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.     

Detection of undiagnosed coagulopathies using routine rapid heparin neutralization.

OBJECTIVE: To determine the most frequent clinical causes of a prolonged activated partial thromboplastin time (APTT) result, and to determine whether a new heparin-removal device (the Hepchek, Pall Biomedical, Glen Cove, NY 11542) is capable of efficiently detecting the causes of these values. DESIGN: A combination of chart review and laboratory testing comparing the criterion standard--the heparin chromogenic substrate assay--with the Hepchek. Laboratory investigations were blinded and controlled. SETTING: Inpatient, acute-care hospital. PATIENTS: A total of 1,000 hospital patients with a variety of hemostatic disorders. MAIN OUTCOME MEASURE: The extent to which the Hepchek accurately identified the etiology of a prolonged APTT result. RESULTS: The APTT was prolonged in 25.2% of samples. The presence of heparin in the sample was confirmed by chromogenic assay or by using the Hepchek heparin-removal filter. The presence of heparin was confirmed in 12.8% of all samples and in more than 50% of all abnormal samples. The cause of the abnormal APTT was often unappreciated by the clinician. Bayesian analysis of the Hepchek's ability to diagnose heparin correctly as the cause of the abnormal APTT showed a sensitivity of 100% and specificity of 99.9%. CONCLUSION: Use of the Hepchek in the routine clinical laboratory is an efficient and rapid method of detecting heparin as a cause of isolated prolonged APTT results, and should reduce demands for unwarranted coagulation analyses and inappropriate treatment with blood products.     

Advances in pharmacotherapy: depression in the elderly– issues and advances in treatment

Depression continues to be a major cause of morbidity and mortality in the elderly. It is estimated that 1–5% of elderly persons who live in the community and 5–43% of nursing–home patients have major depression. Symptoms of depression in the elderly do not differ substantially from younger patients. Tricyclic antidepressants continue to be the drugs of choice in the elderly because of their long record of use with proven efficacy, known adverse effect profile and availability of less expensive generic formulations. The newer secondgeneration antidepressants, including serotonin reuptake inhibitors, appear to offer a major advantage of fewer serious adverse effects in the elderly. This review will highlight recent developments regarding the prevalence and treatment of depression in the elderly.     

Bradykinin-induced release of PGI2 from aortic endothelial cell lines: responses mediated selectively by Ca2+ ions or a staurosporine-sensitive kinase.

1. Bradykinin (100 nM) triggers release of nitric oxide and prostacyclin from both AG07680A and AG04762 bovine cultured aortic endothelial cells. The exposure of these cells to bradykinin is in each case associated with a striking rise in intracellular calcium ion concentration. 2. Exposure of AG07680A cells to 250 nM ionomycin was followed also by a significant release of prostacyclin, whereas 250 nM ionomycin had no capacity to stimulate release of prostacyclin from AG04762 cells. 3. There was a similar concentration-dependent increase in intracellular calcium ion concentration on exposure of AG07680A and AG04762 cells to ionomycin. 4. Exposure of AG04762 cells for 10 min to staurosporine produced a concentration-dependent inhibition (IC50 = 107 +/- 14 nM) in bradykinin-stimulated prostacyclin release. There was no similar inhibitory effect of staurosporine in AG07680A cells. 5. Bradykinin (10 nM) triggered release of nitric oxide from both AG07680A and AG04762 cells, and the effect was not inhibited by 500 nM staurosporine. There was a similar ionomycin-dependent release of nitric oxide from both cell types. 6. These results identify a common pathway for bradykinin-dependent nitric oxide release from both AG07680A and AG04762 cells, involving increases in intracellular calcium ion concentration. In contrast, the bradykinin-dependent release of prostacyclin may involve one of two pathways (involving an increase in intracellular calcium or activation of a staurosporine-sensitive kinase), and the two pathways are selectively exploited in AG07680A and AG04762 cells, respectively.     

Echocontrast agent-enhanced color flow imaging of the breast.

An ultrasound contrast agent which survives transit through the pulmonary circulation after injection into a peripheral vein can also be used for the demonstration of flow characteristics in the peripheral organs. Such a substance (SH U 508 A, Schering AG, Berlin) was tested in the Gynecological Department of the University of Marburg for its suitability for enhancing color-coded Doppler signals in mammary diagnosis in a group of ten women with breast tumors. The observations made are compared with the histologically confirmed diagnoses. The very distinct enhancement of the color-coded Doppler signals allows reliable demonstration of the vascularization characteristics, not only of the malignant, but also of all benign solid tumors examined. Color signals were even recorded from normal mammary tissue, which means that the demonstration of perfusion can no longer be used on its own as a diagnostic criterion. Observation of the characteristics of the blood supply to a tumor might, on the other hand, be an advantage, not only in respect of tumor typing, but also as regards the prognosis of a tumor. In this connection, the registration of the arrival, retention and wash-out phases specific to a contrast agent appears to furnish particularly interesting information about the growth dynamics of a tumor.     

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.     

Serotonin-induced increase in cAMP in ganglia isolated from the myenteric plexus of the guinea pig small intestine: Mediation by a novel 5-HT receptor

Serotonin (5-HT) is a mediator (through 5-HT_1P receptors) of slow EPSPs in myenteric ganglia of the small intestine. The effect of 5-HT can be mimicked by elevating cAMP; therefore, we tested the hypothesis that the slow EPSP-like response to 5-HT is cAMP-mediated. Guinea pig gut was enzymatically dissociated; myenteric ganglia remained intact and were collected by filtration. Neurons in the isolated ganglia retained their ability to manifest the slow EPSP-like response to 5-HT. Exposure to 5-HT raised the ganglionic level of cAMP (ED₅₀ 0.3 μM). This effect was not antagonized by the 5-HT_1P antagonist, N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide (100.0 μM), or mimicked by the 5-HT_1P agonist, 5-hydroxyindalpine (10.0 μM). Increases in cAMP were also evoked by the 5-HT₁ agonist, 5-carboxyamidotryptamine (10.0 μM), the 5-HT₂ agonist, (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI; 1.0–10.0 μM), and by the 5-HT₄ agonists, renzapride (1.0–10.0 μM) and 5-methoxytryptamine (1.0–10.0 μM); however, neither the 5-HT₁/5-HT₂ antagonists, spiperone, methysergide, and methiothepin, nor the 5-HT₄ antagonist, tropisetron (ICS 205–930; 10.0 μM), were able to inhibit the rise in cAMP evoked by these compounds or by 5-HT (0.1–10.0 μM). The 5-HT-evoked elevation of cAMP was antagonized by ketanserin (10.0 μM), which also blocked the effects of 5-methoxytryptamine and DOI, but not those of renzapride. The effective concentration of DOI, however, was higher than that needed for activation of 5-HT₂ receptors, and Northern analysis using a cDNA probe encoding the rat 5-HT₂ receptor failed to reveal the presence of 5-HT₂ mRNA in myenteric ganglia, although it hybridizes with mRNA of the right size in the guinea pig brain. Compounds that failed to change levels of cAMP or to antagonize the action of 5-HT included 8-hydroxy-di-n-propylamino tetralin, R58639, R88226, and sumatriptan. It is concluded that the receptor responsible for the 5-HT-induced rise in cAMP in ganglia isolated from the guinea pig myenteric plexus is not a known subtype of 5-HT receptor. Since the pharmacology of this novel receptor is different from that of the slow EPSP-like response to 5-HT, the receptor probably does not mediate the slow EPSP. © 1993 Wiley-Liss, Inc.     

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.