Hrs, a tyrosine kinase substrate with a conserved double zinc finger domain, is localized to the cytoplasmic surface of early endosomes

Hrs is a 115-kDa double zinc finger protein that is rapidly tyrosine phosphorylated in growth factor-stimulated cells. However, its function remains unknown. Here we show that Hrs is localized to early endosomes. Intracellular localization of endogenous Hrs and exogenously expressed Hrs tagged with the hemagglutinin epitope was examined by immunofluorescence staining using anti-Hrs and anti-hemagglutinin epitope antibodies, respectively. Hrs was detected in vesicular structures and was colocalized with the transferrin receptor, a marker for early endosomes, but only partially with CD63, a marker for late endosomes. A zinc finger domain deletion mutant of Hrs was also colocalized with the transferrin receptor, suggesting that the zinc finger domain is not required for its correct localization. Immunoelectron microscopy showed that Hrs was localized to the cytoplasmic surface of these structures. By subcellular fractionation, Hrs was recovered both in the cytoplasmic and membrane fractions. The membrane-associated Hrs was extracted from the membrane by alkali treatment, suggesting that it is peripherally associated with early endosomes. These results, together with our finding that Hrs is homologous to Vps27p, a protein essential for protein traffic through a prevacuolar compartment in yeast, suggest that Hrs is involved in vesicular transport through early endosomes.     

Human herpesvirus 6 infection as a risk factor for the development of severe drug-induced hypersensitivity syndrome

OBJECTIVES: To test suitability of radiographic evaluation of lung lesions as a substitute for lung lesion scores derived by examination at necropsy in challenge-exposure models of bovine pneumonia. ANIMALS: 10 calves selected by body weight from 20 multiple-source male Holstein calves approximately 1 to 2 months old enrolled in a Pasteurella multocida challenge-exposure study. PROCEDURE: Calves were paired on the basis of weight and randomly assigned within pairs to vaccine or control (saline solution) group. By use of deep tracheal cannulation, calves were challenge exposed with a culture of virulent P multocida, observed for 10 days, euthanatized, and necropsied, and the lungs were scored for pneumonic lesions. Radiographic views of the lung fields of the calves were taken before challenge exposure and before necropsy and were evaluated for alveolar disease by a veterinary radiologist. Lung lesion scores were compared with radiographic evaluations. RESULTS: There was a strong and significant correlation (R2 = 0.91, P < 0.001) between results of the evaluation of postchallenge-exposure radiographs and necropsy results. There also was also strong and significant correlation (R2 = 0.90, P < 0.001) between evaluation of the prechallenge-exposure radiographs and necropsy results. CONCLUSIONS: Radiographic evaluation of lung lesions correlates well with lung lesions found at necropsy. The findings emphasize the need for caution in interpreting the results of challenge-exposure studies of bovine respiratory tract disease in which small numbers of calves are studied.     

Phylogenetic analysis of Rhodococcus erythropolis based on the variation of ribosomal proteins as observed by matrix-assisted laser desorption ionization-mass spectrometry without using genome information

Rhodococcus erythropolis strains characterized as antibiotic producers can be classified into three groups according to their antibiotic spectrum and growth compatibility. Due to their high genotypic similarity, the taxonomic relationship of these strains has not been elucidated. In this study, ribosomal protein profiling using matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) was employed to classify twenty-one strains of R. erythropolis (15 antibiotic producers and 6 non-antibiotic producers). In the first step in this method, a total of 30 intense peaks observed for purified ribosomal subunit proteins of the type strain (R. erythropolis JCM 3201^T) were selected as the reference peaks. The mass spectra observed for the cell lysates of each sample strain were then checked as to whether peaks were observed at the same masses of the reference peaks. The results of peak matching were processed by cluster analysis, generating a dendrogram. Four major clusters of the R. erythropolis strains corresponded to three antibiotic groups and the non-antibiotic group. Furthermore, the topology of the dendrogram was highly comparable with the phylogenetic tree based on DNA gyrase subunit B gene (gyrB) sequencing. These results indicate that our proposed ribosomal protein profiling method using MALDI-MS is a potentially reliable and sufficiently high-throughput technique for the taxonomic analysis of closely related bacterial strains without using DNA sequence information.     

Cellular and transcriptional responses of yeast to the cleavage of cytosolic tRNAs induced by colicin D

Colicin D is a plasmid‐encoded antibacterial protein that specifically cleaves the anticodon loops of four Escherichia coli tRNA^Arg species. Here, we report that the catalytic domain of colicin D, which is expressed in Saccharomyces cerevisiae, impairs cell growth by cleaving specific tRNAs. DNA microarray analysis revealed that mating‐related genes were upregulated, while genes involved in a range of metabolic processes were downregulated, thereby impairing cell growth. The pheromone‐signalling pathway was activated only in α cells by tRNA cleavage, which was not observed in ‘a’ cells or diploid cells. On the basis of these results and on the recent identification of two killer toxins that cleave specific tRNAs, the relationship between tRNA depletion and the resultant cellular response is discussed. Copyright © 2009 John Wiley & Sons, Ltd.     

Coulomb effects on 13C-NMR and polarons in (CH)x and a bound state of solitons

Using the PPP Hamiltonian model of (CH)_x, we study the effects of the Coulomb interaction on the ¹³C-NMR of doped (CH)_x and the electronic and lattice structures of polarons. The broad asymmetric ¹³C-NMR signal is explained by the Coulomb-induced charge density modulation around charged solitons. Polarons accompany the Coulomb-induced charge and spin density modulations which have a dip at the centre and different spreadings, the former being wider. The lattice dimerization in polarons is not only relaxed but also reversed in a central region. A polaron accompanies four midgap levels. With a suitably screened Coulomb interaction, we can get polaron and soliton levels in good agreement with the data of electrochemical doping voltage spectroscopy.The positively and negatively charged solitons make a breather-like bound state, which may explain the self-pinning of photogenerated solitons.     

A flow cytometric method for counting very low levels of white cells in blood and blood components

Reduction of white cells (WBCs) in blood components may reduce the risk of virus transmission and HLA alloimmunization. Filtration provides a means by which to achieve high-efficiency WBC reduction. A method has been developed using flow cytometry to quantitate the number of WBCs in WBC-reduced packed red cells or platelet concentrates. This method uses a detergent and propidium iodide (PI) solution to label the WBC nuclei and incorporates a known amount of fluorescein isothiocyanate (FITC)-labeled chicken red cells (cRBCs) into the mixture as an indicator of the volume examined. The number of observed WBCs per mL is calculated as follows: Number of PI WBC nuclei events/Number of FITC cRBC events x Number of FITC cRBCs added to mixture/Volume of blood in mixture. The method may allow the detection of WBCs at a concentration as low as 0.01 per microliters (10/mL) in a blood sample. It is an efficient method of collecting data, as it requires less than 10 minutes per sample. This flow cytometric technique is suitable for research purposes and for quality control of WBC-reduced blood components, because it is precise and can be used to quantitate WBCs in large or small numbers in a sample.     

Structural analysis of β-form poly(p-xylyene) starting from a high-resolution image

The crystal structure of β-form poly(p-xylylene) is analysed starting from a high-resolution image of a single crystal of this polymer. The high-resolution image corresponding to the projection of molecules onto the ab-plane along the chain axis shows clearly the mutual position of each molecule in a unit cell. The molecules are aligned wavily in the direction along the a-axis and the rough positions of their centres in a unit cell can be determined from the image. The refinement of the structure is carried out by the usual least-squares method using the intensities of electron and X-ray diffractions. The space group of the β-form is trigonal, P3, and the lattice dimensions are a=2.052 nm, c=0.655 nm and γ=120°. The unit cell contains 16 molecules and one of them is considered to occupy statistically one of three equivalent orientations so as to satisfy the P3 symmetry.     

Vitamin E and coenzyme Q concentrations in the thyroid tissues of patients with various thyroid disorders

Chylous fistula complicates 1.1% of all radical neck dissections, and 2.4% of left-sided dissections. The standard treatment of established chylous leak in the reported cases is a pressure dressing applied to the lower neck. Here we present a case of chylous fistula, where conservative methods failed to cope with this complication. The additional application of a fibrin adhesive set was a successful modality of treatment.