New categories designated as healthcare‐associated and community‐associated methicillin‐resistant Staphylococcus pseudintermedius in dogs

The incidence of MRSP has been increasing, and treatment options in veterinary medicine are limited. Few previous studies of MRSP have described the relationships between the genotypes, phenotypes, and clinical backgrounds of the isolates. To gain insight into the associations between the microbiological and clinical characteristics of MRSP, we analyzed 282 Staphylococcus pseudintermedius isolates from dogs. A total of 195 (69.1%) strains were identified as mecA‐positive MRSP and were classified into mainly two genotypes: SCCmec types III (II‐III) (52.8%) and V (37.4%). SCCmec type III MRSP strains were significantly correlated with hospital admission and antimicrobial therapy of the dogs, and exhibited a homogeneous genotype similar to sequence type 71‐MRSP, which is a globally endemic clone in dogs. In contrast, SCCmec type V MRSP strains were not highly correlated with hospital admission and antimicrobial therapy and exhibited genotypic and phenotypic heterogeneity. Properties of MRSP strains SCCmec types III and V were similar to those of HA‐ and CA‐MRSA, respectively. Therefore, we designated these isolates carrying SCCmec types III and V as HA‐MRSP and CA‐MRSP, respectively. Discrimination between HA‐ and CA‐MRSP by oxacillin MIC will provide useful information for treatment and infection control measures for canine MRSP infections.     

Prion infection correlates with hypersensitivity of P2X7 nucleotide receptor in a mouse microglial cell line

We recently established mouse microglial cells persistently infected with mouse-adapted scrapie ME7 (ScMG20/ME7) for in vitro study of prion pathogenesis. Here, we found that ScMG20/ME7 cells were hypersensitive to P2X7 receptor agonists, as demonstrated by sustained Ca(2+) influx, membrane pore formation, cell death, and interleukin-1beta release. P2X7 mRNA expression was upregulated in these cells, and also in scrapie-infected mice brains. Treatment with pentosan polysulfate eliminated the infectivity and disease-related forms of prion protein from ScMG20/ME7 cell cultures, however, hypersensitivity of P2X7 receptors remained. These results suggest that prion infections may strongly affect the P2X7 receptor system in mouse microglial cells.     

Comparative metabolomics of Japanese Black cattle beef and other meats using gas chromatography–mass spectrometry

Progress in metabolomic analysis now allows the evaluation of food quality. This study aims to identify the metabolites in meat from livestock using a metabolomic approach. Using gas chromatography–mass spectrometry (GC/MS), many metabolites were reproducibly detected in meats, and distinct differences between livestock species (cattle, pigs, and chickens) were indicated. A comparison of metabolites between tissues types (muscle, intramuscular fat, and intermuscular fat) in marbled beef of Japanese Black cattle revealed that most metabolites are abundant in the muscle tissue. Several metabolites (medium-chain fatty acids, etc.) involved in triacylglycerol synthesis were uniquely detected in fat tissue. Additionally, the results of multivariate analysis suggest that GC/MS analysis of metabolites can distinguish between cattle breeds. These results provide useful information for the analysis of meat quality using GC/MS-based metabolomic analysis.

ABBREVIATIONS: GC/MS: gas chromatography-mass spectrometry; NMR: nuclear magnetic resonance; MS: mass spectrometry; IS: 2-isopropylmalic acid; MSTFA: N-Methyl-N-trimethylsilyltrifluoroacetamide; CV: coefficient of variation; TBS: Tris-buffered saline; MHC: myosin fast type; PCA: principal component analysis; OPLS-DA: orthogonal partial least-squares discriminant analysis; O2PLS: two-way orthogonal partial least-squares     

Gene structure of human thrombomodulin, a cofactor for thrombin-catalyzed activation of protein C

The gene coding for human thrombomodulin, a thrombin receptor on endothelial cells and a cofactor for the activation of anticoagulant protein C zymogen, was isolated from a human genomic library by employing human thrombomodulin cDNA as a probe. The nucleotide sequences of the gene and the adjacent 5' and 3' flanking regions were then determined. The nucleotide sequence of this gene with approximately 3.7 kilobase pairs was identical to that of the cDNA, indicating that the gene for human thrombomodulin is free of introns. Hybridization data showed that there is only a single thrombomodulin gene in the human genome.     

Induction of cytotoxic T cells to a cross-reactive epitope in the hepatitis C virus nonstructural RNA polymerase-like protein.

Cytotoxic T lymphocytes (CTL) have been found to mediate protection in vivo against certain virus infections. CTL also may play an important role in control of infection by hepatitis C virus (HCV), but no CTL epitopes have yet been defined in any HCV protein. The nonstructural protein with homology to RNA polymerase should be a relatively conserved target protein for CTL. To investigate the epitope specificity of CTL specific for this protein, we used 28 peptides from this sequence to study murine CTL. Mice were immunized with a recombinant vaccinia virus expressing the HCV nonstructural region corresponding to the flavivirus NS5 gene (RNA polymerase), and the primed spleen cells were restimulated in vitro with peptides. CTL from H-2d mice responded to a single 16-residue synthetic peptide (HCV 2422 to 2437). This relatively conserved epitope was presented by H-2d class I major histocompatibility complex (MHC) molecules to conventional CD4- CD8+ CTL but was not recognized by CTL restricted by H-2b. Moreover, exon shuffle experiments using several transfectants expressing recombinant Dd/Ld and Kd demonstrated that this peptide is seen in association with alpha 1 and alpha 2 domains of the Dd class I MHC molecule. This peptide differs from the homologous segments of this nonstructural region from three other HCV isolates by one residue each. Variant peptides with single amino acid substitutions were made to test the effect of each residue on the ability to sensitize targets. Neither substitution affected recognition. Therefore, these conservative mutations affected peptide interaction neither with the Dd class I MHC molecule nor with the T-cell receptor. Because these CTL cross-react with all four sequenced isolates of HCV in the United States and Japan, if human CTL display similar cross-reactivity, this peptide may be valuable for studies of HCV diagnosis and vaccine development. Our study provides the first evidence that CD8+ CTL can recognize an epitope from the HCV sequence in association with a class I MHC molecule.     

Neutrophil transit times through pulmonary capillaries: the effects of capillary geometry and fMLP-stimulation

The deformations of neutrophils as they pass through the pulmonary microcirculation affect their transit time, their tendency to contact and interact with the endothelial surface, and potentially their degree of activation. Here we model the cell as a viscoelastic Maxwell material bounded by constant surface tension and simulate indentation experiments to quantify the effects of (N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)-stimulation on its mechanical properties (elastic shear modulus and viscosity). We then simulate neutrophil transit through individual pulmonary capillary segments to determine the relative effects of capillary geometry and fMLP-stimulation on transit time. Indentation results indicate that neutrophil viscosity and shear modulus increase by factors of 3.4, for 10(-9) M fMLP, and 7.3, for 10(-6) M fMLP, over nonstimulated cell values, determined to be 30.8 Pa.s and 185 Pa, respectively. Capillary flow results indicate that capillary entrance radius of curvature has a significant effect on cell transit time, in addition to minimum capillary radius and neutrophil stimulation level. The relative effects of capillary geometry and fMLP on neutrophil transit time are presented as a simple dimensionless expression and their physiological significance is discussed.     

Oxygen uptake rate of immobilized growing hybridoma cells

Summary The specific oxygen uptake rate of hybridoma cells immobilized in calcium alginate gel particles was measured, and the observed data was compared with those of non-immobilized cells. The uptake rate of the immobilized cells coincided with that of the non-immobilized hybridoma cells just after immobilization, but increased with cell growth. On the other hand, the cellular glucose consumption rate decreased slightly during the experiments. The increased oxygen uptake rate by immobilized cells was closely related to the formation of cell colonies in the gel particles.     

Identification of genes expressed preferentially in the honeybee mushroom bodies by combination of differential display and cDNA microarray

To clarify the molecular basis underlying the neural function of the honeybee mushroom bodies (MBs), we identified three genes preferentially expressed in MB using cDNA microarrays containing 480 differential display-positive candidate cDNAs expressed locally or differentially, dependent on caste/aggressive behavior in the honeybee brain. One of the cDNAs encodes a putative type I inositol 1,4,5-trisphosphate (IP(3)) 5-phosphatase and was expressed preferentially in one of two types of intrinsic MB neurons, the large-type Kenyon cells, suggesting that IP(3)-mediated Ca(2+) signaling is enhanced in these neurons.