Cardiovascular and ventilatory interactions in the facultative air-breathing teleost Pangasianodon hypophthalmus

Journal of Comparative Physiology B - All vertebrates possess baroreceptors monitoring arterial blood pressure and eliciting reflexive changes in vascular resistance and heart rate in response to...     

Conformation of alamethicin in oriented phospholipid bilayers determined by (15)N solid-state nuclear magnetic resonance

The conformation of the 20-residue antibiotic ionophore alamethicin in macroscopically oriented phospholipid bilayers has been studied using (15)N solid-state nuclear magnetic resonance (NMR) spectroscopy in combination with molecular modeling and molecular dynamics simulations. Differently (15)N-labeled variants of alamethicin and an analog with three of the alpha-amino-isobutyric acid residues replaced by alanines have been investigated to establish experimental structural constraints and determine the orientation of alamethicin in hydrated phospholipid (dimyristoylphosphatidylcholine) bilayers and to investigate the potential for a major kink in the region of the central Pro(14) residue. From the anisotropic (15)N chemical shifts and (1)H-(15)N dipolar couplings determined for alamethicin with (15)N-labeling on the Ala(6), Val(9), and Val(15) residues and incorporated into phospholipid bilayer with a peptide:lipid molar ratio of 1:8, we deduce that alamethicin has a largely linear alpha-helical structure spanning the membrane with the molecular axis tilted by 10-20 degrees relative to the bilayer normal. In particular, we find compatibility with a straight alpha-helix tilted by 17 degrees and a slightly kinked molecular dynamics structure tilted by 11 degrees relative to the bilayer normal. In contrast, the structural constraints derived by solid-state NMR appear not to be compatible with any of several model structures crossing the membrane with vanishing tilt angle or the earlier reported x-ray diffraction structure (Fox and Richards, Nature. 300:325-330, 1982). The solid-state NMR-compatible structures may support the formation of a left-handed and parallel multimeric ion channel.     

Viral infection causes rapid sensitization to lipopolysaccharide: central role of IFN-alpha beta

LPS is the major active agent in the pathogenesis of Gram-negative septic shock. In this report we have studied the influence of concurrent viral infection on the outcome of LPS-induced shock. We find that infection with vesicular stomatitis virus sensitizes mice to LPS at an early time point following infection. Treatment of mice with the chemical IFN inducer, polyinosinic:polycytidylic acid, has a similar effect. This hypersensitivity to LPS correlated with hyperproduction of TNF-alpha in vivo. The cellular and molecular mechanisms underlying this phenomenon were investigated using Ab-depleted and gene-targeted mice. Our results revealed that while NK cell depletion and elimination of IFN-gamma partially protected against the sensitizing effects of vesicular stomatitis virus and polyinosinic:polycytidylic acid, the most striking effect was observed in IFN-alphabetaR-deficient mice. Thus hyperproduction of TNF-alpha was completely abrogated in IFN-alphabetaR-deficient mice, indicating that the principal mechanism underlying rapid virus-induced sensitization to LPS is an IFN-alphabeta-mediated priming of mice for an augmented production of TNF-alpha in response to LPS. This conclusion was further supported by the finding that pretreatment of mice with rIFN-alphabeta mimicked the effect of viral infection. In conclusion, our results reveal a previously unrecognized proinflammatory effect of IFN-alphabeta and point to a new pathway through which viral infection may influence the outcome of concurrent bacterial infection.     

Targeted deletions created in yeast vectors by recombinational excision

We have developed a simple method for creating defined deletions in yeast vectors by utilizing the ability of Saccharomyces cerevisiae to perform homologous recombination. Two complementary single-stranded oligonucleotides are designed so that the 5' and 3' halves of the resulting double-stranded oligonucleotide are homologous to the 5' and 3' side of a desired deletion junction, respectively. The sequence to be deleted is cleaved by restriction endonuclease digestion, followed by co-transformation of the linearized plasmid and the oligonucleotide into yeast. By homologous recombination in vivo, a subset of the plasmids will recircularize and simultaneously acquire the deletion as defined by the oligonucleotide.     

Local cooperation dominates over competition between CD4+ T cells of different antigen/MHC specificity

Interactions between CD4(+) T cells in vivo are controlled by a balance between cooperation and competition. In this study the interaction between two populations of CD4(+) T cells of different MHC/peptide specificity was probed at different precursor frequencies, delivering one or both Ags to APC using particle-mediated DNA delivery. Expansion of clonal populations of Ag (OVA and pigeon cytochrome c-specific) CD4(+) T cells was limited at higher precursor frequencies, presumably reflecting intraclonal competition. In contrast, a strong enhancement of the number of cells expressing IFN-gamma, IL-4, and IL-2 was observed in populations of cells at low precursor frequency in the presence of a high frequency of activated cells of a different Ag specificity. The helper effect was most potent when both Ags were delivered to the same dendritic cell (i.e., linked). This reflects the requirement of epicrine or paracrine help for optimal activation of T cell clones at low frequency. A measure of help was also delivered in an endocrine manner (unlinked), especially for Th1 responses, suggesting that there is also limited diffusion of cytokines between dendritic cell clusters. The dominant effects of cooperation over competition between CD4(+) T cells responding to different Ags may have important implications in terms of the efficacy of multivalent vaccines.     

Deficient CD4+ T cell priming and regression of CD8+ T cell functionality in virus-infected mice lacking a normal B cell compartment

In this study, we investigate the state of T cell-mediated immunity in B cell-deficient (B(-/-)) mice infected with two strains of lymphocytic choriomeningitis virus known to differ markedly in their capacity to persist. In B(-/-) C57BL mice infected with the more persisting virus, virus-specific CD8(+) T cells are initially generated that are qualitatively similar to those in wild-type mice. However, although cell numbers are well sustained over time, the capacity to produce cytokines is rapidly impaired. In similarly infected B(-/-) BALB/c mice, virus-specific CD8(+) T cells are completely deleted, indicating that host genotype influences the severity of the T cell defect. In B(-/-) C57BL mice infected with the less persisting virus, CD8(+) T cell dysfunction was not as pronounced, although it was clearly present. Most importantly, the appearance of dysfunctional CD8(+) T cells clearly precedes recrudescence of detectable virus, indicating that the T cell defect is not simply a secondary event due to virus buildup resulting from the failure of B(-/-) mice to produce neutralizing Abs. In contrast with CD8(+) T cells, which initially respond almost as in wild-type mice, the priming of virus-specific CD4(+) T cells was markedly impaired in B(-/-) mice infected with either virus strain. Thus, our results indicate that B cells play an important role in antiviral immunity not only as Ab producers, but also in promoting an optimal and sustained T cell response. The T cell defects are likely to contribute to the chronic course of viral infection in B(-/-) mice.     

Mapping of QTL for Body Conformation and Behavior in Cattle

Genome scans for quantitative trait loci (QTL) in farm animals have concentrated on primary production and health traits, and information on QTL for other important traits is rare. We performed a whole genome scan in a granddaughter design to detect QTL affecting body conformation and behavior in dairy cattle. The analysis included 16 paternal half-sib families of the Holstein breed with 872 sons and 264 genetic markers. The markers were distributed across all 29 autosomes and the pseudoautosomal region of the sex chromosomes with average intervals of 13.9 cM and covering an estimated 3155.5 cM. All families were analyzed jointly for 22 traits using multimarker regression and significance thresholds determined empirically by permutation. QTL that exceeded the experiment-wise significance threshold (5% level) were detected on chromosome 6 for foot angle, teat placement, and udder depth, and on chromosome 29 for temperament. QTL approaching experiment-wise significance (10% level) were located on chromosome 6 for general quality of feet and legs and general quality of udder, on chromosome 13 for teat length, on chromosome 23 for general quality of feet and legs, and on chromosome 29 for milking speed. An additional 51 QTL significant at the 5% chromosome-wise level were distributed over 21 chromosomes. This study provides the first evidence for QTL involved in behavior of dairy cattle and identifies QTL for udder conformation on chromosome 6 that could form the basis of recently reported QTL for clinical mastitis.     

Amino acid changes within conserved region III of the herpes simplex virus and human cytomegalovirus DNA polymerases confer resistance to 4-oxo-dihydroquinolines,a novel class of herpesvirus antiviral agents

The 4-oxo-dihydroquinolines (PNU-182171 and PNU-183792) are nonnucleoside inhibitors of herpesvirus polymerases (R. J. Brideau et al., Antiviral Res. 54:19-28, 2002; N. L. Oien et al., Antimicrob. Agents Chemother. 46:724-730, 2002). In cell culture these compounds inhibit herpes simplex virus type 1 (HSV-1), HSV-2, human cytomegalovirus (HCMV), varicella-zoster virus (VZV), and human herpesvirus 8 (HHV-8) replication. HSV-1 and HSV-2 mutants resistant to these drugs were isolated and the resistance mutation was mapped to the DNA polymerase gene. Drug resistance correlated with a point mutation in conserved domain III that resulted in a V823A change in the HSV-1 or the equivalent amino acid in the HSV-2 DNA polymerase. Resistance of HCMV was also found to correlate with amino acid changes in conserved domain III (V823A+V824L). V823 is conserved in the DNA polymerases of six (HSV-1, HSV-2, HCMV, VZV, Epstein-Barr virus, and HHV-8) of the eight human herpesviruses; the HHV-6 and HHV-7 polymerases contain an alanine at this amino acid. In vitro polymerase assays demonstrated that HSV-1, HSV-2, HCMV, VZV, and HHV-8 polymerases were inhibited by PNU-183792, whereas the HHV-6 polymerase was not. Changing this amino acid from valine to alanine in the HSV-1, HCMV, and HHV-8 polymerases alters the polymerase activity so that it is less sensitive to drug inhibition. In contrast, changing the equivalent amino acid in the HHV-6 polymerase from alanine to valine alters polymerase activity so that PNU-183792 inhibits this enzyme. The HSV-1, HSV-2, and HCMV drug-resistant mutants were not altered in their susceptibilities to nucleoside analogs; in fact, some of the mutants were hypersensitive to several of the drugs. These results support a mechanism where PNU-183792 inhibits herpesviruses by interacting with a binding determinant on the viral DNA polymerase that is less important for the binding of nucleoside analogs and deoxynucleoside triphosphates.