Cleavage of rhesus rotavirus VP4 after arginine 247 is essential for rotavirus-like particle-induced fusion from without

We recently described our finding that recombinant baculovirus-produced virus-like particles (VLPs) can induce cell-cell fusion similar to that induced by intact rotavirus in our assay for viral entry into tissue culture cells (J. M. Gilbert and H. B. Greenberg, J. Virol. 71:4555-4563, 1997). The conditions required for syncytium formation are similar to those for viral penetration of the plasma membrane during the course of viral infection. This VLP-mediated fusion activity was dependent on the presence of the outer-layer proteins, viral protein 4 (VP4) and VP7, and on the trypsinization of VP4. Fusion activity occurred only with cells that are permissive for rotavirus infection. Here we begin to dissect the role of VP4 in rotavirus entry by examining the importance of the precise trypsin cleavage of VP4 and the activation of VP4 function related to viral entry. We present evidence that the elimination of the three trypsin-susceptible arginine residues of VP4 by specific site-directed mutagenesis prevents syncytium formation. Two of the three arginine residues in VP4 are dispensable for syncytium formation, and only the arginine residue at site 247 appears to be required for activation of VP4 functions and cell-cell fusion. Using the recombinant VLPs in our syncytium assay will aid in understanding the conformational changes that occur in VP4 involved in rotavirus penetration into host cells.     

Effect of mild acid treatment on the survival, enteropathogenicity, and protein production in vibrio parahaemolyticus

Vibrio parahaemolyticus is an important food-borne enteropathogen that encounters various adverse conditions in its native environment or during infection. Effects of mild acid treatment on survival under stress conditions, enteropathogenicity, and protein production in this pathogen were investigated. Logarithmically grown cells, at pH 7.5 shifted to pH 5.0 for 30 min, were more resistant to subsequent acid challenge at pH 4.4. A two-phase adaptive procedure (pH 5.8 for 30 min; pH 5.0 for 30 min) was better than a single-phase procedure for enhancing the acid tolerance of this pathogen. The acid-adapted cells were cross-protected against the challenges of low salinity and thermal inactivation. One-dimensional polyacrylamide gel electrophoresis revealed that proteins with molecular masses of 6.4, 9.0, 13.6, 16.3, 18.9, 22.9, 24.4, 28.3, 33. 9, 36.9, 41.2, 47.6, 58.1, 65.6, 80.5, 88.2, and 96.9 kDa were induced or significantly enhanced, while proteins of 25.3, 30.1, 30. 7, and 91.7 kDa were significantly inhibited. Two-dimensional polyacrylamide gel electrophoresis revealed that 20 species of proteins were induced or significantly enhanced, while 26 species were inhibited. In assays conducted using the suckling mouse model, enteropathogenicity of the acid-adapted cells was significantly enhanced in terms of intestine/body weight ratio and in vivo recovery of infected cells.     

Monitoring of lineage-specific chimaerism allows early prediction of response following donor lymphocyte infusions for relapsed chronic myeloid leukaemia

Thrombogenesis in the left atrial appendage (LAA) has been related to the special morphology of this cavity and to its size and degree of dysfunction. However, no study has focused on LAA function in conjunction with left atrial (LA) function in both sinus rhythm (SR) and nonrheumatic idiopathic atrial fibrillation (AF) in relation to clinical status (cardioembolic stroke). Forty-three patients in SR (14 patients with stroke, 29 control subjects) and 45 patients in AF (27 patients with stroke, 18 control subjects) were examined by transthoracic and transesophageal echocardiography. Baseline clinical characteristics and standard transthoracic and transesophageal measurements of the LA and LAA (size, fractional area change, flow measurements, spontaneous echo contrast, and thrombus) were recorded and compared in relation to cardiac rhythm. Patients in the stroke-SR group showed a significant decrease of fractional area change in the LA (32%+/-15%) and LAA (34%+/-15%) in relation to control subjects (43%+/-10%, p = 0.035, 49%+/-13%, p = 0.006, respectively). Patients in the stroke-AF group showed significant reduction of appendage flow measurements (outward velocity = 22+/-13 vs 33+/-19 cm/sec, p = 0.036), whereas no differences were detected in the center of the LA. In multiple regression analysis, the presence of cardioembolic stroke was positively associated with the presence of spontaneous echo contrast (p = 0.0253) and spontaneous echo contrast negatively associated with appendage inward flow velocity (p<0.001). Cardioembolic stroke in patients in SR is associated with a global decrease of shortening in both cavities and in patients with AF, with a reduction of LAA flow parameters. Patients with spontaneous echo contrast, thrombus, or both showed further reduction of shortening and flow velocities in both cavities, indicating a more advanced stage of dysfunction.     

1998 William J. Stickel Bronze Award. Antifungal activity of Melaleuca alternifolia (tea-tree) oil against various pathogenic organisms

Tea-tree oil (oil of Melaleuca alternifolia) has recently received much attention as a natural remedy for bacterial and fungal infections of the skin and mucosa. As with most naturally occurring agents, claims of effectiveness have been only anecdotal; however, several published studies have recently demonstrated tea-tree oil's antibacterial activity. This study was conducted to determine the activity of tea-tree oil against 58 clinical isolates: Candida albicans (n = 10), Trichophyton rubrum (n = 8), Trichophyton mentagrophytes (n = 9), Trichophyton tonsurans (n = 10), Aspergillus niger (n = 9), Penicillium species (n = 9), Epidermophyton floccosum (n = 2), and Microsporum gypsum (n = 1). Tea-tree oil showed inhibitory activity against all isolates tested except one strain of E floccosum. These in vitro results suggest that tea-tree oil may be useful in the treatment of yeast and fungal mucosal and skin infections.     

Transgenic mice secreting coronavirus neutralizing antibodies into the milk

Ten lines of transgenic mice secreting transmissible gastroenteritis coronavirus (TGEV) neutralizing recombinant monoclonal antibodies (rMAbs) into the milk were generated. The rMAb light- and heavy-chain genes were assembled by fusing the genes encoding the variable modules of the murine MAb 6A.C3, which binds an interspecies conserved coronavirus epitope essential for virus infectivity, and a constant module from a porcine myeloma with the immunoglobulin A (IgA) isotype. The chimeric antibody led to dimer formation in the presence of J chain. The neutralization specific activity of the recombinant antibody produced in transiently or stably transformed cells was 50-fold higher than that of a monomeric rMAb with the IgG1 isotype and an identical binding site. This rMAb had titers of up to 10(4) by radioimmunoassay (RIA) and neutralized virus infectivity up to 10(4)-fold. Of 23 transgenic mice, 17 integrated both light and heavy chains, and at least 10 of them transmitted both genes to the progeny, leading to 100% of animals secreting functional TGEV neutralizing antibody during lactation. Selected mice produced milk with TGEV-specific antibody titers higher than 10(6) as determined by RIA, neutralized virus infectivity by 10(6)-fold, and produced up to 6 mg of antibody per ml. Antibody expression levels were transgene copy number independent and integration site dependent. Comicroinjection of the genomic beta-lactoglobulin gene with rMAb light- and heavy-chain genes led to the generation of transgenic mice carrying the three transgenes. The highest antibody titers were produced by transgenic mice that had integrated the antibody and beta-lactoglobulin genes, although the number of transgenic animals generated does not allow a definitive conclusion on the enhancing effect of beta-lactoglobulin cointegration. This approach may lead to the generation of transgenic animals providing lactogenic immunity to their progeny against enteric pathogens.     

A second generation of artificial cornea (Biokpro II)

The properties of a new second-generation colonizable artificial cornea were evaluated in humans. The prosthesis consisted of a peripheral rim of a translucent microporous fluorocarbon polymer (expanded polytetrafluoroethylene) fused with the polymer of the central optic. The optic was made of medical-grade polydimethylsiloxane coated with polyvinylpyrrolidone. Its refractive power was 42.2 diopters and it measured 7.0 mm in diameter and 0.55 mm in thickness. The geometry of the optic was tested by high-frequency ultrasound and intraocular pressure and distensibility were measured in an artificial chamber. Prostheses were implanted in one eye of 13 humans. The average follow-up was 6 months (range 3-9 months). Most of the eyes (11/13) were clinically stable after a 7 months follow up. Seven patients had visual acuity improvements. Mean corrected final visual acuity was 20/200 (range, 20/30 to light perception). Five anatomical failures occured (two extrusions, two retroprosthetic membranes, one endophthalmitis). The new optical core, junction, and surface properties of the polymers offer many advantages, quicker colonization of the supporting skirt, and an optical core with a geometry similar to that of a normal human cornea. Epithelial cells did not migrate over the interface and optical core. It seems that formation of an epithelium over the artificial device is essential for the long-term stability of the implant.     

Wave-front curvature as a cause of slow conduction and block in isolated cardiac muscle

We have investigated the role of wave-front curvature on propagation by following the wave front that was diffracted through a narrow isthmus created in a two-dimensional ionic model (Luo-Rudy) of ventricular muscle and in a thin (0.5-mm) sheet of sheep ventricular epicardial muscle. The electrical activity in the experimental preparations was imaged by using a high-resolution video camera that monitored the changes in fluorescence of the potentiometric dye di-4-ANEPPS on the surface of the tissue. Isthmuses were created both parallel and perpendicular to the fiber orientation. In both numerical and biological experiments, when a planar wave front reached the isthmus, it was diffracted to an elliptical wave front whose pronounced curvature was very similar to that of a wave front initiated by point stimulation. In addition, the velocity of propagation was reduced in relation to that of the original planar wave. Furthermore, as shown by the numerical results, wave-front curvature changed as a function of the distance from the isthmus. Such changes in local curvature were accompanied by corresponding changes in velocity of propagation. In the model, the critical isthmus width was 200 microns for longitudinal propagation and 600 microns for transverse propagation of a single planar wave initiated proximal to the isthmus. In the experiments, propagation depended on the width of the isthmus for a fixed stimulation frequency. Propagation through an isthmus of fixed width was rate dependent both along and across fibers. Thus, the critical isthmus width for propagation was estimated in both directions for different frequencies of stimulation. In the longitudinal direction, for cycle lengths between 200 and 500 milliseconds, the critical width was < 1 mm; for 150 milliseconds, it was estimated to be between 1.3 and 2 mm; and for the maximum frequency of stimulation (117 +/- 15 milliseconds), it was > 2.5 mm. In the transverse direction, critical width was between 1.78 and 2.32 mm for a basic cycle length of 200 milliseconds. It increased to values between 2.46 and 3.53 mm for a basic cycle length of 150 milliseconds. The overall results demonstrate that the curvature of the wave front plays an important role in propagation in two-dimensional cardiac muscle and that changes in curvature may cause slow conduction or block.