Surveillance of biting midges (Culicoides spp.) in Northern Ireland: influence of seasonality, surrounding habitat and livestock housing.

Biting midges, Culicoides spp. (Diptera: Ceratopogonidae), are important vectors of viral pathogens. Following the outbreak of bluetongue serotype 8 in Europe between 2006 and 2009, many Culicoides surveillance programmes were initiated to identify vector-active periods, in accordance with European Commission regulation 2007/1266/EC. This study utilized surveillance data from 4 years of continuous light-trapping at 14 sites in Northern Ireland. The number of captured Culicoides varied from none during the vector-free period (December-April) to more than 36 000 per night during peak activity in the summer. The Obsoletus group represented 75% of Culicoides collected and the Pulicaris group represented 21%. A total of 91% of Culicoides were female, of which 42% were parous. Abundance data, sex ratios and parous rates suggested that both the Obsoletus and Pulicaris groups underwent three generations/year. The Obsoletus group was associated with cattle-rearing habitats and woodland, the Impunctatus group was found in habitats related to sheep rearing and the Pulicaris group were associated with both cattle and sheep. Housing did not reduce incursion of female Obsoletus group Culicoides but it did for males and for the Pulicaris group Culicoides. The influence of housing was strongly affected by time of year, probably reflecting the presence of livestock indoors/outdoors.

Sticky-trapping biting midges (Culicoides spp.) alighting on cattle and sheep: effects of trap colour and evidence for host preference.

Sticky traps were mounted on heifers and sheep to assess Culicoides spp. (Diptera: Ceratopogonidae) host preference. Initially, four coloured 200-cm(2) sticky traps (white, clear, yellow and blue) were attached to the backs of each of ten Friesian heifers that were released into open pasture for 24 h, repeated on six occasions. More Obsoletus group Culicoides were caught on the white and clear traps than on the yellow and blue. Trap position on the right or left flank also affected midge catch, probably due to heifer orientation in the field. Next, six Friesian heifers and six Charollais hoggets each had one clear and one white sticky strap attached to their backs for one 24-h period per week, repeated for 24 weeks. Overall, Obsoletus group Culicoides comprised 91.8% (n = 5, 955) of the midge catch but there was no evidence of host preference, either discounting or including host live weight in the analyses. However, Pulicaris group Culicoides did demonstrate a significant host preference for sheep, providing that the analysis was adjusted for live weight. On heifers, the Pulicaris group comprised 7.5% of biting midges caught, whereas on hoggets, it comprised 12.7%.

The beta subunit of eukaryotic translation initiation factor 2 binds mRNA through the lysine repeats and a region comprising the C2-C2 motif.

Eukaryotic translation initiation factor 2 (eIF2) has been implicated in the selection of the AUG codon as the start site for eukaryotic translation initiation, since mutations in its three subunits in yeast that allow the recognition of a UUG codon by the anticodon of the initiator Met-tRNAMet have been identified. All such mutations in the beta subunit of eIF2 (eIF2beta) mapped to a region containing a putative zinc finger structure of the C2-C2 type, indicating that these sequences could be involved in RNA recognition. Another feature of eIF2beta that could mediate an interaction with RNA is located in the amino-terminal sequences and is composed of three repeats of seven lysine residues which are highly conserved in other species. We show here the ability of eIF2beta, purified from Escherichia coli as a fusion to glutathione S-transferase, to bind mRNA in vitro. Through a deletion analysis, mRNA binding was found to be dependent on the lysine repeats and a region encompassing the C2-C2 motif. Strong mRNA binding in vitro could be maintained by the presence of only one lysine or one arginine run but not one alanine run. We further show that only one run of lysine residues is sufficient for the in vivo function of eIF2beta, probably through charge interaction, since its replacement by arginines did not impair cell viability, whereas substitution for alanines resulted in inviable cells. mRNA binding, but not GTP-dependent initiator Met-tRNAMet binding, by the eIF2 complex was determined to be dependent on the presence of the lysine runs of the beta subunit.

Use of a synthetic lethal screen to identify genes related to TIF51A in Saccharomyces cerevisiae.

The putative eukaryotic translation initiation factor 5A (eIF5A) is an essential protein for cell viability and the only cellular protein known to contain the unusual amino acid residue hypusine. eIF5A has been implicated in translation initiation, cell proliferation, nucleocytoplasmic transport, mRNA decay, and actin polarization, but the precise biological function of this protein is not clear. However, eIF5A was recently shown to be directly involved with the translational machinery. A screen for synthetic lethal mutations was carried out with one of the temperature-sensitive alleles of TIF51A (tif51A-3) to identify factors that functionally interact with eIF5A and revealed the essential gene YPT1. This gene encodes a small GTPase, a member of the rab family involved with secretion, acting in the vesicular trafficking between endoplasmatic reticulum and the Golgi. Thus, the synthetic lethality between TIF51A and YPT1 may reveal the connection between translation and the polarized distribution of membrane components, suggesting that these proteins work together in the cell to guarantee proper protein synthesis and secretion necessary for correct bud formation during G1/S transition. Future studies will investigate the functional interaction between eIF5A and Ypt1 in order to clarify this involvement of eIF5A with vesicular trafficking.

Regulation of hepatic receptor-dependent degradation of LDL by mevinolin in rabbits with hypercholesterolemia induced by a wheat starch-casein diet.

Rabbits fed a wheat starch-casein diet develop a marked hypercholesterolemia and have a slower rate of removal of rabbit 125I-labeled low density lipoproteins (LDL) from plasma. Treating rabbits with mevinolin, a highly potent competitive inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase, at a daily dose of 20 mg per animal prevents the increase in plasma and LDL cholesterol. The mevinolin effect is mediated through an increased rate of removal of rabbit 125I-labeled LDL from plasma. To study the role of mevinolin on the regulation of the hepatic LDL receptor in rabbits, the binding of 125I-labeled LDL and 125I-labeled beta-VLDL (beta-migrating very-low-density lipoproteins) to liver membranes prepared from rabbits fed the wheat starch-casein diet with or without mevinolin was investigated. Liver membranes from wheat starch-casein-fed rabbits have no demonstrable EDTA-sensitive binding activity of 125I-labeled LDL and low (37 ng/mg protein) binding activity of 125I-labeled beta-VLDL. Treatment of the wheat starch-casein fed rabbits with mevinolin results in high levels of specific EDTA-sensitive binding of 125I-labeled LDL (28.7 ng/mg protein) and 125I-labeled beta-VLDL (120 ng/mg protein). To assess the functional role of the hepatic LDL receptor in response to mevinolin, the catabolism of 125I-labeled LDL by perfused rabbit livers was studied. Perfused livers from mevinolin-treated rabbits show a 3.3-fold increase in the rate of receptor-dependent catabolism of 125I-labeled LDL (4.6% X h-1) when compared with that of livers from rabbits not treated with mevinolin (1.4% X h-1). Thus, these studies demonstrate that mevinolin prevents the increase of plasma LDL cholesterol level in rabbits fed a wheat starch-casein diet by regulating the levels of hepatic LDL-binding sites and the rate of receptor-dependent catabolism of LDL by the liver.

Peroxisome proliferator-activated receptors gamma and alpha mediate in vivo regulation of uncoupling protein (UCP-1, UCP-2, UCP-3) gene expression.

A role for peroxisome proliferator-activated receptors, PPAR gamma and PPAR alpha, as regulators of energy homeostasis and lipid metabolism, has been suggested. Recently, three distinct uncoupling protein isoforms, UCP-1, UCP-2, and UCP-3, have also been identified and implicated as mediators of thermogenesis. Here, we examined whether in vivo PPAR gamma or PPAR alpha activation regulates the expression of all three UCP isoforms. Rats or lean and db/db mice were treated with PPAR gamma [thiazolidinedione (TZD)] or PPAR alpha (WY-14643) agonists, followed by measurement of messenger RNAs (mRNAs) for UCP-1, UCP-2, and UCP-3 in selected tissues where they are expressed. TZD treatment (AD 5075 at 5 mg/kg x day) of rats (14 days) increased brown adipose tissue (BAT) depot size and induced the expression of each UCP mRNA (3x control levels for UCP-1 and UCP-2, 2.5x control for UCP-3). In contrast, UCP-2 and UCP-3 mRNA levels were not affected in white adipose tissue or skeletal muscle. Chronic (30 days) low-dose (0.3 mg/kg x day) TZD treatment induced UCP-1 mRNA and protein in BAT (2.5x control). In contrast, chronic TZD treatment (30 mg/kg x day) suppressed UCP-1 mRNA (>80%) and protein (50%) expression in BAT. This was associated with further induction of UCP-2 expression (>10-fold) and an increase in the size of lipid vacuoles, a decrease in the number of lipid vacuoles in each adipocyte, and an increase in the size of the adipocytes. TZD treatment of db/db mice (BRL 49653 at 10 mg/kg x day for 10 days) also induced UCP-1 and UCP-3 (but not UCP-2) expression in BAT. PPAR alpha is present in BAT, as well as liver. Treatment of rats or db/db mice with WY-14643 did not affect expression of UCP-1, -2, or -3 in BAT. Hepatic UCP-2 mRNA was increased (4x control level) in db/db and lean mice, although this effect was not observed in rats. Thus, in vivo PPAR gamma activation can induce expression of UCP-1, -2, and -3 in BAT; whereas chronic-intense PPAR gamma activation may cause BAT to assume white adipose tissue-like phenotype with increased UCP-2 levels. PPAR alpha activation in mice is sufficient to induce liver UCP-2 expression.

Beta-very low density lipoproteins in cholesterol-fed rabbits are of hepatic origin.

When rabbits are fed a cholesterol-rich diet they accumulate beta-migrating very low density lipoprotein (beta-VLDL) in their plasma. beta-VLDL are cholesteryl ester-rich lipoproteins which contain apolipoproteins B and E. There are 2 forms of apolipoprotein B in beta-VLDL. About 90% of apolipoprotein B is present as a 320 000-dalton protein and the remainder is present as a 210 000-dalton protein. These apolipoproteins are tissue specific. Lipoproteins secreted by perfused rabbit livers contain only the 320 000-dalton apolipoprotein B while lipoproteins secreted by the intestine contain only the 210 000-dalton apolipoprotein B. The tissue specificity of apolipoprotein B shows that beta-VLDL is largely of hepatic origin and that only a small fraction is of intestinal origin. The composition of VLDL secreted from the livers of cholesterol-fed rabbits is similar to that of plasma beta-VLDL. Both are cholesteryl ester-rich, in contrast to plasma and perfusate VLDL from normal rabbits which are both triglyceride-rich. This indicates that the cholesteryl ester-rich hepatic VLDL is a direct precursor for plasma beta-VLDL.

Convergence of multiple sensory inputs onto neurons in the dorsolateral medulla in cats.

The dorsolateral medulla, including the nucleus reticularis parvicellularis, the cuneate nucleus, and the external cuneate nucleus, is an integrative region for a variety of sensory inputs. The purpose of this study was to determine whether individual neurons respond to a variety of different sensory modalities. To this end, responses of 40 neurons in the dorsolateral medulla to multiple sources of sensory input were assessed in cats anesthetized with alpha-chloralose. Neurons were located in the nucleus reticularis parvicellularis (24 cells, 60%), the cuneate nucleus (10 cells, 25%), and the external cuneate nucleus (6 cells, 15%). All neurons were tested for responses to: electrical stimulation of afferents coursing through the left stellate ganglion and afferents in the left cervical vagus nerve, and somatic, auditory, and visual stimulation. No neurons responded to all five stimuli. Three cells (7.5%) responded to four stimuli, 11 (27.5%) responded to three stimuli, 10 (25.0%) responded to two stimuli, and 15 (37.5%) responded to only a single stimulus. The remaining cell was unresponsive to any stimulus. As a group, neurons in the nucleus reticularis parvicellularis received input from the greatest number of sensory modalities, and cuneate nucleus neurons received input predominantly from somatosensory afferents. External cuneate nucleus neurons displayed response profiles intermediate between nucleus reticularis parvicellularis and cuneate nucleus. In addition, eight neurons (20% of the total) were sensitive to changes in blood pressure. Results of the present study support the hypothesis that neurons in the nucleus reticularis parvicellularis receive convergent inputs from different sensory modalities.2+ behaviors.

Some central effects in mice of compounds related to nicotine.

1. Some hydroxy-, amino-, and methoxy- phenylalkyltrimethylammonium compounds, beta-pyridylmethyl- dimethylamine and pyrrolidine, and beta-pyridylethyltrimethylammonium, were tested on avoidance learning in mice and their effects were compared with those of (-)-nicotine.2. The o- and m- hydroxybenzyl-, o-hydroxyphenethyl- and m-hydroxyphenylpropyl- trimethylammonium compounds improved performance; (-)-nicotine, in one-quarter of the dose, had similar effects. The m- and p-hydroxyphenethyl-, o-hydroxyphenylpropyl- and o- and p- aminobenzyl, and o-, m-, and p- aminophenethyl-trimethylammonium compounds impaired performance.3. (-)-Nicotine and m-hydroxyphenylpropyltrimethylammonium appeared also to enhance memory consolidating processes.4. The central actions of some of the compounds suggest that the possibility that they can penetrate into the central nervous system should not be ruled out even though they are quaternary salts.5. No correlation was found between the effects of the compounds on avoidance learning and on the frog rectus muscle. Though the differences may be due to differences in access to the central nervous system, it is also possible that the receptors associated with learning processes are different from those in the frog rectus and possibly more specialized.

The affinity and activity of compounds related to nicotine on the rectus abdominis muscle of the frog (Rana pipiens).

1. Series of pyridylalkyl- and substituted phenylalkyl-trimethylammonium salts, triethylammonium salts, diethylamines and di-n-propylamines have been made. The substituents in the benzene ring were nitro, chloro, bromo, methoxy, hydroxy and amino groups and the alkyl residues had one, two, or three methylene groups separating the aromatic nucleus from the cationic head.2. Most of the trimethylammonium compounds caused a contracture of the frog rectus muscle, but some were partial agonists and a few were antagonists. The di-n-propylamines were all antagonists, as were most of the diethylamines and triethylammonium compounds, though some of these were partial agonists and a few triethylammonium compounds were agonists. The affinities of the antagonists and partial agonists for the receptors stimulated by beta-pyridylmethyltrimethylammonium (and by nicotine) were measured. The equipotent molar ratios of all the agonists were measured relative to beta-pyridylmethyltrimethylammonium.3. The dissociation constants of the pyridylmethyldiethylamines and substituted benzyldiethylamines were measured. The effects of substituents on the pK(a) of benzyldiethylamine were similar to their effects on the pK(a) of aniline, though there were differences with some of the o-substituted compounds, which could be attributed to internal hydrogen-bond formation.4. There is no obvious correlation between the effects of a substituent on the pK(a) of benzyldiethylamine and its effects on affinity. Although increasing the size of the cationic group usually increased affinity, it did not always do so. The compounds with the highest affinity, p-hydroxybenzyldiethylamine (log K, 5.90) had about half the affinity of (+)-tubocurarine (log K, 6.11), but the triethylammonium analogue (log K, 4.17) had only about one-fiftieth of the affinity of the tertiary base. The binding of the drug to the receptor appears to involve many factors which include the size of the groups as well as their electron-releasing or withdrawing nature and other properties, such as their polar and lipophilic or lipophobic character.5. There is no obvious correlation between the effects of a substituent on the affinity of the diethylamino or triethylammonium compounds and its effects on the activity of the trimethylammonium analogue. The most active compounds contain hydroxy- and amino-, phenyl or beta-pyridyl groups, m-hydroxyphenyl-propyltrimethylammonium being about 50 times as active as nicotine, but the corresponding diethylamino or triethylammonium compounds do not have high affinity. There does not seem necessarily to be an inverse relationship between activity and affinity, however, because some m-nitro and m-chloro trimethylammonium compounds have considerable activity and the analogous triethylammonium compounds have considerable affinity.6. It is suggested that ability to activate these receptors is associated with the presence of substituents which can interact with water molecules which may be involved in the action of the drug at the receptor.

Ophthalmologic features of thallium poisoning.

Thallium intoxication is characterized by the development of painful peripheral neuropathy, alopecia, mental disorders, and in severe cases, respiratory failure and death. Toxic optic neuropathy is also a feature. Ophthalmologic features of thallium poisoning include optic neuropathy, blepharoptosis, lens opacities, and ophthalmoplegia. A 44-year-old man with criminal sublethal thallium poisoning was examined one month after he was seen in the neurology department with classic systemic features. He was found to have diminished contrast sensitivity, a tritan defect in color vision, and a relative cecocentral scotoma before he developed optic atrophy.

Mutation analysis of the Cys-X2-Cys-X19-Cys-X2-Cys motif in the beta subunit of eukaryotic translation initiation factor 2.

Recessive lethal mutations in the beta subunit of eIF-2 that restore HIS4 expression in the absence of an AUG start codon were isolated from diploid Saccharomyces cerevisiae strains. DNA sequence analysis of these alleles and of eIF-2 beta suppressor alleles isolated from haploid strains, identified point mutations that altered one of six amino acids that map to a Cys-X2-Cys-X19-Cys-X2-Cys "zinc finger" motif and immediately adjacent residues. Five of the affected amino acids are identical in the human and yeast eIF-2 beta protein. Together with earlier studies (Donahue et al., 1988), these point mutations implicate the zinc finger domain of eIF-2 beta in start-site selection during the scanning process. We have supplemented the mutations obtained by genetic selection with an additional set of constructed mutations in this region. Our studies indicate that the cysteine residues and the intervening amino acids of this motif are essential for eIF-2 beta function in translation initiation in vivo. However, the effects observed in cells containing a copy of eIF-2 beta with a deletion of this motif suggest that this mutated form is still able to associate with other components of the initiation complex, imparting defects on translation initiation. Thus, this motif may be required only for later events that lead to initiator codon recognition. Alterations in defined positions, as found in our suppressor alleles, could lead to recognition of non-AUG codons.

Morphological changes in the human corneal epithelium associated with surgical corneal clouding.

Light microscopy and electron microscopy were used to define morphological changes occurring in human corneal epithelium during surgery. No correlation was established between preoperative medication or peroperative fluids and corneal epithelial clouding. It is suggested that the changes we observed in relation to epithelial clouding were the direct result of disturbance to endothelial pump functions.

Radiation retinopathy following treatment of posterior nasal space carcinoma.

Posterior nasal space carcinoma has a high mortality and most patients are treated with radiotherapy. Radiation retinopathy was encountered in 7 out of 10 survivors included in this study. Five of the affected patients lost vision as a result of the retinopathy. One patient required laser photocoagulation and responded well to this treatment. There was a variation in the severity of the retinopathy among the patients studied despite the fact that all patients received a similar dose of radiotherapy. We suspect that previously unrecognised factors in the planning of radiotherapy fields may explain this difference.

Cytological factors relating to posterior capsule opacification following cataract surgery.

Simulated extracapsular cataract extractions on cadaver eyes were performed which demonstrated that the cells of the anterior capsule remain largely intact and that only a small amount of cortical lens matter remains postoperatively. Human lens epithelial cells from normal and cataractous lenses were grown in culture. There was no appreciable difference in growth rate between cells from normal and those from cataractous lenses or between equatorial and central capsule cells. The cells grew from the cut edges of the capsule, suggesting that release from contact inhibition is an important factor in stimulating proliferation. The daughter cells became increasingly abnormal and metaplastic in successive generations, but there was no evidence of differentiation into fibroblasts within the 35-day culture period, suggesting that a retinal growth factor may be involved with the fibrosis occurring in opacification of the posterior capsule. A small anterior capsulotomy will release fewer cells from contact inhibition and hence reduce cell proliferation after extracapsular cataract extraction.

The human anterior lens capsule--an attempted chemical debridement of epithelial cells by ethylenediaminetetracetic acid (EDTA) and trypsin.

The addition of edetic acid (EDTA) or trypsin to the infusion during a simulated extracapsular cataract extraction on cadaver eyes facilitates the removal of lens epithelial cells from the anterior capsule. Modification of the chemical composition of infusions used during extracapsular surgery may maximise lens epithelial cell removal and hence reduce the incidence of opacification of the posterior capsule after cataract extraction.

Desensitization of beta3-adrenergic receptor-stimulated adenylyl cyclase activity and lipolysis in rats.

The beta3-adrenergic receptor is an integral membrane protein consisting of seven transmembrane domains. Unlike the beta1 and beta2 receptors, this subtype lacks the consensus phosphorylation sites required for desensitization by serine kinases. Using the rodent specific beta3 agonist BRL 35135, our initial data indicated that beta3 receptor-mediated glycerol levels progressively decreased following daily oral doses of 5 mg/kg. Therefore, we initiated studies designed to delineate the possible mechanism(s) for this decreased response. Within 3 hours following a single oral dose of BRL 35135, serum glycerol levels and UCP (uncoupling protein) RNA levels were significantly increased whereas beta3 RNA levels were significantly decreased. Rats were dosed daily for 5 days with either vehicle or BRL 35135 (5 mg/kg, p.o.) and blood samples were collected for glycerol analysis. Adipose tissue was excised for lipolysis and adenyl cyclase measurements. In addition, UCP and beta3 receptor RNA levels were assessed. No effect on adipocyte BRL 37344-stimulated adenylyl cyclase activity was observed 3 hours following the initial dose of BRL 35135. Although a slight decrease (approximately 25%) in adenylyl cyclase activity could be observed 24 hours following the initial dose, it wasn't until day 4 of dosing that a significant decrease (50%) was observed. In contrast, beta3- stimulated lipolysis in adipocytes from BRL 35135-treated rats was decreased 85% within 24 hours and this decrease persisted through four days of treatment. These data indicate that the lipolytic response to beta3 receptor activation is decreased after only a single oral dose of BRL 35135, whereas receptor-mediated adenylyl cyclase activation, although initially unaffected, also desensitizes by day four of treatment.

An outbreak of methicillin resistant Staphylococcus aureus on a burn unit: potential role of contaminated hydrotherapy equipment.

OBJECTIVE: To report a multi-institution outbreak caused by a single strain of methicillin-resistant Staphylococcus aureus (MRSA). OUTBREAK: Between September 19 and November 20, 1996 an index case and five secondary cases of nosocomial MRSA occurred on a 26 bed adult plastic surgery/burn unit (PSBU) at a tertiary care teaching hospital. Between November 11 and December 23, 1996, six additional cases were identified at a community hospital. One of the community hospital cases was transferred from the PSBU. All strains were identical by pulsed-field gel electrophoresis. MRSA may have contributed to skin graft breakdown in one case, and delayed wound healing in others. Patients required 2 to 226 isolation days. CONTROL MEASURES: A hand held shower and stretcher for showering in the hydrotherapy room of the PSBU were culture positive for the outbreak strain, and the presumed means of transmission. Replacement of stretcher showering with bedside sterile burn wound compresses terminated the outbreak. The PSBU was closed to new admissions and transfers out for 11 days during the investigation. Seven of 12 patients had effective decolonization therapy. CONCLUSION: Environmental contamination is a potential source of nosocomial MRSA transmission on a burn unit. Notification among institutions and community care providers of shared patients infected or colonized with an antimicrobial resistant microorganism is necessary.