on food intake. Lastly, data assessing the effects of CRH administration on central neurotransmitter levels are presented and compared with levels seen in clinical populations. The effect of CRH on food intake seen in animals is consistent with a putative role for CRH in clinical syndromes where appetite suppression is apparent. Since some of the effects of CRH on food intake are subject to pharmacological intervention, strategies directed at peptidergic mechanisms of psychiatric disorders should be explored.     

Effect of substrate on Ca2(+)-concentration required for activity of the Ca2(+)-dependent proteinases, mu- and m-calpain

The Ca2+ concentrations required for half-maximal activity of mu- and m-calpain purified from bovine skeletal muscle were tested using four different protein substrates and three different synthetic peptide substrates. Hammersten casein, the commonly used substrate for measuring mu- and m-calpain activity, required 2.5 microM Ca2+ for half-maximal activity of mu-calpain and 290 microM Ca2+ for half-maximal activity of m-calpain. When Hammersten casein was dialyzed against 8 M urea and 10 mM EDTA to remove all endogenous Ca2+, it required 1.9 and 290 microM Ca2+ for half-maximal activity of mu- and m-calpain, respectively. Rabbit skeletal muscle myofibrils and rabbit skeletal muscle troponin required 65 microM and 24 microM Ca2+ for half-maximal activity of mu-calpain and 380 microM and 580 microM Ca2+ for half-maximal activity of m-calpain, respectively. The three synthetic substrates tested, Suc-Leu-Tyr-MCA, Boc-Leu-Thr-Arg-MCA, and Suc-Leu-Leu-Val-Tyr-MCA, required 1.6 microM to 3.7 microM Ca2+ for half-maximal activity of mu-calpain and 200 to 560 microM Ca2+ for half-maximal activity of m-calpain.     

Induction by ionizing radiation of the gadd45 gene in cultured human cells: lack of mediation by protein kinase C.

The effect of ionizing radiation on the expression of two DNA-damage-inducible genes, designated gadd45 and gadd153, was examined in cultured human cells. These genes have previously been shown to be strongly and coordinately induced by UV radiation and alkylating agents in human and hamster cells. We found that the gadd45 but not the gadd153 gene is strongly induced by X rays in human cells. The level of gadd45 mRNA increased rapidly after X rays at doses as low as 2 Gy. After 20 Gy of X rays, gadd45 induction, as measured by increased amounts of mRNA, was similar to that produced by the most effective dose of the alkylating agent methyl methanesulfonate. No induction was seen after treatment of either human or hamster cells with 12-O-tetradecanoylphorbol-13-acetate, a known activator of protein kinase C (PKC). Therefore, gadd45 represents the only known mammalian X-ray-responsive gene whose induction is not mediated by PKC. However, induction was blocked by the protein kinase inhibitor H7, indicating that induction is mediated by some other kinase(s). Sequence analysis of human and hamster cDNA clones demonstrated that this gene has been highly conserved and encodes a novel 165-amino-acid polypeptide which is 96% identical in the two species. This gene was localized to the short arm of human chromosome 1 between p12 and p34. When induction in lymphoblast lines from four normal individuals was compared with that in lines from four patients with ataxia telangiectasia, induction by X rays of gadd45 mRNA was less in the cell lines from this cancer-prone radiosensitive disorder. Our results provide evidence for the existence of an X-ray stress response in human cells which is independent of PKC and which is abnormal in taxia telangiectasia.     

Enzyme-linked immunosorbent assays for detecting antibodies to Shiga-like toxin I,Shiga-like toxin II,andEscherichia coli O157:H7 lipopolysaccharide in human serum

Shiga-like toxin-producingEscherichia coli O157:H7 are important causes of bloody diarrhea and hemolytic uremic syndrome. To facilitate the epidemiologic study of these organisms, we developed enzyme-linked immunosorbent assays (ELISAs) for antibodies to Shiga-like toxin I (SLT I), Shiga-like toxin II (SLT II), andE. coli O157 lipopolysaccharide (LPS). We tested serum samples from 83 patients in two outbreaks ofE. coli O157:H7 diarrhea and from 66 well persons. Forty-three patients (52%) had at least one serum sample positive for anti-O157 LPS antibodies; among 26 culture-confirmed patients, 24 (92%) had at least one positive serum sample. Two (3%) of 66 control sera had positive anti-O157 LPS titers. ELISA results for SLT I and II were compared with those of HeLa cell cytotoxicity neutralization assays on both patient and control sera. Neutralization assays detected anti-SLT I antibodies in at least one serum sample from each of 17 (20%) patients and 7 (10.6%) controls, while 16 (19%) patients and 7 controls had positive titers by anti-SLT I ELISA. Although all serum samples, including control sera, showed nonspecific neutralization of SLT II, no antibody titers to SLT II were detected by either neutralization or ELISA. These results indicate that ELISAs for SLT I and SLT II antibodies are comparable to HeLa cell cytotoxicity neutralization assays. Both the ELISAs and neutralization assays are insensitive in detecting infected patients. However, the ELISA for antibodies toE. coli O157 LPS is both sensitive and specific, and may be more useful than assays for antitoxic antibodies in detecting persons withE. coli O157:H7 infection.     

Ananain: a novel cysteine proteinase found in pineapple stem

A previously unknown cysteine proteinase, named ananain, has been isolated from crude commercial pineapple stem bromelain. The purification procedure involved affinity chromatography on Sepharose-Gly-Phe-glycinaldehyde semicarbazone, and cation-exchange chromatography. The relative molecular mass of ananain was very similar to that of bromelain (25,000 and 26,000, respectively), but ananain differed greatly in specificity for hydrolysis of peptide and protein substrates. The new enzyme behaved as a typical cysteine proteinase in showing strong inhibition by chicken cystatin, whereas bromelain was scarcely affected. Ananain was also shown to be immunologically distinct from bromelain. The significance of the discovery of ananain for the interpretation of previous work on "bromelain" is pointed out.     

Interactions between the benzodiazepine receptor antagonist Ro 15-1788 (flumazepil) and the inverse agonist beta-CCE: behavioral studies with squirrel monkeys

The effects of Ro 15-1788 and ethyl-beta-carboline-3-carboxylate (beta-CCE) were studied alone and in combination on the behavioral performances of squirrel monkeys. Under one procedure, performances maintained by food were suppressed by electric shock presentation (punishment or "conflict" procedure). Under a second procedure, responding was maintained either by food or electric shock delivery under a 5-min fixed-interval schedule. Doses of beta-CCE between 0.1 and 3.0 mg/kg, i.m., produced graded decreases in punished responding which were reversed by pretreatment with Ro 15-1788 (1.0 - 10.0 mg/kg, i.m.). Low doses of beta-CCE (0.03 - 0.3 mg/kg, i.m.) increased responding of monkeys maintained by shock presentation, but did not affect food-maintained responding; higher doses of beta-CCE decreased responding under both schedules. These effects of beta-CCE are opposite those produced by the benzodiazepines under this procedure. Ro 15-1788 (1.0 mg/kg i.m.) antagonized the effects of beta-CCE, producing a shift to the right in the dose-response curves. These findings provide further support for the view that beta-CCE and Ro 15-1788 produce effects mediated by the same benzodiazepine receptor recognition site.     

Human liver cathepsin L.

Cathepsin L was purified to apparent homogeneity from human liver obtained post mortem. It was necessary to treat the homogenate at pH 4.2 and 37 degrees C to release active enzyme. The purification procedure involved ion-exchange chromatography on carboxymethyl-Sephadex and the Mono S column of a Pharmacia fast-protein-liquid-chromatography system. The enzyme was found to consist of two polypeptide chains of Mr 25 000 and 5000. The larger chain was shown to contain the active-site cysteine residue. Human cathepsin L proved to be similar to the rat and rabbit enzymes in regard to kinetic constants for the substrate benzyloxycarbonylphenylalanylarginine 7-(4-methyl)coumarylamide and rates of inactivation by the active-site-directed reagents benzyloxycarbonylphenylalanylphenylalanyldiazomethane and benzyloxycarbonylphenylalanylalanyldiazomethane. Thus clear characteristics of cathepsin L are now emerging, and these should simplify the identification of the enzyme in other tissues and species.     

Differential recovery of circulating T cell subsets after nodal irradiation for Hodgkin's disease

To better understand the immunologic effects of lymphoid irradiation (LI), blood levels of T cell subsets were sequentially monitored in 15 patients before, during, and after irradiation treatment for Hodgkin's disease. Blood levels of all lymphocytes, T cells, and T cell subsets (defined by OKT4 and OKT8) fell dramatically and in similar proportions during early therapy, reaching levels less than 20 to 25% of control by the completion of mantle irradiation, and continuing at very depressed levels through the completion of therapy. Blood levels of OKT8-reactive (OKT8+) cells returned to pretreatment levels (402 +/- 38/mm3 vs 360 +/- 32/mm3 pretreatment) between 6 to 8 mo after LI, whereas blood levels of OKT4-reactive (OKT4+) cells returned to only 42% of previous values (242 +/- 22/mm3 vs 584 +/- 34/mm3 pretreatment) over the same period. The pre-LI ratio of OKT4+ to OKT8+ cells was 1.85 +/- 0.24 and fell to 0.65 +/- 0.05 between 6 to 8 mo after LI. During the recovery period, discrepancies of 208 +/- 32 cells/mm3 (3 to 5 months post LI) and 198 +/- 32 cells/mm3 (6 to 8 mo post LI) developed between the blood levels of OKT3+ cells and the sum of OKT4+ and OKT8+ cells. This suggests the emergence of OKT4+/OKT3-, OKT8+/OKT3-, and/or OKT4+/OKT8+ cells. In five patients, the sum of OKT4+ and OKT8+ cells was compared with the number of cells simultaneously co-stained by OKT4 and OKT8. It appeared that a significant proportion of the cells were OKT4+/OKT3- and OKT8+/OKT3- lymphocytes. We concluded that LI is similarly cytotoxic to peripheral blood T cell subpopulations. The reversed ratio after LI is a result of a slower repopulation of the peripheral blood by OKT4+ cells relative to OKT8+ cells. T cells after LI show a high degree of antigenic immaturity. It is postulated that the bone marrow that lies outside the fields of treatment and contains predominantly immature OKT8+/OKT3- cells is a major source of T cells repopulating the peripheral blood after LI.