In vitro hypoglycemic and antimicrobial activities of Senecio leucanthemifolius Poiret.

This study reports on the alpha-amylase inhibitory and antimicrobial activities of Senecio leucanthemifolius Poiret. Extracts of S. leucanthemifolius were tested for their antimicrobial and antifungal activities against seven different pathogenic microorganisms using the microdilution technique. The ethyl acetate extract exhibited a strong antibiotic activity against Staphylococcus aureus with a MIC value of 31.25 microg mL(-1), while the n-hexane extract showed a significant activity against dermatophytic fungi. S. leucanthemifolius extracts were tested also for their potential hypoglycemic activity through the in vitro inhibition of alpha-amylase. The dichloromethane extract inhibited alpha-amylase with a value of 56.6% at 0.05 mg mL(-1) and the n-butanol extract showed a value of 89.2% at 1 mg mL(-1).

Adsorption of radioiodine on platinum: a fast and simple column method to obtain concentrated and pure radioiodide in either water or anhydrous solvents.

Radioiodine can be adsorbed on a small column filled with platinum powder from an acidified aqueous solution. The adsorption is nearly quantitative, irrespective of the oxidation state of the iodine used. With an alternated flow of hydrogen gas and solvent, the iodine can be desorbed from the platinum into an aqueous or organic solvent. Depending on the solvent used, the desorption process is also nearly quantitative, and the eluate obtained contains almost pure radioiodide. Using this method, labelling reactions with radioiodide are no longer restricted to water-stable substrates and catalysts.

Persistent efficacy of doramectin topical solution against induced infestations of Bovicola bovis and Solenopotes capillatus.

A study was conducted in two locations, Wyoming and Wisconsin, USA, to evaluate the persistent efficacy of doramectin topical solution at a dose rate of 500 microg/kg body weight against artificially induced infestations of Bovicola bovis and Solenopotes capillatus on cattle. At each location, lice-free beef calves were individually housed and randomly allocated to treatment. Both B. bovis and S. capillatus were transferred from untreated donor animals to doramectin-treated cattle at the end of 35, 63, 91 or 126 day post-treatment periods. Cattle treated with a saline pour-on served as the control. Based on the geometric means of lice counts 2 weeks following transfer, the persistent efficacy of a single treatment with doramectin topical solution against induced infestations of B. bovis was 100.0, 100.0, 99.5, and 100.0% at post-treatment days of 35, 63, 91, and 126, respectively. Persistent efficacy against induced infestations of S. capillatus, for the same intervals, were 100.0, 94.9, 86.3, and 74.9%.

The effectiveness of doramectin pour-on in the control of gastrointestinal nematode infections in cow-calf herds.

Two field studies were conducted in the USA to determine the efficacy of a single strategically-timed dose of doramectin pour-on in the control of gastrointestinal nematodosis in beef cow-calf herds and the resultant effects on calf productivity. One study was carried out between May and October 1996 in a spring-calving herd at a site located in Idaho (ID) and the other between January and July 1997 in a fall-calving herd at a site located in Mississippi (MS). In each study, cow-calf pairs were randomly allotted by sex of calf to pastures and one of two treatment groups (doramectin pour-on at the recommended dose rate of 500 microg/kg body weight or untreated control). There were four pasture replicates per treatment at each site. Each pasture contained 12 cow-calf pairs at the ID site and 15 cow-calf pairs at the MS site. Treatment was administered to cows and calves on 21 May 1996 (ID) or 23 January 1997 (MS). Following treatment, cow-calf pairs were assigned to their designated pastures where they remained until the calves were weaned 140 (ID) or 168 (MS) days later. Cow and calf fecal egg counts and calf body weights were recorded on treatment day and then at monthly intervals until study termination. Doramectin treatment reduced nematode egg output in cows and calves over the entire grazing season compared to untreated controls and resulted in calf weight gain improvements of 9.8kg (p=0.295) at the ID site and 17.4kg (p=0.0002) at the MS site.

The effectiveness of a single dose of doramectin pour-on in the control of gastrointestinal nematodes in yearling stocker cattle.

Three field studies were conducted to determine the efficacy of a single dose of doramectin pour-on in the control of gastrointestinal nematode infections in yearling stocker calves on pasture. These 140-day studies were carried out between October 1995 and March 1996 in Tennessee (TN), between January and June 1997 in Louisiana (LA), and between May and September 1997 in Wisconsin (WI). Calves with patent nematode infections were equally allocated to treatments (doramectin pour-on, at 500 microg/kg body weight or untreated control) and pastures as randomized complete-block designs (LA and TN studies) or completely at random (WI study). There were six pasture replicates per treatment at each site, with each pasture replicate accommodating six calves at the TN site (36 calves per treatment), five calves at the LA site (30 calves per treatment), and seven calves at the WI site (42 calves per treatment). Fecal samples for nematode egg counts were collected on Day 0, and at 28-day intervals thereafter. Body weights were recorded on Day 0 and at 28-day intervals until study termination. Nematode egg output of the doramectin-treated groups was reduced over the entire grazing period compared to those in the untreated control groups, resulting in average daily weight-gain advantages of 0.055 kg (p < or = 0.05) for the TN study, 0.208 kg (p < or = 0.05) for the LA study, and 0.116 kg (p < or = 0.05) for the WI study.

A capsaicin-receptor homologue with a high threshold for noxious heat.

Pain-producing heat is detected by several classes of nociceptive sensory neuron that differ in their thermal response thresholds. The cloned capsaicin receptor, also known as the vanilloid receptor subtype 1 (VR1), is a heat-gated ion channel that has been proposed to mediate responses of small-diameter sensory neurons to moderate (43 degrees C) thermal stimuli. VR1 is also activated by protons, indicating that it may participate in the detection of noxious thermal and chemical stimuli in vivo. Here we identify a structurally related receptor, VRL-1, that does not respond to capsaicin, acid or moderate heat. Instead, VRL-1 is activated by high temperatures, with a threshold of approximately 52 degrees C. Within sensory ganglia, VRL-1 is most prominently expressed by a subset of medium- to large-diameter neurons, making it a candidate receptor for transducing high-threshold heat responses in this class of cells. VRL-1 transcripts are not restricted to the sensory nervous system, indicating that this channel may be activated by stimuli other than heat. We propose that responses to noxious heat involve these related, but distinct, ion-channel subtypes that together detect a range of stimulus intensities.

Comparison of the persistent efficacy of the injectable and pour-on formulations of doramectin against artificially-induced infection with Dictyocaulus viviparus in cattle.

The persistent efficacy of the injectable and topical formulations of doramectin was compared against experimental challenges with infective larvae of Dictyocaulus viviparus in two separate studies. Four groups of 10 randomly-assigned calves, negative for lungworm larvae by the Baermann technique, were used in each study. Calves were treated subcutaneously in the midline of the neck or poured down the midline of the back with saline (1 ml/50 kg. injection: 1 ml/10 kg. pour-on) on Day 0 or doramectin (200 microg/kg = 1 ml/50 kg. injection: 500 microg/kg = 1 ml/10 kg. pour-on) on Day 0, 7, or 14. Two additional calves from the same pool of animals were randomly assigned as larval-viability monitors and received no treatment. Calves were inoculated daily with a gavage of approximately 100 larvae of D. viviparus from days 35 to 49 for the injectable study and days 28 to 42 for the pour-on study. The two larval viability monitor calves received approximately 3000 infective larvae in the same manner on Day 49 or 42 for the injectable and pour-on studies, respectively. Equal numbers of calves from each treatment group as well as the larval viability monitor calves were necropsied on days 14 and 15 after the last lungworm inoculation to enumerate the worm burden. The worms recovered were quantified and identified. For each study, geometric mean worm recoveries for each treatment group were back transformed from the natural log-transformed data (worm count +1) and were used to estimate percentage reduction. Doramectin injectable solution was 100.0% efficacious against lungworms for up to 49 days and the pour-on formulation was 100.0%, 93.1% and 81.5% effective in reducing lungworm infection resulting from challenge infection for up to 28, 35, and 42 days post-treatment, respectively.

ATP receptors in sickness, pain and death.

Extracellular ATP elicits biological responses ranging from cell death to synaptic transmission. Recent gene-cloning efforts have uncovered a family of cell-surface ATP receptors, which are potential targets for the development of novel drugs to treat airway and cardiovascular diseases, inflammation and pain.

Signaling by extracellular nucleotides.

ATP and other nucleotides can be released from cells through regulated pathways, or following the loss of plasma membrane integrity. Once outside the cell, these compounds take on new roles as intercellular signaling molecules that elicit a broad spectrum of physiological responses through the activation of numerous cell surface receptor subtypes. This review summarizes recent advances in the molecular characterization of ATP receptors and discusses roles for cloned receptors in established and novel physiological processes.

New structural motif for ligand-gated ion channels defined by an ionotropic ATP receptor.

The adenosine-5'-triphosphate (ATP) molecule is an extracellular messenger in neural and non-neural tissues, where it activates several cell-surface-receptor subtypes, including G-protein-coupled receptors and ligand-gated ion channels. ATP-gated channels (termed P2x receptors) have been characterized on smooth muscle cells and autonomic and sensory neurons, where they mediate membrane depolarization and, in some cases, Ca2+ entry. P2x receptors are functionally heterogeneous, but resemble acetylcholine- and serotonin-gated channels with respect to ion selectivity and kinetic parameters of channel gating. We report here that despite such close functional similarities, the deduced sequence of a cloned P2x receptor predicts an unusual subunit structure resembling voltage-insensitive cation channels. Thus, the P2x receptor provides a striking example of convergent evolution, whereby proteins have been fashioned with similar functional properties from subunits having very different structural characteristics. There is sequence similarity between the ATP receptor and RP-2, a gene activated in thymocytes undergoing programmed cell death. RP-2 may encode a receptor for ATP or another metabolite released during apoptosis.

Utilization of cryopreserved semen in tumor patients.

Treatment with cytotoxic drugs or with radiation in order to manage a tumor or another life-threatening disease involves a potential hazard to male fertility. In these cases cryopreservation of semen is recommended. However, the number of patients requesting the subsequent insemination of their partner is rather low. It would be of interest if patients with a high probability for desiring use of the cryodeposit for insemination could be identified. During the years 1985-1992 we performed cryopreservation in 29 patients attending our department. One year following cryopreservation the utilization of the cryodeposit was analyzed: 29 patients were not interested in further maintenance, 2 patients died, 3 patients requested use for insemination, 31 patients decided to maintain the semen further in a commercial cryobank, 17 patients had a complete restitution of spermatogenesis within the observation period. In 7 patients the interval is yet below 1 year. The different modes of utilizing the cryodeposit were analyzed in relation to the semen quality, age, status, kind of disease and primary treatment. None of these factors possibly influencing the utilization showed differences between the groups. We conclude that it is impossible to predict the probability of the use of a cryodeposit of semen based on the examined patient characteristics at the time of preservation. We plan to further on offer semen preservation to all patients requiring it in a situation of threatened fertility, bearing in mind that the relative costs of the cryopreservation are low.

The variability of measuring sperm concentration and motility as determined by computer assisted image analysis and visual estimation.

Systems of computer-assisted image processing (CAIP) offer good reproducibility and low intra-assay variability between replicate analyses of the same sample. The correlation between these values and those obtained by directly microscopic observation can be well demonstrated. The comparability between systems of different manufacturers is sufficient and satisfactory. Thus, they are well-suited for the routine work in the andrologic laboratory. Since sperm parameters are used for the prediction of male fertility, it is important to know whether computer-assisted analysis improves the predictive power as compared to visual estimation. In our study we compared the variation of semen parameters between two semen samples of the same individual taken in an interval of at least 3 months, by using the CAIP and visual methods. Significantly close correlations between the two values were obtained by both methods. Values of cell concentration showed the highest correlation in both methods (r = 0.75). The lowest correlation, but also statistically significant, was observed in the values of locally motile cells (r = 0.54 for CAIP and r = 0.30 for visual determination). Although apparently higher in CAIP within method correlation coefficients did not show statistically meaningful differences between both methods. We conclude from our study that if values of sperm concentration and motility as measured by CAIP are compared to those obtained by visual measurement, the first method is not superior to the latter in terms of retest reliability.

Expression cloning of an ATP receptor from mouse neuroblastoma cells.

Extracellular ATP activates cell-surface metabotropic and ionotropic nucleotide (P2) receptors in vascular, neural, connective, and immune tissues. These P2 receptors mediate a wealth of physiological processes, including nitric oxide-dependent vasodilation of vascular smooth muscle and fast excitatory neurotransmission in sensory afferents. Although ATP is now recognized as a signaling molecule, the cellular and molecular mechanisms underlying its actions have been difficult to study due to the absence of selective P2 receptor antagonists and cloned receptor genes. Nonetheless, five mammalian P2 receptor subtypes have been tentatively assigned based solely on agonist specificity and signaling properties. Here we report the cloning of a mouse cDNA encoding a P2 receptor that shares striking homology with several G protein-coupled peptide receptors. When expressed in Xenopus laevis oocytes, the cloned receptor resembles a metabotropic P2U receptor; activation by either ATP or UTP elicits the mobilization of intracellular calcium. mRNA encoding the P2U purinergic receptor is found in neural and nonneural tissues.

Digestion, feed intake, and live weight gain by cattle consuming bermudagrass and supplemented with different grains.

The objectives of this study were to determine whether type of supplemental cereal grain, with a relatively high level of supplementation, affects feed intake, characteristics of digestion, and live weight gain by cattle consuming bermudagrass (BER). In Exp. 1, five beef steers (423 +/- 22 kg average BW) with cannulas in the rumen and duodenum were used in a Latin square design experiment. Steers consumed BER hay (1.5% BW; 10.1% CP, 75% NDF, and 6% ADL) alone (Control) or with approximately .7% BW (DM) of ground corn (GC), whole corn (WC), ground sorghum grain (SG), or ground wheat (W). At 8 h after supplementation, ruminal pH was lower for W than for Control, WC, and SG (P < .05). True ruminal OM digestion was lowest for SG and highest for W (P < .05; 49.4, 50.7, 51.0, 42.0, and 57.3% for Control, GC, WC, SG, and W, respectively). In Exp. 2, five Holstein steer calves (187 +/- 9 kg average BW) were used in a Latin square design. Bermudagrass hay (9.7% CP, 72% NDF, and 6% ADL) was consumed ad libitum alone (Control) or with approximately 1% BW of grain (same as in Exp. 1). Digestible OM intake was similar among grain treatments. In Exp. 3, 96 crossbred beef steers (256 +/- 2 kg initial BW) grazed BER (clipped forage samples: 13 to 16% CP, 68 to 73% NDF, and 4 to 5% ADL) for 85 d and received the same grain treatments as in Exp. 2 plus a barley (B) treatment. Live weight gain was .47, .84, .80, .68, .81, and .51 kg/d for Control, GC, WC, B, SG, and W, respectively (SE = .028). In conclusion, when growing cattle grazing BER were supplemented once daily with grain at approximately 1.0% BW, grain that degraded in the rumen slowly (GC, WC, and SG) resulted in live weight gain greater than that resulting from grain that degraded rapidly (B and W).

Primary structure and functional expression of the 5HT3 receptor, a serotonin-gated ion channel.

The neurotransmitter serotonin (5HT) activates a variety of second messenger signaling systems and through them indirectly regulates the function of ion channels. Serotonin also activates ion channels directly, suggesting that it may also mediate rapid, excitatory responses. A complementary DNA clone containing the coding sequence of one of these rapidly responding channels, a 5HT3 subtype of the serotonin receptor, has been isolated by screening a neuroblastoma expression library for functional expression of serotonin-gated currents in Xenopus oocytes. The predicted protein product has many of the features shared by other members of the ligand-gated ion channel family. The pharmacological and electrophysiological characteristics of the cloned receptor are largely consistent with the properties of native 5HT3 receptors. Messenger RNA encoding this receptor is found in the brain, spinal cord, and heart. This receptor defines a new class of excitatory ligand-gated channels.

Relationships of body weight, forage composition, and corn supplementation to feed intake and digestion by Holstein steer calves consuming bermudagrass hay ad libitum.

Holstein steer calves (101 to 350 kg BW) consumed bermudagrass hay ad libitum without or with supplemental ground corn up to 1.0% of BW. As BW increased, total DMI increased quadratically (-2.459 + .05448 [BW]-.000073 [BW2] + .540 [corn DMI]; R2 = .83, sy.x = .655). Each kilogram of corn DM decreased bermudagrass DMI by .46 kg. Total digestible OM intake (kg) increased with BW and corn supplementation (.314 + .0127 [BW] + .441 [corn OM intake]; R2 = .79, sy.x = .444). Feed intake level accounted for approximately 2.5 times more variability in total digestible OM intake than digestion did. Corn supplementation decreased digestion of bermudagrass NDF (62.50 - 8.468 [corn DMI, % BW]; R2 = .13, sy.x = 8.121), with a similar decrease across BW. Increasing bermudagrass DMI (% of BW) decreased bermudagrass NDF digestion slightly, but variation accounted for was only 33% of that attributable to corn DMI (% of BW). Concentrations of common fiber fractions (NDF, ADF, cellulose, hemicellulose, and ADL) in bermudagrass explained very little variation in feed intake and digestion, indicating considerable influence of other factors. Bermudagrass intake and digestion were not related, and no substantial interactions were observed among steer BW, corn level, and bermudagrass composition.

cDNA and gene structure for a human subtilisin-like protease with cleavage specificity for paired basic amino acid residues.

A cDNA encoding the human fur gene product was isolated from a human hepatoma cell line. The cDNA encodes a protein with significant amino acid sequence identity to the prokaryotic subtilisin family of serine proteases. More extensive sequence identity was found when the protein was compared with eukaryotic proteases such as PRB1 of Saccharomyces cerevisiae, and with PC2 and PC3, the only other known mammalian subtilisin-like proteases. In contrast to these proteins, however, the fur gene product shares a more extensive topographic and functional homology with the KEX2 endoprotease of S. cerevisiae. Each protease contains a signal peptide, a glycosylated extra cytoplasmic domain, a hydrophobic membrane-spanning region, and a short, hydrophilic "tail" sequence. As with KEX2, the expressed human protease was shown to cleave mammalian proproteins at their paired basic amino acid processing sites. We have, therefore, proposed the function-based acronym PACE (paired basic amino acid cleaving enzyme) for this prototypic mammalian proprotein processing enzyme.

Expression of a human proprotein processing enzyme: correct cleavage of the von Willebrand factor precursor at a paired basic amino acid site.

Intracellular proteolytic processing of precursor polypeptides is an essential step in the maturation of many proteins, including plasma proteins, hormones, neuropeptides, and growth factors. Most frequently, propeptide cleavage occurs after paired basic amino acid residues. To date, no mammalian propeptide processing enzyme with such specificity has been purified or cloned and functionally characterized. We report the isolation and functional expression of a cDNA encoding a propeptide-cleaving enzyme from a human liver cell line. The encoded protein, called PACE (paired basic amino acid cleaving enzyme), has structural homology to the well-characterized subtilisin-like protease Kex2 from yeast. The functional specificity of PACE for mediating propeptide cleavage at paired basic amino acid residues was demonstrated by the enhancement of propeptide processing of human von Willebrand factor when coexpressed with PACE in COS-1 cells.