Amphiphysin is necessary for organization of the excitation-contraction coupling machinery of muscles, but not for synaptic vesicle endocytosis in Drosophila.

Amphiphysins 1 and 2 are enriched in the mammalian brain and are proposed to recruit dynamin to sites of endocytosis. Shorter amphiphysin 2 splice variants are also found ubiquitously, with an enrichment in skeletal muscle. At the Drosophila larval neuromuscular junction, amphiphysin is localized postsynaptically and amphiphysin mutants have no major defects in neurotransmission; they are also viable, but flightless. Like mammalian amphiphysin 2 in muscles, Drosophila amphiphysin does not bind clathrin, but can tubulate lipids and is localized on T-tubules. Amphiphysin mutants have a novel phenotype, a severely disorganized T-tubule/sarcoplasmic reticulum system. We therefore propose that muscle amphiphysin is not involved in clathrin-mediated endocytosis, but in the structural organization of the membrane-bound compartments of the excitation-contraction coupling machinery of muscles.

Control of hepatitis B in the United Kingdom.

Control of hepatitis B in the UK is based upon selective vaccination of persons in high-risk groups. To assess the likely cost-effectiveness of changes to this policy, information on the current burden of HBV infection in the UK is required. Laboratory reports of acute hepatitis B suggest that the vast majority of new hepatitis B infections acquired in the UK occur in adults, even after adjustment for unapparent infection. In childhood, perinatal transmission remains the most significant known risk factor. Universal antenatal screening has the potential to prevent perinatal infections in UK births and a substantial proportion of those UK acquired infections which lead to carriage. In addition, to antenatal screening, universal infant vaccination (at 2, 3 and 4 months) can, in the short term, only prevent the small number of infections acquired in childhood. Economic analysis using current surveillance data is required to assess the possible cost-benefit of universal vaccination. Regardless of this, there is an urgent need to improve selective vaccination and to ensure that a high proportion of antenatal carriers is identified.

Wild type and constitutively activated forms of the Drosophila Toll receptor have different patterns of N-linked glycosylation.

Toll is a Drosophila membrane protein related in sequence to the mammalian platelet glycoprotein 1B and to the interleukin-1 receptor. It mediates a signal transduction pathway leading to the development of dorsoventral polarity in the Drosophila embryo. In this paper we show that a constitutively activated mutant receptor, Toll10B, is processed into a distinct isoform of slower electrophoretic mobility when compared with the wild type molecule in both cell lines and the embryo. The wild type protein can also be processed into this form if over-expressed but in the embryo is present as the smaller species. We show that the decrease in the mobility of Toll10B and over-expressed wild type receptors is caused by altered patterns of N-linked glycosylation and that both forms are secreted to the cell surface. On the basis of these results, we propose that the Toll10B receptor is unable to associate with a limiting co-factor which when bound directly or indirectly masks supplementary N-linked glycosylation sites.

n-3 fatty acid deficiency increases brain protein synthesis in the free-moving adult rat.

The autoradiographic method with L-[35S]methionine was used to determine the effects of an n-3 fatty acid deficiency on brain protein synthesis. Brain protein synthesis was significantly increased (from 50 to 150%) in 45 of the 52 brain structures studied in n-3 fatty acid-deficient rats as compared with control animals. Biochemical analysis confirmed the increase in overall rate of protein synthesis in brain as a whole.

Bovine plasma vitamin A responses to selected nutrients, monensin, and endophytic fescue.

Previous research at this station adapted a maximal dose response (MDR) method of evaluating vitamin A status and utilization for use in beef cattle. This method was used in two experiments. In this first experiment, forty-eight crossbred steers (average weight, 284 kg) were fed diets supplemented with salt, monensin or both, and injected with vitamin E, zinc or selenium. Steers receiving monensin had higher (38.5 micrograms/dl, monensin; 31.0 micrograms/dl, controls) initial plasma concentrations of vitamin A (P = .14). However, monensin did not affect post-dosing (MDR) vitamin A concentrations. None of the other dietary treatments or injections affected either pre- or post-dosing concentrations of vitamin A in the plasma. In a second experiment 23 lactating multiparous beef cows (average weight, 500 kg) grazing either fungal endophyte-infected or endophyte-free tall fescue were used to assess possible influences of infected fescue upon vitamin A metabolism. Fungal endophyte infection did not affect either pre- (44.9 micrograms/dl, end-noninf.; 47.7 micrograms/dl, end-inf.) or post-dosing (57.2 micrograms/dl, end-noninf.; 59.3 micrograms/dl, end-inf.) vitamin A concentrations.

Progressive increases of protein synthesis in the circumventricular organs during chronic dehydration in rats.

The quantitative autoradiographic method with L-(35S)methionine was applied to investigate the effect of chronic dehydration on rates of protein synthesis in circumventricular organs (CVOs). Water deprivation for 1, 2 and 3 days causes progressive increases of protein synthesis in the subfornical organ (SFO), the area postrema, the organum vasculosum laminae terminalis and the neurohypophysis. Chronic salt ingestion with 2% NaCl in drinking water for 3 days resulted in increases of protein synthesis in the CVOs similar to those found after 3 days water deprivation, with only one exception, the SFO, in which the rise in protein synthesis was of lower amplitude after 3 days salt ingestion as compared to 3 days water deprivation. These results suggest that several circulating factors related to intracellular dehydration and the high plasma levels of the neurohormones vasopressin and oxytocin are probably important determinants of the rise of protein synthesis in circumventricular organs. Alternatively, the elevated level of blood-borne angiotensin II may well explain the higher metabolic response of the SFO following water deprivation compared to salt ingestion.

Induction of tall fescue toxicosis in heat-stressed cattle and its alleviation with thiamin.

Livestock grazing endophyte (Acremonium coenophialum)-infected tall fescue (Festuca arundinacea Schreb. cv. Kentucky 31) at high ambient temperatures may suffer from fescue toxicosis. Adult Angus cows (Bos taurus) were fed 0 to 1 kg/d of 70% infected tall fescue seed containing about 4.4 g of loline alkaloids in factorial combination with thiamin at 0 or 1 g/d. Cows assigned to the zero level of tall fescue seed received a supplement of equivalent energy and protein. Ingestive behavior was measured at 1330 to 1430 EDT during two 4-d periods in two consecutive weeks in August on alfalfa (Medicago sativa L.) pastures using a tethered grazing system. Alfalfa intake per measured grazing session of cows given tall fescue seed declined linearly (P less than .01) as air temperatures during grazing increased above 25 degrees C, largely because of shorter grazing meals. Thiamin increased alfalfa intake per measured grazing session by extending grazing time. Alkaloids in ingested endophyte-infected tall fescue induce thiamin deficiencies in cattle that result in symptoms of tall fescue toxicosis.

Decreased protein synthesis in hypothalamic nuclei following L-5-hydroxytryptophan in intact and p-chlorophenylalanine-pretreated rats.

The rate of protein synthesis was estimated in individual hypothalamic nuclei by a quantitative autoradiographic technique with L-[35S]methionine. The i.v. administration of 60 mg/kg L-5-hydroxytryptophan (40 min before) resulted in a 45-55% decrease of overall protein synthesis rate in all the hypothalamic nuclei examined. In rats pretreated (42 h before) with a single i.v. injection of 280 mg/kg p-chlorophenylalanine, a drug which is known to deplete brain serotonin concentration, the administration of 60 mg/kg L-5-hydroxytryptophan resulted in a 50-75% decrease of protein synthesis rates in the hypothalamic nuclei. These results suggest that the systemic administration of large doses of L-5-hydroxytryptophan may inhibit protein synthesis in hypothalamic nuclei directly or indirectly after the conversion of this compound to serotonin.

Bioavailability of magnesium in beef cattle fed magnesium oxide or magnesium hydroxide.

Two experiments were conducted to compare Mg bioavailability from Mg oxide (MgO) vs Mg hydroxide (Mg(OH)2) fed in either a completely mixed diet or a mineral supplement. In Exp. 1, these Mg sources were incorporated into completely mixed diets and offered to 15 steers (282 kg) allotted to three treatments: control diet containing .19% Mg, control plus .2% added Mg as MgO, or control plus .2% added Mg as Mg(OH)2. Each calf was fed 5 kg/d of the respective diet during 10-d adjustment and 7-d collection periods. Blood samples were collected on d 1, 3 and 7. Mg supplementation increased (P less than .01) fecal and urinary Mg excretions, whereas apparent Mg absorption (%) and retention were similar (P greater than .10) for all treatments. Plasma Mg concentrations were similar (P less than .10) for calves supplemented with MgO and Mg(OH)2 but were higher (P less than .05) for Mg supplemented than for control calves on d 7. In Exp. 2, these Mg sources were incorporated into mineral supplements and offered free choice to 30 spring-calving beef cows gazing tetany-inducing pastures from March 6 to May 1. Each of three groups of 10 cows was assigned to a 5.7-ha tall fescue pasture and offered either a control supplement or a supplement containing 40% MgO or Mg(OH)2. Blood samplers were collected on d 0, 7, 14, 28, 42 and 56. Plasma Mg concentrations were not different (P greater than .10) for cows offered MgO and Mg(OH)2 but were higher (P less than .01) for Mg-supplemented than for control cows on d 28, 42 and 56.(ABSTRACT TRUNCATED AT 250 WORDS)

Performance and plasma amino acids of growing calves fed corn silage supplemented with ground soybeans, fishmeal and rumen-protected lysine.

A 100-d growth study was conducted to evaluate performance and plasma amino acid (AA) responses of 96 crossbred beef calves (220 kg) with ad libitum access to corn silage and supplemented with ground soybeans (GSB) with or without added fishmeal (FM) and (or) rumen-protected lysine (Lys). Calves were allotted by breed, sex and weight to four treatments with three replicate pens of eight calves per pen. The treatments were: GSB, GSB + Lys, GSB + FM and GSB + FM + Lys. The isonitrogenous supplements were top-dressed on corn silage once daily at a level of 2.27 kg/hd, with FM providing one-half of the supplemental N in FM-containing supplements. The Lys-containing supplements provided a daily intake of 6.0 g/hd of rumen-protected Lys. Dry matter intake was similar (P less than .10) for all treatments. Overall ADG and feed efficiency of GSB calves averaged .83 kg/d and 7.39 kg feed/kg gain, respectively, and were 14% lower than the mean of calves fed supplements containing FM and(or) Lys. Lysine was not the principal factor limiting growth because the inclusion of Lys alone in the GSB-containing supplements did not improve (P greater than .10) ADG, feed efficiency or plasma AA concentrations. In contrast, FM supplementation increased (P less than .05) ADG, feed efficiency and plasma concentrations of total AA, total essential AA and total nonessential AA. The inclusion of Lys in the GSB + FM-containing supplement resulted in no further improvement (P greater than .10) in performance.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasma vitamin A levels in cattle in response to large doses of vitamin A.

A dose response procedure was developed for evaluating vitamin A status and utilization in cattle. This could be useful for evaluating diets, vitamin A stores and homeostatic control of vitamin A. Three experiments were designed to determine: 1) size of vitamin A dose required to increase plasma vitamin A concentrations; 2) time after dosing when plasma vitamin A concentrations peak, and 3) if changes in plasma vitamin A concentrations are useful in assessing dietary vitamin A utilization. Using twenty-four steers and heifers, the first two experiments showed that 30X or 40X daily vitamin A requirement (daily requirement calculated as X = body weight in kilograms times 55 International Units of vitamin A) given orally was sufficient to cause plasma vitamin A concentrations to rise 20 hours after dosing. Plasma vitamin A concentrations increased (P less than .01) from 57 to 81 micrograms/dl and 64 to 84 micrograms/dl after a 30X or 40X treatment, respectively. Twenty-X daily requirement resulted in a slight increase after 20 hours (P greater than .05). Seventy-two feedlot steers were used in experiment three to determine if diet affects vitamin A dose response. Variations in feed intake, monensin in the diet or a single dose of vitamin E did not cause significant changes in plasma vitamin A response to vitamin A dosage. Steers receiving monensin had higher initial plasma vitamin A concentrations, but no differences were observed after dosing. Results indicate that dose responses may be useful in assessing dietary vitamin A utilization.

Long-lasting insomnia induced by preoptic neuron lesions and its transient reversal by muscimol injection into the posterior hypothalamus in the cat.

In order to analyse the role of the anterior hypothalamus in the regulation of the sleep-waking cycle we made bilateral neuronal lesions at different levels of the anterior hypothalamus in cats, by means of microinjections of a cell-specific neurotoxin:ibotenic acid. These lesions resulted in severe insomnia in eight cats. This insomnia was characterized by a large decrease or even disappearance of paradoxical sleep and deep slow wave sleep and, to a lesser extent, by a decrease of light slow wave sleep, for 2-3 weeks. In the other five animals, we observed a large reduction of deep slow wave sleep (0-40% of control level), but a less intensive decrease of time spent in paradoxical sleep (50-75% of control level) and no marked effect on light slow wave sleep. During the first 3-6 postoperative days we also noticed hyperthermia in all cats; thereafter, the animals presented only a slight increase in brain temperature which did not appear to trigger the sleep impairment. Histological analysis of the different lesions revealed that the insomnia could be attributed to neuronal cell body destruction in the mediobasal part of the anterior hypothalamus covering; the medial preoptic area and a narrow portion of the lateral preoptic area as well as a restricted part of the anterior hypothalamic nucleus. In order to investigate the putative role of the posterior hypothalamic structures in the mechanism of insomnia after lesion of the mediobasal preoptic area neurons we injected an agonist of GABA into the ventrolateral part of the posterior hypothalamus to locally depress the neuronal activity. The bilateral intracerebral microinjection of muscimol (0.5-5 micrograms) induced a transient intensive hypersomnia (slow wave sleep and paradoxical sleep). These findings indicate that neuronal cell loss in the mediobasal preoptic area induced a long lasting insomnia. Thus, it may be hypothesized that the integrity of this structure is necessary for sleep appearance. Finally, our data are in keeping with an intrahypothalamic regulation of the sleep-waking cycle.

Primary structure and subunit stoichiometry of F1-ATPase from bovine mitochondria.

The enzyme complex F1-ATPase has been isolated from bovine heart mitochondria by gel filtration of the enzyme released by chloroform from sub-mitochondrial particles. The five individual subunits alpha, beta, gamma, delta and epsilon that comprise the complex have been purified from it, and their amino acid sequences determined almost entirely by direct protein sequence analysis. A single overlap in the gamma-subunit was obtained by DNA sequence analysis of a complementary DNA clone isolated from a bovine cDNA library using a mixture of 32 oligonucleotides as the hybridization probe. The alpha, beta, gamma, delta and epsilon subunits contain 509, 480, 272, 146 and 50 amino acids, respectively. Two half cystine residues are present in the alpha-subunit and one in each of the gamma- and epsilon-chains; they are absent from the beta- and delta-subunits. The stoichiometry of subunits in the complex is estimated to be alpha 3 beta 3 gamma 1 delta 1 epsilon 1 and the molecular weight of the complex is 371,135. Mild trypsinolysis of the F1-ATPase complex, which has little effect on the hydrolytic activity of the enzyme, releases peptides from the N-terminal regions of the alpha- and beta-chains only; the C-terminal regions are unaffected. Sequence analysis of the released peptides demonstrates that the N terminals of the alpha- and beta-chains are ragged. In 65% of alpha-chains, the terminus is pyrrolidone carboxylic acid; in the remainder this residue is absent and the chains commence at residue 2, i.e. lysine. In the beta-subunit a minority of chains (16%) have N-terminal glutamine, or its deamidation product, glutamic acid (6%), or the cyclized derivative, pyrrolidone carboxylic acid (5%). A further 28% commence at residue 2, alanine, and 45% at residue 3, serine. The delta-chains also are heterogeneous; in 50% of chains the N-terminal alanine residue is absent. The sequences of the alpha- and beta-chains show that they are weakly homologous, as they are in bacterial F1-ATPases. The sequence of the bovine delta-subunit of F1-ATPase shows that it is the counterpart of the bacterial epsilon-subunit. The bovine epsilon-subunit is not related to any known bacterial or chloroplast H+-ATPase subunit, nor to any other known sequence. The counterpart of the bacterial delta-subunit is bovine oligomycin sensitivity conferral protein, which helps to bind F1 to the inner mitochondrial membrane.(ABSTRACT TRUNCATED AT 400 WORDS)

Labile magnesium reserves in beef cows subjected to different prepasture supplementation regimens.

Thirty pregnant beef cows were utilized to determine the effects of winter Mg supplementation regimen on blood minerals after cows were turned to a spring tetanigenic tall fescue pasture. The winter Mg treatments were (1) tall fescue hay with free access to a Mg-deficient salt-mineral mix, (2) 6.4 kg of corn silage dry matter plus 114 g MgO/d and (3) tall fescue hay with free access to a salt-mineral mix containing 40% MgO from January 1 to February 15. All cows were then placed on the same tetanigenic pasture from February 15 to April 17 with free access to a Mg-deficient salt-mineral supplement. Forage Mg remained below .2% during the experiment. Forage Ca, P and Al changed throughout the spring, attaining maximum values of .35%, .46% and 415 ppm, respectively. Forage N and K also increased throughout the spring, reaching values of 3.5 and 3.8%, respectively, at the April 3 sampling. The forage K:(Ca + Mg) ratio approached 2.2 by March 26, which coincided closely with the average tetany date (March 29). Serum Mg averaged 1.97, 3.58 and 2.06 mg/dl for treatments 1, 2 and 3, respectively, on February 15 before turning cows to pasture. There were no treatment differences for serum Ca, P and K during the experiment. Eight cows exhibited symptoms of grass tetany (collapse) on an average date of March 29. Winter Mg supplementation provided little long-term protection against hypomagnesemia after turning cows to tetanigenic pasture, indicating that cows must have a supplemental source of Mg during this critical period.

Selenium and sulfur supplementation to steers grazing tall fescue.

Forty steer calves averaging 257 kg were allotted to a randomized complete block design experiment containing 10 animals per treatment. Four tall fescue pastures of 5.7 ha each were utilized during the 84-d grazing study. Each pasture treatment block contained 10 calves. Calves were fed a control supplement of 96.9% ground corn and 3.1% trace mineral salt in two pastures, or a sulfur supplement that contained 94.3% ground corn, 3.1% trace mineral salt, and 2.5% elemental sulfur in the remaining two pastures. Each supplement was fed at the rate of 0.45 kg/head daily. One-half of the calves in each pasture were injected with 15 mg selenium (Se) as sodium selenite initially and at 28-d intervals throughout the trial. The treatments were as follows: (1) control-no sulfur, no selenium; (2) selenium, no sulfur; (3) sulfur, no selenium and; (4) selenium plus sulfur. Calf weights were monitored and certain blood parameters measured every 28 d. Average plasma Se values were increased (P < 0.001) from 0.021 ppm in calves not receiving Se to 0.043 ppm in those receiving Se. Plasma urea nitrogen levels were not affected by Se administration, but were lowered (P <0.05) from 16.6 to 15.8 mg/100 mL by sulfur administration. All plasma amino acids measured, except methionine, phenylalanine, argi-nine, and citrulline, were cleared from the blood in response to Se supplementation as reflected by their lower concentrations when compared with other treatment groups. Overall average daily gains were not significantly (P > 0.10) increased by sulfur supplementation, but were increased (P < 0.05) from 0.33 kg/d in calves not receiving Se to 0.43 kg/d in those receiving Se injections. Significant interactions between fulfur and Se were not noted.