Isolation and partial characterization of human platelet vinculin.

A 130,000 Mr protein was isolated from human platelets by sequential DEAE-Sephacel and Sepharose Cl-4B chromatography. Low shear viscometric measurements showed that the enriched protein after DEAE-Sephacel chromatography inhibited actin polymerization. This effect was somewhat greater in the presence of EGTA than in the presence of calcium. Further purification by Sepharose Cl-4B chromatography resulted in a complete loss of this inhibitory effect. Studies with fluorescent actin detected no nucleation or "+" end capping activity in either the DEAE-Sephacel- or Sepharose Cl-4B-purified vinculin. Antibodies raised in mice against the 130,000-mol-wt protein were shown to cross-react with chicken gizzard vinculin and a similar molecular weight protein was detected in WI38 cells and, Madin-Darby canine kidney cells. Lysis experiments with the Madin-Darby canine kidney cells indicated that most of the vinculin was soluble in Triton X-100, although some was found associated with the insoluble cytoskeletal residue. By immunofluorescence, vinculin in WI38 cells was localized to adhesion plaques as described by others. Discrete localization in platelets was also detected and appeared to depend on their state of adhesion and spreading. The results of these experiments suggest that human platelets contain a protein similar to vinculin. It is not clear if platelet vinculin is associated with structures analogous to adhesion plaques found in other cell types. The data indicate that the previously reported effects of nonmuscle vinculins on actin polymerization may be due to a contaminant or contaminants.

14-3-3 proteins: a highly conserved, widespread family of eukaryotic proteins.

A family of proteins known as 14-3-3 is currently receiving increased attention by investigators studying a broad range of biological systems, including plants and invertebrates. The outstanding feature of this family is the extraordinarily high sequence conservation observed. Current thinking indicates that these proteins may function as regulators in signal transduction/phosphorylation mechanisms.

Peripheral mechanisms of intestinal dysmotility in the morphine tolerant and dependent rats.

Changes of intestinal motility and transit produced by tolerance to and dependence upon morphine have been partly attributed to peripheral mechanisms. We evaluated the effect of chronic peripheral morphine administration and peripheral mu-receptor blockade on vagal afferent activity (VAA) and c-Kit positive intramuscular cells of Cajal (ICCs). Ten rats were subjected to chronic subcutaneous morphine infusion for 72 h with subsequent VAA recording. Potential frequency was evaluated within recordings before and after mu receptor blockade by (D)-Phe -Cys -Tyr -(D)-Trp -Orn -Thr -Phe -Thr (CTOP) i.p. injections. Afterwards the rats were sacrificed and intramuscular c-Kit antigen expression was assessed by image analysis within removed fragments of duodenum and ascending colon. An equal group of rats served as a control for VAA and c-Kit expression. Analysis of VAA revealed similar frequencies of potentials in morphine tolerant / dependent rats before CTOP and in the controls. CTOP increased potential frequency in the morphine group which effect was visible mostly within the first 20 minutes (p=0.01). The morphine infused animals presented also higher c-Kit expression in both the duodenum (p<0.001) and the ascending colon (p<0.001) in comparison to the control group. Results of our study may indicate the involvement of both the intestinal wall and the long vago-vagal reflexes in tolerance to and dependence upon opioids.

courtless, the Drosophila UBC7 homolog, is involved in male courtship behavior and spermatogenesis.

The courtless (col) mutation disrupts early steps of courtship behavior in Drosophila males, as well as the development of their sperm. Most of the homozygous col/col males (78%) do not court at all. Only 5% perform the entire ritual and copulate, yet these matings produce no progeny. The col gene maps to polytene chromosome band 47D. It encodes two proteins that differ in their carboxy termini and are the Drosophila homologs of the yeast ubiquitin-conjugating enzyme UBC7. The col mutation is caused by an insertion of a P element into the 3' UTR of the gene, which probably disrupts translational regulatory elements. As a consequence, the homozygous mutants exhibit a six- to sevenfold increase in the level of the COL protein. The col product is essential, and deletions that remove the col gene are lethal. During embryonic development col is expressed primarily in the CNS. Our results implicate the ubiquitin-mediated system in the development and function of the nervous system and in meiosis during spermatogenesis.

Cholecystokinin-induced antinociception is not blocked by CCK-A or CCK-B receptor antagonists.

To determine the relative importance of CCK-A, CCK-B, and opioid receptors in mediating the antinociceptive actions of cholecystokinin, we evaluated the actions of selective agonists and antagonists in the mouse hot plate assay. The agonists used were CCK (1-30 nmol i.c.v.), a CCK-A receptor agonist (SNF9019; 0.3-10 nmol i.c.v.), and a CCK-B receptor agonist (SNF9007; 0.3-10 nmol i.c.v.). The antagonists used were the CCK-A receptor antagonist, L364,718 (12.5 nmol i.c.v.), CCK-B receptor antagonist, L365,260 (2.5-25 nmol i.c.v.), and the nonselective opioid receptor antagonist naloxone (1 mg/kg s.c.). CCK and its receptor-selective analogues, SNF9019 and SNF9007, resulted in antinociception that was blocked by naloxone, but was not antagonized by L364,718 or L365,260. In contrast, in positive control experiments, the inhibitory effects of CCK, SNF9019, and SNF9007 on gastrointestinal propulsion in mice were antagonized by identical i.c.v. doses of L364,718 and L365,260. We conclude that centrally administered CCK produces antinociception in the mouse hot plate assay via opioid receptors, but independent of CCK-A or CCK-B receptors. It is necessary to speculate that other CCK receptors, not antagonized by currently available selective antagonists, may exist.

Inhibition of adenylyl cyclase activity by the cholecystokinin analog SNF 9007 in neuroblastoma x glioma NG108-15 hybrid cells.

The effect of the cholecystokininB (CCKB) receptor-selective cholecystokinin octapeptide (CCK-8) analog SNF 9007 on forskolin-stimulated adenylyl cyclase activity in NG108-15 hybrid cells was measured. The activity of SNF 9007 was compared to the delta opioid agonists D-Pen2-D-Pen5-enkephalin (DPDPE, delta 1 receptor-selective) and Tyr-D-Ala-Phe-Glu-Val-Val-Gly-NH2, (D-Ala2-deltorphin II, delta 2-receptor-selective) because SNF 9007 binds with moderate affinity to delta opioid receptors. SNF 9007 inhibited forskolin-stimulated adenylyl cyclase activity with efficacy similar to DPDPE. IC50 determinations showed that D-Ala2-deltorphin II was the most potent, followed by DPDPE, then SNF 9007 (IC50 values = 0.013, 0.21 and 4.8 microM, respectively). CCK-8 had no effect on adenylyl cyclase activity. The delta 1 receptor-selective antagonist 7-benzylidenenaltrexone hydrochloride (BNTX, 10 nM) had no effect on the activity of any of these agonists, but the delta 2 receptor-selective antagonist naltriben methanesulfonate (NTB, 10 nM) increased IC50 values of all the agonists. Combinations of BNTX and NTB (10 nM each) increased the D-Ala2-deltorphin II IC50 value 12-fold, the DPDPE IC50 value 18-fold and the SNF 9007 IC50 value 26-fold. The effect of the combined delta antagonists on SNF 9007 activity was different from the effect on DPDPE or D-Ala2-deltorphin II activity. These data suggest that the interaction of the CCK-8 analog SNF 9007 with opioid receptors in NG108-15 hybrid cells is different from the interaction of opioid peptides with these receptors.

Pirenzepine (LS 519): a weak inhibitor of acid secretion by isolated rat parietal cells.

Carbamylcholine-stimulated potentiation of dibutyryl cyclic AMP-induced acid secretion by isolated rat parietal cells was specifically inhibited by the muscarinic cholinergic antagonist pirenzepine at an IC50 of 1.1 microM. In contrast to the weak inhibition by pirenzepine, the potentiation was inhibited by atropine at an IC50 of 4.6 nM. In vivo, pirenzepine is one-tenth as potent as atropine as an inhibitor of acid secretion. The weak activity of pirenzepine shown in this study suggests that its in vivo inhibitory activity is likely due to an interaction with a site involved in the regulation of gastric acid secretion which has higher-affinity muscarinic receptors for pirenzepine than those associated with parietal cells.

Scholarship in teaching: an imperative for the 21st century.

At some medical schools broader definitions of scholarship have emerged along with corresponding changes in their academic reward systems. Such situations are not common, however. The definition of scholarship generally applied by medical schools is unnecessarily narrow and excludes areas of legitimate academic activity and productivity that are vital to the fulfillment of the school's educational mission. The authors maintain that creative teaching with effectiveness that is rigorously substantiated, educational leadership with results that are demonstrable and broadly felt, and educational methods that advance learners' knowledge are consistent with the traditional definition of scholarship. Faculty whose educational activities fulfill the criteria above are scholars and must be recognized by promotion. The authors specifically address scholarship in education, focusing on teaching and other learning-related activities rather than on educational research, which may be assessed and rewarded using the same forms of evidence as basic science or clinical research. They build on Boyer's work, which provides a vocabulary for discussing the assumptions and values that underlie the roles of faculty as academicians. Next, they apply Glassick et al.'s criteria for judging scholarly work to faculty members' educational activities to establish a basis for recognition and reward consistent with those given for other forms of scholarship. Finally, the authors outline the organizational infrastructure needed to support scholars in education.

Behavioral analysis of Drosophila mutants displaying abnormal male courtship.

We describe six recessive autosomal male sterile mutations in Drosophila, generated by mobilization of single P-elements, exhibiting abnormal male courtship behavior. Detailed analysis of courtship behavior elicited by virgin wild type females indicated that five of the six mutants are affected in the early steps of courtship. The sixth mutant is blocked at the step of attempted copulation which occurs later in the courtship sequence. All of the mutants have normal olfactory responses and normal locomotor activity. No defect in the visual modality has been observed for the five mutants affected in the initiation of courtship. The mutant blocked at attempted copulation lacks the 'on' and 'off' transients, but this appears to be due to genetic background rather than the mutation itself. Abnormal spermatogenesis was observed in five of the mutants. Spermatogenic defects vary and include lesions in the proliferation of the germline, in meiosis, and in the differentiation and maturation of the spermatids into motile sperm.