Inhibition of nicotinic responses of bovine adrenal chromaffin cells by the protein kinase C inhibitor, Ro 31-8220

1. The effects of the protein kinase C inhibitor, Ro 31-8220, on the responses of cultured bovine adrenal chromaffin cells to nicotine, phorbol 12, 13-dibutyrate (PDBu) and K+ have been investigated. 2. Tyrosine hydroxylase activity was measured in situ in intact cells by measuring 14CO2 evolved following the hydroxylation and rapid decarboxylation of [14C]-tyrosine offered to the cells. Secretion of endogenous adrenaline and noradrenaline was measured by use of h.p.l.c. with electrochemical detection. Cyclic AMP levels were measured in cell extracts by RIA. 3. Ro 31-8220 produced a concentration-dependent inhibition of 300 nM PDBu-stimulated tyrosine hydroxylase activity with an IC50 of < 2 microM and complete inhibition at 10 microM. It had no effect on the responses to forskolin. 4. Ro 31-8220 produced a concentration-dependent inhibition of 5 microM nicotine-stimulated tyrosine hydroxylase activity, adrenaline and noradrenaline secretion and cellular cyclic AMP levels, with an IC50 of about 3 microM and complete inhibition by 10 microM. At concentrations up to 10 microM, Ro 31-8220 had little or no effect on the corresponding responses to 50 mm K+. 5. A structural analogue of Ro 31-8220, bisindolylmaleimide V, that lacks activity as a protein kinase C inhibitor, had no effect up to 10 microM on PDBu-stimulated tyrosine hydroxylase activity or on nicotine-stimulated cyclic AMP levels or noradrenaline secretion and only marginal inhibitory effects on nicotine-stimulated tyrosine hydroxylase activity and adrenaline secretion. 6. A structurally related protein kinase C inhibitor, bisindolylmaleimide I, inhibited PDBu-stimulated tyrosine hydroxylase activity with an IC50 of < 1 microM and complete inhibition by 3 microM, but had essentially no effect on nicotine stimulated tyrosine hydroxylase activity or catecholamine secretion. 7. The results suggest that Ro 31-8220 is not only a protein kinase C inhibitor but is also a potent inhibitor of nicotinic receptor responses in adrenal chromaffin cells by a mechanism unrelated to protein kinase C inhibition. The results are consistent with Ro 31-8220 being a nicotinic receptor antagonist.     

Local infiltration of epinephrine and bupivacaine before tonsillectomy

The effects of peritonsillar injections of epinephrine and local anesthetics before tonsillectomy on blood loss and postoperative pain were evaluated in a prospective, randomized double-blind trial on 103 children. Patients were randomly assigned into one of three groups: controls given injections of 0.9% NaCl (n = 34), patients injected with 0.4 ml/kg (1:200,000) epinephrine combined with 0.25% bupivacaine (n = 33) and patients given only 1:200,000 epinephrine (n = 36). All injections and operations were performed by the same surgeon (KS). Blood loss was calculated by weighing all blood aspirated perioperatively and swabs used during surgery. Postoperative pain was assessed at regular intervals by using three methods: (1) use of a visual analogue scale by parents and nurses to estimate pain; (2) postoperative need for nalbuphine as analgesic; (3) the Hannallah-Broadman semi-objective pain score (including crying, anxiety, restlessness, and changes in blood pressure). The mean blood loss in the control group (given NaCl) was 132 g, which was significantly increased when compared with the epinephrine/bupivacaine group (85 g) and the group treated with only epinephrine (90 g). However, analysis of the postoperative pain scores did not reveal any significant differences among groups. These findings indicate that the peritonsillar injection of bupivacaine does not decrease postoperative pain, but peritonsillar injections of epinephrine will significantly reduce blood loss during tonsillectomy.     

Analytical methods and stability assessment of liquid yeast derived sucrase

Two independent analytical methods for determining the activity and stability profile of liquid yeast derived sucrase (YS) were established and validated in order to conduct preliminary stability studies as a function of temperature. The methods included a hexokinase-based (HK) enzymatic assay for determining the formation of glucose upon hydrolysis of sucrose by YS, and a direct polarimetric procedure to quantitate YS hydrolysis of sucrose. Both assays were validated with respect to YS dilution, incubation time, sucrose or glucose concentration, linearity of response and within- and between-day variability. A preliminary stability study was conducted over a 24 week period with liquid YS samples stored at -20, 4, 30, 40 and 50 degrees C. Enzymatic activity was monitored as a function of time using both the HK and polarimetric assays. Liquid YS samples stored at -20, 4 and 30 degrees C retained 100% activity after 24 weeks storage, while the samples stored at 40 degrees C lost approximately 70% activity over the same storage period and samples stored at 50 degrees C lost approximately 95% activity after 12 weeks storage. The two methods of analysis gave consistent results over the course of the study.     

Results of a genome-wide genetic screen for panic disorder

There is some controversy over whether or not a discrete site that integrates vomiting activities in somatic and autonomic nerves is present in the medulla oblongata. On the basis of our previous studies, we hypothesized that the temporal patterns of muscle contractions in vomiting are generated by a central pattern generator in the retrofacial area of the rostral medulla. To investigate this hypothesis further, the effects of electrical and chemical lesions of the medullary area were observed in decerebrate paralyzed dogs. Efferent activities of the phrenic and abdominal muscle nerves were recorded to recognize fictive vomiting. The right half of the medulla oblongata was transversely severed about 3 mm rostral to the obex. Fictive vomiting responses to vagal stimulation still appeared after hemisection in all 11 dogs. In addition, stimulation of the contralateral reticular area dorsomedial to the retrofacial nucleus produced fictive vomiting even after hemisection. An electrical lesion or injection of kainic acid (0.5-1.0 microl) was applied at the point where reticular stimulation induced fictive vomiting. After this destruction, no activities that corresponded to fictive vomiting could be induced by stimulation of vagal afferents or the reticular site. Salivation was decreased by hemisection, and decreased further, but was not completely abolished, with destruction of the reticular area. Kainic acid is known to selectively destroy neural cell bodies. Therefore, we concluded that neuronal somata in the reticular formation dorsomedial to the retrofacial nucleus play an essential role in the central patterning of vomiting activities in peripheral motor nerves.     

Production of androgenetic haploids in zebrafish with ultraviolet light

Anal cancer is more commonly found in homosexual and bisexual men than cervical cancer is in women. Invasive anal cancer may be preceded by anal squamous intraepithelial lesions (ASIL), and treatment of ASIL may prevent the development of anal cancer. We characterized the prevalence and risk factors for ASIL in 346 HIV-positive and 262 HIV-negative homosexual men. Anal cytology, biopsy of visible anal lesions, and human papillomavirus (HPV) tests were performed, and data on HIV serostatus, CD4 count, and medical and lifestyle history were collected. ASIL was diagnosed in 36% of HIV-positive men and 7% of HIV-negative men (relative risk [RR] = 5.7; 95% confidence interval [CI], 3.6-8.9). Among HIV-positive men, the RR for ASIL increased with lower CD4 levels but was elevated even in men with CD4 levels >500/mm3 (RR = 3.8; 95% CI, 2.1-6.7) when compared with HIV-negative men. High-level HPV infection, as measured by detection of both hybrid capture (HC) group A and group B types, was another significant risk factor for ASIL in both HIV-positive men (RR = 8.8; 95% CI, 2.3-35) and HIV-negative men (RR = 20; 95% CI, 5.5-71) when compared with HC-negative men. HIV-negative men with anal HPV infection and HIV-positive men, regardless of CD4 level, are at high risk for ASIL.     

Residents'' corner. Answer to case of the month #58. Megacystis-microcolon-intestinal hypoperistalsis syndrome

The aim of this study was to compare the predictive value of umbilical artery Doppler velocimetry in women in the supine position with that in women in the complete left lateral position as a screening test for abnormal obstetric outcomes. Umbilical artery resistance index (RI) was measured at 27-29 weeks and 35-37 weeks in 202 pregnant women. The measurements were performed with the mother in the supine position in 100 cases (supine group), and in the complete left lateral position in 102 cases (lateral group). Predictive values of the tests for abnormal outcomes (small for gestational age, fetal distress, pregnancy-induced hypertension) were compared between both groups. When abnormal RIs were defined as being greater than the 90th centile in the supine group, the sensitivities for any of the abnormal outcomes at 27-29 weeks were 18% in the supine group and 6% in the lateral group; the positive predictive values were 30% and 25%, respectively. For measurement at 35-37 weeks, the sensitivity and positive predictive value were 29% and 45%, respectively in the supine group, and 0% in both cases in the lateral group. When abnormal RIs were defined as being greater than the 90th centile in the lateral group, the sensitivities at 27-29 weeks were 41% in the supine group and 6% in the lateral group; the positive predictive values were 44% and 8%, respectively. At 35-37 weeks, the sensitivity and positive predictive value were 53% and 43% in the supine group, and 6% and 8% in the lateral group. Umbilical artery Doppler velocimetry when the mother was in the complete left lateral position was of little value as a screening test. However, when the mother is in the supine position, it may serve as a kind of stress test and disclose latent obstetric abnormalities in certain cases.     

Evaluation of mild lameness in horses trotting on a treadmill by clinicians and interns or residents and correlation of their assessments with kinematic gait analysis

OBJECTIVE: To estimate sensitivity and accuracy of subjective evaluation of mild lameness in horses during treadmill locomotion and to correlate subjective evaluation with kinematic analysis. ANIMALS: 19 lame and 5 clinically normal horses. PROCEDURE: Lameness was evaluated by subjective score and kinematic analysis before and after palmar digital nerve block (PDNB). Evaluations were made by 6 clinicians and 7 interns or residents. Within- and between-observer agreement analyses (kappa values) were calculated and compared, using a Student's t-test. Pearson's product-moment correlation coefficients were calculated between clinician's change in score and the change in kinematic variables after PDNB. RESULTS: Within-observer agreement was within the range expected for conditions of moderate diagnostic difficulty. Within-observer agreement was higher for clinicians than for interns or residents. Between-observer agreement was acceptable for scores within 1 value of each other. Between-observer agreement of change in lameness score after PDNB was poor. When kinematic variables were ranked with each clinician's subjective change in score, only 2 were among the top 3 for the majority of clinicians. Asymmetry of vertical head movement between contralateral forelimb stance phases and the point of maximum hoof height during swing decreased as lameness subjectively improved. CONCLUSION: Mild lameness may be difficult to evaluate during treadmill locomotion. Although clinicians were more repeatable in their subjective evaluation of lameness than interns or residents, they were not more reliable at detecting the true state of lameness. CLINICAL RELEVANCE: Lack of agreement between clinician scoring of mild lameness emphasizes the need to use more objective measures for quantifying lameness.     

The role of protonation and metal chelation preferences in defining the properties of mercury-binding coiled coils

To define the delicate interplay between metal chelation, protein folding and function in metalloproteins, a family of de novo-designed peptides was synthesized that self-assemble in aqueous solution to form two and three-stranded alpha-helical coiled coils. Each peptide contains a single Cys residue at an a or d position of the heptad repeat. Peptide association thus produces a Cys-rich coordination environment that has been used to bind Hg(II) ions. These peptides display a pH-dependent association, with trimers observed above the pKa of Glu side-chains and dimers below this value. Finite-difference Poisson-Boltzmann calculations suggest that the dimeric state decreases the unfavorable electrostatic interactions between positively charged Lys side-chains (relative to the trimer). The Cys-containing peptides bind Hg(II) in a position-dependent fashion. Cys at a positions form three-coordinate Hg complexes at high pH where the trimeric aggregation state predominates, and two-coordinate complexes at lower pH. A d position Cys, however, is only able to generate the two-coordinate complex, illustrating the difference in coordination geometry between the two positions in the coiled coil. The binding of Hg(II) was also shown to substantially increase the stability of the helical aggregates.