K+ channel cAMP activated in guinea pig gallbladder epithelial cells

In guinea pig gallbladder epithelial cells, an increase in intracellular cAMP levels elicits the rise of anion channel activity. We investigated by patch-clamp techniques whether K(+) channels were also activated. In a cell-attached configuration and in the presence of theophylline and forskolin or 8-Br-cAMP in the cellular incubation bath, an increase of the open probability (P(o)) values for Ca(2+)-activated K(+) channels with a single-channel conductance of about 160 pS, for inward current, was observed. The increase in P(o) of these channels was also seen in an inside-out configuration and in the presence of PKA, ATP, and cAMP, but not with cAMP alone; phosphorylation did not influence single-channel conductance. In the inside-out configuration, the opioid loperamide (10(-5) M) was able to reduce P(o) when it was present either in the microelectrode filling solution or on the cytoplasmic side. Detection in the epithelial cells by RT-PCR of the mRNA corresponding to the alpha subunit of large-conductance Ca(2+)-activated K(+) channels (BK(Ca)) indicates that this gallbladder channel could belong to the BK family. Immunohistochemistry experiments confirm that these cells express the BK alpha subunit, which is located on the apical membrane. Other K(+) channels with lower conductance (40 pS) were not activated either by 8-Br-cAMP (cell-attached) or by PKA + ATP + cAMP (inside-out). These channels were insensitive to TEA(+) and loperamide. The data demonstrate that under conditions that induce secretion, phosphorylation activates anion channels as well as Ca(2+)-dependent, loperamide-sensitive K(+) channels present on the apical membrane.     

Rational design of a CD4 mimic that inhibits HIV-1 entry and exposes cryptic neutralization epitopes

The conserved surfaces of the human immunodeficiency virus (HIV)-1 envelope involved in receptor binding represent potential targets for the development of entry inhibitors and neutralizing antibodies. Using structural information on a CD4-gp120-17b antibody complex, we have designed a 27-amino acid CD4 mimic, CD4M33, that presents optimal interactions with gp120 and binds to viral particles and diverse HIV-1 envelopes with CD4-like affinity. This mini-CD4 inhibits infection of both immortalized and primary cells by HIV-1, including primary patient isolates that are generally resistant to inhibition by soluble CD4. Furthermore, CD4M33 possesses functional properties of CD4, including the ability to unmask conserved neutralization epitopes of gp120 that are cryptic on the unbound glycoprotein. CD4M33 is a prototype of inhibitors of HIV-1 entry and, in complex with envelope proteins, a potential component of vaccine formulations, or a molecular target in phage display technology to develop broad-spectrum neutralizing antibodies.     

POCUS: mining genomic sequence annotation to predict disease genes

Here we present POCUS (prioritization of candidate genes using statistics), a novel computational approach to prioritize candidate disease genes that is based on over-representation of functional annotation between loci for the same disease. We show that POCUS can provide high (up to 81-fold) enrichment of real disease genes in the candidate-gene shortlists it produces compared with the original large sets of positional candidates. In contrast to existing methods, POCUS can also suggest counterintuitive candidates.     

A novel disorder caused by defective biosynthesis of N-linked oligosaccharides due to glucosidase I deficiency

Glucosidase I is an important enzyme in N-linked glycoprotein processing, removing specifically distal alpha-1,2-linked glucose from the Glc3Man9GlcNAc2 precursor after its en bloc transfer from dolichyl diphosphate to a nascent polypeptide chain in the endoplasmic reticulum. We have identified a glucosidase I defect in a neonate with severe generalized hypotonia and dysmorphic features. The clinical course was progressive and was characterized by the occurrence of hepatomegaly, hypoventilation, feeding problems, seizures, and fatal outcome at age 74 d. The accumulation of the tetrasaccharide Glc(alpha1-2)Glc(alpha1-3)Glc(alpha1-3)Man in the patient's urine indicated a glycosylation disorder. Enzymological studies on liver tissue and cultured skin fibroblasts revealed a severe glucosidase I deficiency. The residual activity was <3% of that of controls. Glucosidase I activities in cultured skin fibroblasts from both parents were found to be 50% of those of controls. Tissues from the patient subjected to SDS-PAGE followed by immunoblotting revealed strongly decreased amounts of glucosidase I protein in the homogenate of the liver, and a less-severe decrease in cultured skin fibroblasts. Molecular studies showed that the patient was a compound heterozygote for two missense mutations in the glucosidase I gene: (1) one allele harbored a G-->C transition at nucleotide (nt) 1587, resulting in the substitution of Arg at position 486 by Thr (R486T), and (2) on the other allele a T-->C transition at nt 2085 resulted in the substitution of Phe at position 652 by Leu (F652L). The mother was heterozygous for the G-->C transition, whereas the father was heterozygous for the T-->C transition. These base changes were not seen in 100 control DNA samples. A causal relationship between the alpha-glucosidase I deficiency and the disease is postulated.     

Tubulin carboxypeptidase assay based on the action of the enzyme on [14C]tyrosinated tubulin bound to nitrocellulose membrane

We have developed a method for the determination of tubulin carboxypeptidase activity which is based on the action of the enzyme on the substrate, [14C]tyrosinated tubulin, previously adsorbed on nitrocellulose membrane. In addition to being two to three times more sensitive than previous carboxypeptidase assays, this method allows the determination of dilute enzyme preparations even containing high salt (inhibitory) concentrations. This is a valuable property specially under circumstances in which numerous high salt-containing fractions with scarce activity should be analyzed (for example after certain chromatographic stages during enzyme purification). Our method is simpler, less time-consuming, and suitable for multiple, simultaneous determinations and the substrate bound to nitrocellulose can be stored for several months without significant alteration of its properties. Peptidases other than tubulin carboxypeptidase can act on [14C]tyrosinated tubulin bound to nitrocellulose, solubilizing radioactive compounds, suggesting the eventual applicability of this method to assay proteases in general. Other features and advantages of the assay as well as its limitations are discussed.     

Multicenter evaluation of the phosphorylcholine-coated biodivYsio stent in short de novo coronary lesions: The SOPHOS study

AIMS: The BiodivYsio trade mark stent (Biocompatibles Ltd, Farnham, UK) is coated with a phosphorylcholine (PC)-containing copolymer to confer biocompatibility. The SOPHOS (Study Of PHosphorylcholine coating On Stents) study was designed to assess the safety and efficacy of this novel coronary stent and by indirect comparison to indicate equivalence with other formal stent studies. METHODS AND RESULTS: Patients with angina and a single short (#x2A7F;12 mm) de novo lesion in a native coronary artery of >/=2.75 mm diameter were included. A total of 425 patients were allocated in 24 centers. Clinical data were collected at one-, six- and nine-month follow-up. Angiography was performed before and after the stent implantation. In addition, in the first 200 patients (SOPHOS A) angiography was routinely performed at six months. The following 225 patients (SOPHOS B) were merely followed up clinically. The primary end-point of the study, the six-month MACE-rate (MACE = Major Adverse Cardiac Events) was 13.4% (two cardiac death; five Q-wave/nine non-Q-wave myocardial infarctions (MI); nine CABG and 32 target lesion revascularization (TLR), which is similar to the calculated 15% MACE-rate in comparable reference studies. Secondary end-points included among others restenosis at six months in the SOPHOS A population. The target vessel diameter was 2.98 +/- 0.48 mm. Minimal lumen diameter pre/post procedure and at follow-up was 1.00 +/- 0.32, 2.69 +/- 0.37, 1.91 +/- 0.71 mm, respectively. The binary restenosis rate (>/=50% diameter stenosis at follow-up) was 17.7%. CONCLUSION: The coronary BiodivYsio stent is safe and effective as a primary device for the treatment of native coronary artery lesions in patients with stable or unstable angina pectoris. Clinical and angiographic results are in the statistical range of equivalence with comparable studies with other current stents.     

A laser-based method for measuring thermal nociception of cattle

We describe a method for measuring nociception in cattle using a CO(2) laser aimed at the caudal aspect of the metatarsi. In Experiment 1, infrared thermography showed that calves responded by lifting their legs when skin temperatures reached 45-55 degrees C. In Experiment 2a, the validity of the method was tested by comparing the response latencies of 14 calves to two power settings (2.25 W vs. 4.5 W) with each setting being applied six times. We found that both leg-lift latencies and tail-flick latencies were lower at the higher power setting, and the calves were more likely to respond by kicking than by simply moving the leg. The standard deviations between and within calves were smaller at the higher power setting, and the large within-calf variation means that at least three tests were required to obtain reliable measures that could differentiate between calves. In Experiment 2b, application of the laser at a range of power settings (2.0, 3.0, 4.0, 4.5, 5.0 and 5.5 W) on 16 calves showed that response latencies decreased as power increased up to 4.5 W, after which no further change occurred. In Experiment 3, the repeatability of the method was evaluated on nine measures with the high power setting (4.5 W). The coefficient of variation associated with repetition of the measures was 36%. In general, we found little change in response latencies with repeated use of the laser, except that responses on the second test tended to be shorter. Experiment 4 showed that ambient temperatures between 16 degrees C and 27 degrees C did not affect response latencies, but these were longer at temperatures of 7 degrees C. We suggest that the method is a useful way of measuring cattle's sensitivity to nociception as the animals need not be restrained and the distance to the animal need not be closely controlled. However, to obtain accurate, valid and reliable measures it is necessary to use a high power setting (4.5 W) and take at least three consecutive measures of the response latency.     

Plasma levels of GH and PRL and concentrations in the fluids of bovine ovarian cysts and follicles

Prolactin and GH have been detected within the ovary, and it has become increasingly evident that they have a role as intrafollicular regulatory factors. The aim of the present work was to gain an insight into the elements influencing intraovarian GH and PRL in bovine species and to see whether cystic degeneration was accompanied by abnormal bovine GH (bGH) and PRL (bPRL) plasma patterns. We followed the relationships between plasma and ovarian fluid bGH and bPRL concentrations over an entire year in Friesian cows whose ovaries showed distinct types of structures. To assess the presence of bGH and bPRL within ovarian cells, we assayed selected ovarian structures by immunohistochemistry. The results demonstrated that: 1) plasma and ovarian fluid hormonal concentrations were independent, and their ratio was independent of the ovarian structure classes, subclasses and period of the year; 2) in the majority of the cows the concentration of bGH in ovarian fluid was no more than 80% of the level in plasma, whereas in about half the animals bPRL concentrations were higher in the ovary than in peripheral plasma; 3) mean bPRL concentrations in ovarian fluids were significantly higher in summer than in winter; 4) immunoreactive bGH and bPRL were present within granulosa and luteal cells. Thus, it is suggested that in the cow bGH and bPRL levels in the ovary might be regulated in some way independently of the pituitary.     

INCREASED HARASSMENT OF RIGHT WHALES (EUBALAENA AUSTRALIS) BY KELP GULLS (LARUS DOMINICANUS) AT PENÍNSULA VALDÉS, ARGENTINA

Kelp gulls at Península Valdés, Argentina, have recently developed the habit of feeding on pieces of skin and blubber that they gouge from the backs of southern right whales. In response, the whales flinch violently, submerge, and swim rapidly away underwater. The level of harassment in 1995 was almost five times higher than when first studied in 1984 by Thomas (1988). In 1995, 67% of attacks were aimed at large white lesions on the whales' backs. The proportion of whales with lesions increased from 0.01 in 1974 to 0.32 in 1990. Mother-calf pairs that were attacked traveled at medium and fast speeds for 3.1 h per day, compared to 0.8 h for undisturbed pairs. Mother-calf pairs are estimated to spend approximately 24% of their daylight hours in states of gullinduced disturbance. Little food is available at Península Valdés, so mothers must rely on blubber reserves to support their calves' growth, behavioral development, and migration to the feeding grounds. Even when undisturbed by gulls, mothers often curtail their calves' play and nursing bouts, suggesting that their energy reserves are limited. Increasingly intense harassment by gulls may therefore compromise calf development and might even induce right whales to abandon Península Valdés for other calving grounds.