The Presence of Insulin-degrading Enzyme in Human Ileal and Colonic Mucosal Cells

The aim of this research is to characterize the presence of insulin-degrading enzyme in human colon and ileal mucosal cells. Biochemical studies, including the activity-pH profiles, the effects of enzyme inhibitors, immunoprecipitation and western blots, were conducted. The majority of insulin-degrading activity in colon mucosal cells was localized in the cytosol. In both colon and ileum, cytosolic insulin-degrading activities had a pH optimum at pH 7.5, and were extensively inhibited by each of N-ethylmaleimide, p-chloromercuribenzoate, and 1,10-phenanthroline, but were very weakly affected by each of leupeptin, chymostatin, diisopropyl phosphofluoridate and soybean trypsin inhibitor. In the colon and ileum, more than 93% and 96%, respectively, of cytosolic insulin-degrading activities were removed by the mouse monoclonal antibody to human RBC insulin-degrading enzyme, as compared with less than 20% by the normal mouse IgG for both tissues. Further, a western blot analysis revealed that a cytosolic protein of 110 kD, in both human colon and ileum, reacted with the monoclonal antibody to insulin-degrading enzyme. It is concluded that insulin-degrading enzyme is present in the cytosol of human colon and ileal mucosal cells.     

Dual effects of chlorobutanol on secretory response and intracellular Ca2+ dynamics in isolated pancreatic acini of the rat.

1. The effects of chlorobutanol, a widely used drug preservative, on exocrine response and intracellular Ca2+ dynamics were examined in isolated pancreatic acini of the rat. 2. Chlorobutanol (1 mg ml-1) markedly inhibited the secretory response to cholecystokinin octapeptide (CCK-8), carbamylcholine chloride (carbachol), or sodium fluoride, a direct G-protein activator. However, chlorobutanol itself induced a maximal release of amylase when the dose was increased to 4 mg ml-1. 3. An oscillatory fluctuation of cytoplasmic Ca2+ concentration, [Ca2+]c, induced by 5 pM CCK-8 or 0.3 microM carbachol was totally abolished in the presence of 1 mg ml-1 chlorobutanol. 4. A biphasic change in [Ca2+]c induced by 100 pM CCK-8, a rapid rise followed by a gradual decay, was transformed to an oscillatory fluctuation by the preservative. 5. Chlorobutanol inhibited 13 pM [125I]-CCK-8 or 0.5 nM [3H]-methylscopolamine chloride binding to the acinar cells in a dose-dependent manner. 6. These results indicate that chlorobutanol produces discernible pharmacological effects on the secretory response in rat pancreatic acinar cells through changes in the Ca2+ dynamics. Possible sites of action could be at a binding process of secretagogues to their receptors, at an activation process of a G-protein located in the plasma membrane, or at the processes following G-protein activation. However, the possibility that the preservative may distort the Ca(2+)-transport function of the plasma membrane or the membrane of intracellular organella, especially Ca(2+)-sequestering pools, cannot be excluded.     

Bicycle helmet use by adults: the impact of companionship.

Most of the nearly 1,000 fatal bicycle-related injuries annually could be prevented if riders used safety helmets. Helmet use by adult bicyclists has received relatively little attention because educational campaigns to promote helmet use generally focus on children. Helmet use by adult and child bicyclists at 120 suburban and rural sites in three Maryland counties was observed on two Saturdays in 1990-91 during an evaluation of the impact of a mandatory helmet law. Concordance or discordance of helmet use within various groups of bicyclists--adults only, adults with children, and children only--was recorded. Helmet use among 2,068 adult bicyclists was 49 percent, 51 percent, and 74 percent in the three counties. In two counties combined, 52 percent (365 of 706) of solo adult bicyclists wore helmets compared with only 5 percent (5 of 94) of solo child bicyclists (P < .001). Helmet use or nonuse was concordant among 87 percent of 277 adult-adult pairs, 94 percent of 50 child-child pairs, and 91 percent of 32 adult-child pairs of bicyclists observed. Concordance rates of helmet use or nonuse were similarly high among pairs of adult bicyclists of the same or mixed sexes. These data are consistent with the concept that both adults and children tend to adopt the helmet-wearing behaviors of their companions. Public health efforts focused on adults should encourage helmet use by adult bicyclists both to prevent head injuries and to provide a role model for children.     

The Perceived Influence of Clinical Pharmacy Services on Physician Prescribing Behavior: A Matched-Pair Comparison of Pharmacists and Physicians

In contrast with cross-sectional designs used in previous studies, this exploratory study compared survey data from 127 matched pairs of clinical pharmacists and physicians working together. Physicians' perceptions of the importance of clinical pharmacy activities for patient care and the competence of pharmacists performing the activities were examined for their influence on prescribing behavior in an institutional setting. Data from a national survey showed that physicians rated pharmacists higher regarding recommendations based on drug use evaluations (p = 0.004) and competency to provide all clinical pharmacy services. Scores for pharmacokinetics ratings were similar between pharmacists and physicians (p = 0.168). Pharmacists rated the importance of recommendations based on cost-effectiveness higher than physicians (p = 0.012). Overall, physicians' perceptions of activity importance for patient care and pharmacist competency appear to dictate pharmacists' influence on physician prescribing behavior (R = 0.723).     

Age-related Changes in Bovine α-crystallin and High-molecular-weight Protein

The high-molecular-weight (HMW) protein from the lens is composed mostly of α-crystallin in a highly aggregated state. Bovine HMW protein was carefully separated from α-crystallin by size-exclusion chromatography. α-Crystallin has chaperone-like ability whereby it stabilizes other proteins under conditions of stress (e.g. heat). Comparison of bovine HMW protein and α-crystallin shows that the HMW protein has a markedly reduced chaperone ability compared to α-crystallin. However, in contrast to the results of other workers, we observe no alteration with age in the ability of α-crystallin to act as a chaperone. Using electrospray ionisation mass spectrometry, changes in the phosphorylation of the α-crystallin subunits with age have been quantified. Phosphorylation of α-crystallin occurs early in life but does not alter in proportion after about three years of age. In addition, phosphorylation of the A subunit of α-crystallin has little effect on its chaperone ability. As is found in the artificially prepared HMW complex of α- and γ-crystallin, NMR spectroscopy shows that in the naturally occurring HMW protein, the short C-terminal extension of the α_Bsubunit has lost its flexibility whereas the α_Asubunit extension is still flexible. Post-translational modifications therefore seem to have little effect on the chaperone action of α-crystallin, but alterations in the quaternary structure of α-crystallin via incorporation into the HMW aggregate, lead to major changes in the chaperone ability of the protein. The results are consistent with the notion that one of the contributing factors to cataract formation in the lens is the depletion of α-crystallin with age as it is converted into the HMW protein.     

Alpha-melanocyte-stimulating hormone reduces endotoxin-induced liver inflammation.

Alpha-Melanocyte-stimulating hormone (MSH) is a potent anti-inflammatory agent in many models of inflammation, suggesting that it inhibits a critical step common to different forms of inflammation. We showed previously that alpha-MSH inhibits nitric oxide (NO) production in cultured macro-phages. To determine how alpha-MSH acts in vivo, we induced acute hepatic inflammation by administering endotoxin (LPS) to mice pretreated with Corynebacterium parvum, alpha-MSH prevented liver inflammation even when given 30 min after LPS administration. To determine the mechanisms of action of alpha-MSH, we tested its influence on NO, infiltrating inflammatory cells, cytokines, and chemokines. Alpha-MSH inhibited systemic NO production, hepatic neutrophil infiltration, and increased hepatic mRNA abundance for TNF alpha, and the neutrophil and monocyte chemokines (KC/IL-8 and MCP-1). We conclude that alpha-MSH prevents LPS-induced hepatic inflammation by inhibiting production of chemoattractant chemokines which then modulate infiltration of inflammatory cells. Thus, alpha-MSH has an effect very early in the inflammatory cascade.     

Changes in the Subcellular Distribution of the Rat Uterus Oestrogen Receptor as Induced by Oestradiol,Tamoxifen and ZD 182,780

The aim of this work was to compare the subcellular distribution of the oestrogen receptor from the uteri of rats treated with vehicle alone (control group), oestradiol or one of the antio-estrogenic drugs tamoxifen and ZD 182,780. The nuclear, microsomal and cytosolic oestrogen receptor contents were evaluated by an immunoenzymatic method (“ER-EIA” kit from Abbott Laboratories) and the results in each fraction were expressed as a percentage of the total number of receptors. Parallel studies were performed to assess the uterotrophic effect of these drugs and to assess that they had reached the uterus. In the control group, we found that the oestrogen receptor was distributed mainly between the microsomal (29.1 ± 1.3%) and cytosolic (68.1 ±0.9%) fractions, with only a small amount located in the nucleus (2.8 ± 0.5%). When oestradiol was administered, the oestrogen receptor distribution was: nuclear 11.7 ± 2.0, microsomal 15.5 ± 1.3 and cytosolic 72.8 ± 3.3% and, in the tamoxifen group, the results were: nuclear 18.5 ± 1.5, microsomal 26.0 ± 31 and cytosolic 55.5 ± 3.4%, which shows a relative shift both to the control and the oestradiol-treated groups. In the uteri of rats treated with ZD 182,780 the results were very similar to those obtained in the control group. Our results indicate that the subcellular distribution of the oestrogen receptor varies according to the drug administered and that this receptor may not be located in a single subcellular compartment. Moreover, the nuclear uptake of the ZD 182,780-oestrogen receptor complex seems to be blocked, possibly due to impaired receptor dimerization. In the case of tamoxifen, the intracellular transport of the receptor also seems to be blocked, probably due to the nuclear retention of the receptor, thus suggesting that tamoxifen must impair the oestrogen receptor function on a step subsequent to the receptor dimerization.     

Differential distribution of N-acetylaspartylglutamate and N-acetylaspartate immunoreactivities in rat forebrain

Summary Contradictory immunohistochemical data have been reported on the localization of N-acetylaspartylglutamate in the rat forebrain, using different carbodiimide fixation protocols and antibody purification methods. In one case, N-acetylaspartylglutamate immunoreactivity was observed in apparent interneurons throughout all allocortical and isocortical regions, suggesting possible colocalization with GABA. In another case, strong immunoreactivity was observed in numerous pyramidal cells in neocortex and hippocampus, suggesting colocalization with glutamate or aspartate. Reconciling these disparate findings is crucial to understanding the role of N-acetylaspartylglutamate in nervous system function. Antibodies to N-acetylaspartylglutamate and a structurally related molecule, N-acetylaspartate, were purified in stages, and their cross-reactivities with protein conjugates of N-acetylaspartylglutamate and N-acetylaspartate were monitored at each stage by solidphase immunoassay. Reduction of the cross-reactivity of the anti-N-acetylaspartylglutamate antibodies to N-acetylaspartateprotein conjugates to about 1% eliminated significant staining of most pyramidal neurons in the rat forebrain. Utilizing highly purified antibodies, the distributions of N-acetylaspartylglutamate and N-acetylaspartate were examined in several major telencephalic and diencephalic regions of the rat, and were found to be distinct. N-acetylaspartylglutamate-immunoreactivity was observed in specific neuronal populations, including many groups thought to use GABA as a neurotransmitter. Among these were the globus pallidus, ventral pallidum, entopeducular nucleus, thalamic reticular nucleus, and scattered non-pyramidal neurons in all layers of isocortex and allocortex. N-acetylaspartate-immunoreactivity was more broadly distributed than N-acetylaspartylglutamate-immunoreactivity in the rat forebrain, appearing strongest in many pyramidal neurons. Although N-acetylaspartate-immunoreactivity was found in most neurons, it exhibited a great range of intensities between different neuronal types.