Molecular cloning and functional characterization of KCC3, a new K-Cl cotransporter

We isolated and characterized a novelK-Cl cotransporter, KCC3, from human placenta. The deduced proteincontains 1,150 amino acids. KCC3 shares 75-76% identity at theamino acid level with human, pig, rat, and rabbit KCC1 and 67%identity with rat KCC2. KCC3 is 40 and 33% identical to twoCaenorhabditis elegans K-Cl cotransporters and ~20%identical to other members of the cation-chloride cotransporter family(CCC), two Na-K-Cl cotransporters (NKCC1, NKCC2), and the Na-Clcotransporter (NCC). Hydropathy analysis indicates a typical KCCtopology with 12 transmembrane domains, a large extracellular loopbetween transmembrane domains 5 and 6 (unique to KCCs), and largeNH₂ and COOH termini. KCC3 is predominantly expressed inkidney, heart, and brain, and is also expressed in skeletal muscle,placenta, lung, liver, and pancreas. KCC3 was localized to chromosome15. KCC3 transiently expressed in human embryonic kidney (HEK)-293cells fulfilled three criteria for increased expression of K-Clcotransport: stimulation of cotransport by swelling, treatment withN-ethylmaleimide, or treatment with staurosporine.

     

The intactness and orientation of acetylcholine receptor-rich membrane from Torpedo californica electric tissue

By a mild and highly reproducible fractionation of Torpedo californica electric tissue, we prepared membrane which was 30 times enriched in nicotinic acetylcholine receptor (AChR). This preparation was neither alkali-stripped nor reconstituted and consequently contained nu (43-kDa protein), which is associated with the cytoplasmic aspect of the receptor. We tested this membrane for the presence of sealed vesicles and determined the orientation of these vesicles by combining three methods. Two of these methods were based on the accessibilities, in the presence and absence of detergent, of the extracellular acetylcholine binding site to alpha-bungarotoxin and of the intracellular nu to trypsin. These two methods are specific for AChR-containing membrane. The third method was morphometry of electron micrographs, by which we estimated the proportion of sequestered membrane. These methods taken together indicated that approximately 45% of the AChR-containing membrane was in the form of leaky vesicles or sheets, 33% was sealed right-side-out vesicles, 11% was sealed inside-out vesicles, and 11% was sequestered within multilamellar or multivesicular vesicles. The complexity of this membrane needs to be taken into account in sidedness studies of the AChR.     

Effect of route of atropine delivery on bronchospasm from cold air and methacholine

We undertook a study to determine whether the apparent disparity between the dose of inhaled atropine required to inhibit the bronchoconstriction induced by inhaled methacholine and the dose required to inhibit the bronchoconstriction induced by eucapnic hyperpnea with cold air is a function of the route of administration of atropine. In six subjects with asthma, we constructed dose-response curves to inhaled methacholine and to eucapnic hyperpnea with cold air after treatment with inhaled atropine (0.5 mg delivered) and intravenous placebo, with inhaled placebo and intravenous atropine (0.5 mg injected), and with inhaled and intravenous placebos. Atropine by either route shifted the dose-response curves to both cold air and to methacholine to the right. In every subject, however, inhaled atropine caused a markedly greater rightward shift of the inhaled methacholine dose-response curve than did intravenous atropine, whereas inhaled and intravenous atropine had similar effects on the cold air dose-response curve. These findings suggest that the apparent disparity between the doses of atropine required to inhibit methacholine- and cold air-induced bronchoconstriction may be a function of the route of administration of atropine and thus does not imply a nonmuscarinic action of atropine. The findings support the view that cold air causes bronchoconstriction via muscarinic pathways.     

Dental Hygienists' and Dental Hygiene Students' Attitudes Toward the Elderly

Dental hygiene students' and graduates' attitudes toward the elderly were evaluated in two different countries representing two different cultures. Five dental hygiene programs in the U.S. and the only dental hygiene program in Israel were selected to participate in addition to graduates from both Colorado-U.S. and Jerusalem-Israel. A total of 180 dental hygiene students of which 44 were Israelis, 70 U.S. graduates, and 52 Israeli graduates, participated. Both American graduates and American dental hygiene students were found, based on the usage of the Rosencranz-McNevin measure, to have significantly more positive attitudes toward the elderly as compared to the Israelis. Israeli and American dental hygiene students had a mean score of 120.92 and 105.96 respectively, Israeli graduates scored 122.39 and American graduates had a mean score of 105.09. The differences in culture, geriatric education in schools, and response to the survey according to what is most socially acceptable in the U.S. were the main reasons for these differences. Yet, it is hypothesized that these gaps in attitudes would decrease with the years, following the slow but stable “Americanization” of the Israeli society.