Effects of diet source and vaccination for porcine circovirustype 2 and Mycoplasma hyopneumoniae on nursery pig performance.

Two experiments were conducted to evaluate the effect of nursery diet sources, porcine circovirustype 2 (PCV2) and Mycoplasma hyopneumoniae (M. hyo) vaccines, and vaccination timing on pig (Sus scrofa) performance. In Exp. 1, a total of 400 pigs (5.6 BW, 1.03 kg SD) were used in a 20-d study. Treatments were arranged in a 4 × 2 factorial in a blocked design (5 pigs/pen and10 pens/treatment), with main effects of diet manufacturing source (A, B, C, or D) and vaccination timing (d 0 or 8). On either d 0 (weaning) or 8, pigs received 2 vaccines (Circumvent PCV, Intervet/Schering-Plough Animal Health, Millsboro, DE; and RespiSure One, Pfizer Animal Health, New York, NY). A pre-determined amount of segregated early weaning (SEW) diet (0.45 kg/pig) was fed followed by a transition diet until d 8, and a common diet from d 8 to 20. Diet source affected (P < 0.001) ADG during the first 4 d and affected (P ≤ 0.02) ADG and ADFI from d 4 to 8. There were no differences (P ≥ 0.18) among diet sources once pigs were fed a common diet (d 8 to 20). Overall, diet source did not affect ADG; but ADFI tended (P = 0.06) to be decreased for pigs fed Diet C compared with those fed Diets A, B, and D. Pigs vaccinated on d 0 had decreased (P ≤ 0.01) ADG and ADFI (d 4 to 8 and d 0 to 8), resulting in lighter (P = 0.003) BW on d 8 than those of pigs not yet vaccinated (d 8). However, overall ADG was not affected by vaccination timing. In Exp. 2, 360 pigs (5.9 SD, 0.91 kg BW) were used in a 35-d trial to evaluate the effects of different vaccines. Treatments were arranged in a 3 by 2 factorial in a blocked design (5 pigs/pen and 12 pens/treatment). Main effects included PCV2 vaccine (none; CircoFLEX, Boehringer Ingelheim Vetmedica Inc., St. Joseph, MO; or Circumvent PCV); with or without M. hyo vaccine (RespiSure, Pfizer Animal Health, New York, NY). Overall, pigs vaccinated with Circumvent PCV had decreased (P < 0.02) ADG and ADFI compared with CircoFLEX-vaccinated or control pigs. On d 35, pigs vaccinated with Circumvent PCV weighed less (P < 0.01) than CircoFLEX-vaccinated or control pigs. RespiSure-vaccinated pigs had decreased (P ≤ 0.05) ADG compared with control pigs from d 14 to 21 and d 21 to 29. On d 35, RespiSure-vaccinated pigs tended (P = 0.06) to weigh and consume less than control pigs. These data indicate diet source and vaccination timing affects pig performance after weaning. Vaccination for PCV2 and M. hyo independently reduced ADG and ADFI, but the effect was product-dependent.

Regulation of myosin light chain phosphorylation by RhoB in neuronal cells.

The phosphorylation of myosin light chain (MLC) is a key regulatory point in the control of cellular morphology. Evidence suggests that RhoA-a member of the Rho GTPase family-regulates MLC phosphorylation via Rho kinase (ROK). Neurones display subtle alterations in their cytoarchitecture during the synaptic plasticity following high-frequency stimulation. We have recently demonstrated that RhoB, and not RhoA, is activated in neurones by high-frequency stimulation. However, the downstream consequences of RhoB activation in cells are unclear. In this study, we tested the hypothesis that RhoB might stimulate neuronal MLC phosphorylation. Transfection of PC12 cells with constitutively active RhoB increased MLC phosphorylation. Conversely, dominant-negative RhoB vectors reduced MLC phosphorylation. The effect of RhoB was attenuated by pretreatment with a selective ROK inhibitor. This confirms that Rho GTPases are important regulators of MLC phosphorylation, but suggests that, in neuronal cells, the control is exerted via RhoB rather than RhoA.

Increased long-term potentiation in the CA1 region of rat hippocampus via modulation of GTPase signalling or inhibition of Rho kinase.

There is accumulating evidence that Ras, and Ras-related GTPases of the Rho family, such as RhoA, RhoB and Rac1, are involved in synaptic plasticity in brain regions such as the hippocampus. We have recently shown that Rho family GTPases are activated by synaptic transmission in the CA1 region of the hippocampus. Since the function of these GTPases is dependent on post-translational isoprenylation by either farnesyl or geranylgeranyl transferases, we tested the hypothesis that inhibition of isoprenylation would modify long-term potentiation (LTP). Farnesyl transferase inhibition, which suppressed activation of RhoB and Ras but not RhoA or Rac1, reduced the magnitude of LTP, while geranylgeranyl transferase inhibition, which inhibited RhoA and Rac1 but not RhoB, increased the magnitude of LTP. In addition, Y-27632, a specific inhibitor of a downstream effector of Rho GTPases-Rho-kinase-also increased the magnitude of LTP. This provides strong evidence that GTPases are important mediators of synaptic plasticity, and demonstrates that Rho-kinase acts to reduce the degree of plasticity at hippocampal synapses during LTP. Rho-kinase inhibitors have the unusual property of increasing the magnitude of LTP, and so may be potential cognitive enhancers.

Activation of Rho GTPases by synaptic transmission in the hippocampus.

Ras-related GTPases of the Rho family, such as RhoA and RhoB, are well-characterised mediators of morphological change in peripheral tissues via their effects on the actin cytoskeleton. We tested the hypothesis that Rho family GTPases are involved in synaptic transmission in the CA1 region of the hippocampus. We show that GTPases are activated by synaptic transmission. RhoA and RhoB were activated by low frequency stimulation, while the induction of long-term potentiation (LTP) by high frequency stimulation was associated with specific activation of RhoB via NMDA receptor stimulation. This illustrates that these GTPases are potential mediators of synaptic transmission in the hippocampus, and raises the possibility that RhoB may play a role in plasticity at hippocampal synapses during LTP.

Distribution of Rho family GTPases in the adult rat hippocampus and cerebellum.

Small GTPases are monomeric guanine nucleotide binding proteins of 20-25 kDa mass. Rho GTPases belong to the Ras superfamily of small GTPases. The small GTPases of the Rho family have been shown to participate in the organisation of the actin cytoskeleton and signal transduction pathways leading to gene transcription. Recent evidence suggests that Rho family GTPases may play an important role in synaptic communication in the brain, and particularly in synatic plasticity. In this study the distribution of RhoA, RhoB, RhoG, Cdc42, and Rac1 was investigated in hippocampal and cerebellar tissue of adult rat brain using immunohistochemical techniques. Previous studies suggest that distribution of Rho family mRNA is uniform throughout these structures. Here we provide evidence for differences in expression of these proteins between different regions of the hippocampus, and between the molecular and granular layers in the cerebellum. These differences may prove important with regard to the physiological functions of Rho family GTPases.

Characterisation of ATP-induced facilitation of transmission in rat hippocampus.

Superfusion of rat hippocampal slices with ATP induces a form of facilitation that has been poorly characterised. The present study has confirmed that at low concentrations of ATP (10 microM or less), an initial depression of evoked potential size is followed by a rebound facilitation which is not reproduced by alphabeta-methyleneATP, betagamma-methyleneATP, or the dinucleotide P1,P6-diadenosine hexaphosphate. The post-ATP facilitation could be prevented by the adenosine A1 receptor antagonists 8-phenyltheophylline or 1,3-dipropyl-8-cyclopentyltheophylline (50 nM), or superfusion of adenosine deaminase. The adenosine A2A receptor antagonist 8-(chlorostyryl)-caffeine did not affect the inhibition but prevented the post-ATP facilitation. The NMDA receptor antagonist 2-amino-5-phosphonopentanoic acid prevented the establishment of post-ATP facilitation. The post-ATP facilitation was also blocked by suramin at a concentration (50 microM) that does not block glutamate receptors. Suramin prevented the induction but not the maintenance phase of the post-ATP facilitation. The repeated induction of post-ATP facilitation by bursts of electrical stimulation designed to saturate the normal mechanisms of long-term potentiation prevented the induction of post-ATP facilitation. However, repeated applications of ATP to achieve saturation of its receptor did not prevent the subsequent induction of electrically evoked long-term potentiation. It is concluded that ATP can induce a form of synaptic facilitation which resembles only partially that induced by electrical stimulation and which may require the simultaneous activation of P1 and P2 receptors.

Presynaptic P2 receptors?

Although the emphasis in ATP research has been on postjunctional receptors, there is also evidence for presynaptic receptors regulating transmitter release in the autonomic nervous system. Recent work has attempted to identify similar mechanisms in the central nervous system. Some of the existing results can be explained by the metabolism of nucleotides to adenosine or adenosine 5'-monophosphate (AMP). However, studies of presynaptic effects using sensitive electrophysiological tests such as paired-pulse interactions indicate that nucleotides can act at presynaptic sites, but that their effects may be mediated by a release of adenosine. Results are also described which indicate that, under some conditions, nucleotides can mediate phenomena such as long-term potentiation, which probably involves a significant presynaptic element. In part these effects may involve a nucleotide-induced release of adenosine and the simultaneous activation of P1 and P2 receptors.

Virologic responses to a ritonavir--saquinavir-containing regimen in patients who had previously failed nelfinavir.

OBJECTIVES: The effectiveness of a second protease inhibitor in patients who failed an initial protease inhibitor is unclear but believed to be low. It has been postulated, however, that patients who fail nelfinavir may respond differently. We therefore assessed the virologic response to a ritonavir-saquinavir-containing regimen in patients who had previously failed nelfinavir. METHODS: A total of 26 patients enrolled in the nelfinavir clinical trials AG506 and AG511 at our two sites who failed (two consecutive HIV viral loads > 5000 copies/ml; branched DNA assay) were switched to a combination of stavudine 40 mg twice daily, lamivudine 150 mg twice daily, ritonavir 400 mg twice daily and saquinavir 400 mg twice daily. RESULTS: The mean viral load at enrollment in this study was 46 674 copies/ml (range, 1075-146400 copies/ml). The median CD4 cell count was 222 x 10(6)/l (range, 82-448 x 10(6)/l). The median duration of nelfinavir use with a detectable viral load before the switch occurred was 48 weeks. Two patients discontinued the study at 3 weeks. All of the remaining patients (n = 24) reached undetectable viral loads (< 500 copies/ml) that were sustained at week 24 in 17 (71%) out of 24 subjects. The most frequent baseline mutations in the protease gene prior to switching were D30N (13 out of 18), N88D (eight out of 18) and M36I (eight out of 18). The presence or absence of these mutations was not predictive of a short-term virologic response. CONCLUSIONS: Most patients who failed a nelfinavir-containing regimen responded to a switch to a combination regimen with saquinavir-ritonavir.

Interaction between adenosine A1 and A2 receptor-mediated responses in the rat hippocampus in vitro.

Previous work has been carried out on the effects of adenosine on transmitter release and on the excitability of postsynaptic neurones, but little is known about the effects of adenosine on the coupling between the two. In this study, we examine the effects of specific adenosine receptor agonists and antagonists on the population excitatory postsynaptic potential (population EPSP) slope, the population spike amplitude, and the relationship between the two (E-S coupling) in the CA1 area of rat hippocampus. Activation of adenosine A1 receptors by adenosine or the selective agonist N6-cyclopentyladenosine resulted in a decrease of the population spike amplitude by a greater extent than could be accounted for by the decrease in population EPSP slope, resulting in a dissociation in the E-S relationship, reflected as a right-shift in the E-S curve. Activation of adenosine A2A receptors by the selective agonist 2-p-(2-carboxyethy)phenethylamino-5'-N-ethylcarboxamidoadeno sine (CGS 21680), or blockade by antagonists ZM 241385 and CP 66713 had no effect on evoked responses. However, when both adenosine A1 and A2A receptors were activated at the same time, a significant attenuation of the inhibitory effects of N6-cyclopentyladenosine on population spike amplitude was observed, resulting in a left-shift in the E-S curve. Intracellular recording indicated that N6-cyclopentyladenosine raised the threshold for spike induction by pulses of depolarising current, even at a concentration which did not produce hyperpolarisation of the neurone. At 30 nM, CGS 21680 prevented this effect of N6-cyclopentyladenosine, and this apparent antagonism was prevented by the A2A receptor antagonist ZM 241385. The results show that adenosine A1 receptors change the coupling between presynaptic transmitter release and postsynaptic cell firing, and that this effect is attenuated by A2A receptor activation.

Adenosine N1-oxide inhibits vaccinia virus replication by blocking translation of viral early mRNAs.

Adenosine N1-oxide (ANO) is a potent and highly selective inhibitor of vaccinia virus replication. We examined the impact of ANO on vaccinia virus macromolecular synthesis during synchronous infection of BSC40 cells. Viral DNA replication and viral late protein synthesis were blocked completely by ANO, effects that were attributable to a defect in the expression of viral early genes. Vaccinia virus early proteins were not synthesized in the presence of ANO, even though vaccinia virus early mRNAs were produced. Cellular protein synthesis was unaffected by ANO, and virus infection in the presence of the drug did not elicit the normal shutoff of host protein synthesis. Adenosine N1-oxide triphosphate (ANO-TP), the predominant metabolite of the drug in vivo, could substitute for ATP in RNA synthesis by purified vaccinia virus RNA polymerase. ANO-TP could support early transcription by purified virions if dATP was provided as an energy source. ANO-TP did not inhibit early transcription in the presence of ATP. These findings suggest a novel antiviral mechanism whereby incorporation of a modified nucleotide into viral mRNAs might selectively block viral gene expression at the level of translation. We believe that ANO merits consideration as an antipoxvirus drug for topical treatment of molluscum contagiosum in humans.

Vaccinia virus morphogenesis is blocked by a temperature-sensitive mutation in the I7 gene that encodes a virion component.

The ts16 mutation of vaccinia virus WR (R. C. Condit, A. Motyczka, and G. Spizz, Virology 128:429-443, 1983) has been mapped by marker rescue to the I7L open reading frame located within the genomic HindIII I DNA fragment. The I7 gene encodes a 423-amino-acid polypeptide. Thermolabile growth was attributed to an amino acid substitution, Pro-344-->Leu, in the predicted I7 protein. A normal temporal pattern of viral protein synthesis was elicited in cells infected with ts16 at the nonpermissive temperature (40 degrees C). Electron microscopy revealed a defect in virion assembly at 40 degrees C. Morphogenesis was arrested at a stage subsequent to formation of spherical immature particles. Western immunoblot analysis with antiserum directed against the I7 polypeptide demonstrated an immunoreactive 47-kDa polypeptide accumulating during the late phase of synchronous vaccinia virus infection. Immunoblotting of extracts of wild-type virions showed that the I7 protein is encapsidated within the virus core. The I7 polypeptide displays amino acid sequence similarity to the type II DNA topoisomerase of Saccharomyces cerevisiae.

Temperature-sensitive mutations in the vaccinia virus H4 gene encoding a component of the virion RNA polymerase.

Four previously isolated temperature-sensitive (ts) mutants of vaccinia virus WR (ts1, ts31, ts55, and ts58) comprising a single complementation group (R. C. Condit, A. Motyczka, and G. Spizz, Virology 128:429-443, 1983) have been mapped by marker rescue to the H4L open reading frame located within the genomic HindIII-H DNA fragment. The H4 gene is predicted to encode a 93.6-kDa polypeptide expressed at late times during infection. Nucleotide sequence alterations responsible for thermolabile growth lead to amino acid substitutions in the H4 gene product. All four ts alleles display "normal" patterns of early and late viral protein synthesis at the nonpermissive temperature (40 degrees C). Mature virion particles, microscopically indistinguishable from wild-type virions, are produced in the cytoplasm of cells infected with ts1 at 40 degrees C. Western immunoblot analysis localizes the H4 protein to the virion core. After solubilization from cores, the H4 protein is associated during purification with transcriptionally active vaccinia virus DNA-dependent RNA polymerase.

Mapping the active-site tyrosine of vaccinia virus DNA topoisomerase I.

Site-directed mutagenesis of the vaccinia virus gene encoding a type I DNA topoisomerase implicates Tyr-274 as the active-site residue that forms a covalent adduct with DNA during cycles of DNA-strand breakage and reunion. Replacement of Tyr-274 by phenylalanine results in loss of the ability of the enzyme to relax negatively supercoiled DNA as well as to form the covalent DNA-protein intermediate. Substitution of phenylalanine for tyrosine at nine other sites in the protein has no apparent effect on enzyme activity. Amino acid sequence alignment reveals Tyr-274 to be homologous to Tyr-727 and Tyr-771, respectively, of the type I topoisomerases from Saccharomyces cerevisiae and Saccharomyces pombe; Tyr-727 and Tyr-771 have been shown to represent the active-site tyrosines of those enzymes. Sequence comparison of the active-site regions defines a motif Ser-Lys-Xaa-Xaa-Tyr common to the viral and cellular type I topoisomerases, including the human enzyme.