The refined crystal structure of cowpea mosaic virus at 2.8 A resolution

Comoviruses are a group of plant viruses in the picornavirus superfamily. The type member of comoviruses, cowpea mosaic virus (CPMV), was crystallized in the cubic space group I23, a = 317 A and the hexagonal space group P6(1)22, a = 451 A, c = 1038 A. Structures of three closely similar nucleoprotein particles were determined in the cubic form. The roughly 300-A capsid was similar to the picornavirus capsid displaying a pseudo T = 3 (P = 3) surface lattice. The three beta-sandwich domains adopt two orientations, one with the long axis radial and the other two with the long axes tangential in reference to the capsid sphere. T = 3 viruses display one or the other of these two orientations. The CPMV capsid was permeable to cesium ions, leading to a disturbance of the beta-annulus inside a channel-like structure, suggesting an ion channel. The hexagonal crystal form diffracted X rays to 3 A resolution, despite the large unit cell. The large ( approximately 200 A) solvent channels in the lattice allow exchange of CPMV cognate Fab fragments. As an initial step in the structure determination of the CPMV/Fab complex, the P6(1)22 crystal structure was solved by molecular replacement with the CPMV model determined in the cubic cell.     

Use of PCR to study epidemiology of Serratia marcescens isolates in nosocomial infection.

A method to characterize strains of Serratia marcescens based on the PCR amplification of enterobacterial repetitive intergenic consensus sequences has been developed. The PCR fingerprints were generated from boiled supernatants prepared directly from bacterial colonies without the need for DNA extraction. The technique was applied to isolates obtained during an outbreak of pneumonia from seven mechanically ventilated patients, and its result indicated that the outbreak was due to the spread of two epidemic strains. This technique was validated by comparison with rRNA gene restriction analysis. There was complete concordance between these two techniques in discriminating the outbreak-related strains from epidemiologically unrelated isolates. Typing with both biochemical profile and antibiogram profile, though simple, was found to be less reliable than genotyping. The results show that this enterobacterial repetitive intergenic consensus PCR provides a rapid and simple means of typing S. marcescens isolates for epidemiologic studies.     

Evaluation of MicroScan Rapid Pos Combo panels for identification of staphylococci.

MicroScan Rapid Pos Combo panels (Baxter Diagnostics, Inc., MicroScan, West Sacramento, Calif.) contain substrates conjugated with fluorophores and substrates with a fluorescent pH indicator. AutoSCAn W/A, an automated panel processor equipped with a fluorometer, reads the panels after 2 h of incubation and can identify staphylococci to the species level. We tested 239 strains belonging to 17 species of staphylococci. All the strains were identified by conventional methods (W.E. Kloos and K.H. Schleifer, J. Clin. Microbiol. 1:82-88, 1975) and by the MicroScan Rapid ID system. The system correctly identified 219 (91.6%) strains; nine (3.8%) identification results were probably correct, and six (2.5%) results were incorrect. The system designated five (2.1%) strains as rare biotypes. The automated MicroScan Rapid ID system is useful and reliable in identifying most human isolates of staphylococci encountered in the clinical laboratory.     

Detection and specificity of antibodies secreted by spleen cells in mice immunized with Streptococcus mutans.

Immune responses of mice to Streptococcus mutans serotype c were analyzed by means of the enzyme-linked immunospot assay to determine the predominant specificities of the antibodies developed. In general, the numbers of splenic antibody-secreting cells correlated with serum antibody levels. A low dose (10(8) CFU) of killed whole cells injected twice intraperitoneally induced antibodies mainly against surface protein antigen I/II. A higher dose (10(9) CFU) given two to six times also resulted in a predominance of antigen I/II antibody-secreting cells and, in addition, antibody responses to surface protein antigen III and lipoteichoic acid occurred. Cells producing antibodies to serotype c polysaccharide were elicited only on repeated immunization. These results agreed with the development of antibodies in rabbits repeatedly immunized intravenously with killed whole cells of S. mutans, S. rattus, and S. sobrinus, which induced specific antibodies in accordance with the surface antigens that they express. Mice immunized twice with the same dose of purified antigens I/II and III developed greater numbers of antigen I/II splenic antibody-forming cells than antigen III splenic antibody-forming cells and higher serum antibody levels to antigen I/II than to antigen III. Furthermore, a single injection of antigen I/II but not of antigen III was sufficient to induce a strong specific-antibody response. Some evidence was also obtained for weak polyclonal stimulation of spleen cells by S. mutans cells and by antigen I/II, a result which could be relevant to the induction by S. mutans of antibodies reactive with mammalian tissues. It was concluded that for the antigens examined, S. mutans elicited the strongest antibody response against antigen I/II, which was also highly immunogenic in purified form.     

The role of HIV-related chemokine receptors and chemokines in human erythropoiesis in vitro

In order to better define the role of HIV-related chemokines in human erythropoiesis we studied: A) the expression of chemokine receptors, both on human CD34(+) cells which include erythroid progenitors and on more mature erythroid cells; B) the functionality of these receptors by calcium flux, chemotaxis assay and phosphorylation of mitogen-activated protein kinases (MAPK) p42/44 (ERK1/ERK2) and AKT, and finally C) the influence of chemokines on BFU-E formation. We found that HIV-related chemokine receptor CXCR4, but not CCR5, is detectable on human CD34(+) BFU-E cells. CXCR4 surface expression decreased during erythroid maturation, although CXCR4 mRNA was still present in cells isolated from differentiated erythroid colonies. SDF-1, a CXCR4 ligand, induced calcium flux and phosphorylation of MAPK (p42/44) and AKT in CD34(+)KIT(+) bone marrow mononuclear cells which contain BFU-E, as well as chemotactic activity of both human CD34(+) BFU-E progenitors and erythroid cells isolated from day 2-6 BFU-E colonies. Responsiveness to SDF-1 decreased when the cells differentiated to the point of surface expression of the erythroid-specific marker Glycophorin-A. In contrast, the CCR5 ligands (macrophage inflammatory protein-1alpha [MIP-1alpha], MIP-1beta, and RANTES) did not activate calcium flux, MAPK and AKT phosphorylation or chemotaxis of CD34(+)KIT(+) cells or cells isolated from the BFU-E colonies. Interestingly, none of the chemokines tested in this study had any effect on BFU-E colony formation. In conclusion, only CXCR4 is functional, and its specific ligand SDF-1 may therefore play an important role in the homing and/or retention of early erythroid precursors in the bone marrow environment.     

Gender paradox in cardiac calcium deposits in middle-aged and elderly patients: mitral annular and coronary calcifications interrelationship

OBJECTIVES: mitral annular calcification (MAC) occurs mainly in middle-aged and elderly patients and can lead to serious clinical consequences. Male predominance in the prevalence of coronary disease is well-established. Paradoxically, the prevalence of MAC, which is theoretically based on the same etiological mechanisms as coronary atherosclerosis, seems to be predominant in postmenopausal women. The goal of this work was to investigate gender influences on interrelationship between MAC and coronary calcifications (CC) in the same population of middle-aged and elderly patients with increased cardiovascular risk. METHODS: the study comprised 522 patients (284 men and 238 postmenopausal women, aged 52-80 years, mean 65+/-6), who were recruited to the International Nifedipine GITS Study of Intervention as a Goal in Hypertension Treatment (INSIGHT) study in our region. They underwent both fast spiral computed tomography of the heart and echo-Doppler. MAC was defined as advanced when its thickness was > or =5mm; otherwise it was defined as trivial. RESULTS: there were 37 (16%) women and 25 (9%) men with advanced MAC (AMAC), 97 (41%) women and 118 (42%) men with trivial MAC and 104 (44%) women and 141 (50%) men without MAC. The prevalence of any type of CC was significantly higher among men (P=0. 001). In sharp contrast to the distinct male predominance in coronary disease, AMAC was more prevalent among women. In patients without CC prevalence was 9 and 4%, increasing to 16 and 8% in those with nonsevere CC and to 38 and 14% in patients with severe CC, respectively (P=0.001). Multivariate analysis showed that AMAC can predict the presence of severe CC in women and men, with OR of 4.1 and 2.6 (CI 1.2-14.8 and 1.0-10.6) and coronary disease with OR of 2. 5 and 2.5 (CI 0.6-10.6 and 1.0-6.4), respectively. CONCLUSIONS: AMAC signifies a high probability of coronary atherosclerosis in patients of both genders. The inverted gender predominance in the prevalence of annular calcification and CC could be explained by additional etiological (likely osteoporotic) mechanisms of MAC development among postmenopausal women.     

Attenuation of pulmonary neuroendocrine differentiation in mice lacking Clara cell secretory protein

During development and injury, pulmonary neuroendocrine (NE) cells may transiently express Clara cell 10 kD protein (CC10), a major product of the nonciliated progenitor cells for normal and neoplastic airway epithelia suggesting a close relationship between the cells. To assess the role of CC10 during NE differentiation, we studied CC10-deficient mouse lungs by immunohistochemistry and digital imaging. The knockout model revealed a lack of the disrupted gene product in the lung. Because NE cells, which occur as solitary cells or in neuroepithelial bodies (NEBS), comprise less than 1% of airway epithelia, we counted foci positive for each of the three NE markers--synaptophysin, calcitonin gene-related peptide (CGRP), and protein gene product (PGP) 9.5--and developed a method to analyze numerous airways in serial sections. Digitized images of slides were segmented with Photoshop imaging software. The length of airway epithelium and total section areas were then measured using MetaMorph image analysis software. A comparable range of NE foci was observed regardless of CC10 expression patterns with all three markers, suggesting that CC10 is not critical for NE ontogenesis. However, discrimination according to size revealed that wild-type lungs harbored 30% to 40% greater synaptophysin- and CGRP-containing NEBs relative to CC10 deficient lungs. We posit that an attenuation of pulmonary NE differentiation afflicts the CC10-deficient state. Our imaging application greatly facilitates the acquisition and analysis of complex structures such as the lung and promises to be a widely applicable technique for assessments of tissue sections.     

A novel two colour ELISPOT assay. I. Simultaneous detection of distinct types of antibody-secreting cells

A novel assay system has been developed which is based on the ELISPOT methodology and employs a combination of two immunoenzyme visualization systems yielding distinct colour products. This variation permits the simultaneous enumeration of two different types of cell secreting antigenically distinct products. Optimal conditions for the concurrent detection of human mononuclear cells secreting IgG or IgA antibodies are described.