A mouse-adapted SARS-coronavirus causes disease and mortality in BALB/c mice

No single animal model for severe acute respiratory syndrome (SARS) reproduces all aspects of the human disease. Young inbred mice support SARS-coronavirus (SARS-CoV) replication in the respiratory tract and are available in sufficient numbers for statistical evaluation. They are relatively inexpensive and easily accessible, but their use in SARS research is limited because they do not develop illness following infection. Older (12- to 14-mo-old) BALB/c mice develop clinical illness and pneumonitis, but they can be hard to procure, and immune senescence complicates pathogenesis studies. We adapted the SARS-CoV (Urbani strain) by serial passage in the respiratory tract of young BALB/c mice. Fifteen passages resulted in a virus (MA15) that is lethal for mice following intranasal inoculation. Lethality is preceded by rapid and high titer viral replication in lungs, viremia, and dissemination of virus to extrapulmonary sites accompanied by lymphopenia, neutrophilia, and pathological changes in the lungs. Abundant viral antigen is extensively distributed in bronchial epithelial cells and alveolar pneumocytes, and necrotic cellular debris is present in airways and alveoli, with only mild and focal pneumonitis. These observations suggest that mice infected with MA15 die from an overwhelming viral infection with extensive, virally mediated destruction of pneumocytes and ciliated epithelial cells. The MA15 virus has six coding mutations associated with adaptation and increased virulence; when introduced into a recombinant SARS-CoV, these mutations result in a highly virulent and lethal virus (rMA15), duplicating the phenotype of the biologically derived MA15 virus. Intranasal inoculation with MA15 reproduces many aspects of disease seen in severe human cases of SARS. The availability of the MA15 virus will enhance the use of the mouse model for SARS because infection with MA15 causes morbidity, mortality, and pulmonary pathology. This virus will be of value as a stringent challenge in evaluation of the efficacy of vaccines and antivirals.     

Protein S-guanylation by the biological signal 8-nitroguanosine 3',5'-cyclic monophosphate

The signaling pathway of nitric oxide (NO) depends mainly on guanosine 3',5'-cyclic monophosphate (cGMP). Here we report the formation and chemical biology of a nitrated derivative of cGMP, 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP), in NO-mediated signal transduction. Immunocytochemistry demonstrated marked 8-nitro-cGMP production in various cultured cells in an NO-dependent manner. This finding was confirmed by HPLC plus electrochemical detection and tandem mass spectrometry. 8-Nitro-cGMP activated cGMP-dependent protein kinase and showed unique redox-active properties independent of cGMP activity. Formation of protein Cys-cGMP adducts by 8-nitro-cGMP was identified as a new post-translational modification, which we call protein S-guanylation. 8-Nitro-cGMP seems to regulate the redox-sensor signaling protein Keap1, via S-guanylation of the highly nucleophilic cysteine sulfhydryls of Keap1. This study reveals 8-nitro-cGMP to be a second messenger of NO and sheds light on new areas of the physiology and chemical biology of signal transduction by NO.     

A novel spectrofluorimetric method based on a reaction with an azoisoxazoles–benzenesulfonamide derivative for determination of uranium (VI) ions in water samples

Selective fluorometric detection and determination of uranium ions is provided here using a novel fluorescent reagent, namely (E)-4-([4-hydroxynaphthalen-1-yl]diazenyl)-N-(5-methyleisoxazol-3-yl) benzenesulfonamide (U^VI reagent). The U^VI reagent offers a selective fluorescence enhancement behaviour at emission wavelength = 557 nm. The parameters affecting fluorometric detection of uranium ions, such as the pH, solvent type, ligand concentration, interaction time, and interfering ions, were investigated and adjusted. The proposed U^VI reagent can detect and determine uranium ions even at low concentrations, for which the obtained limit of detection was 0.1 ppm. Additionally, this proposed determination protocol was successfully used to detect, monitor, and determine uranium ions in actual water samples.     

Microvascular anastomosis in an optical cavity: is it possible?

Microsurgery is one of the highly interesting surgical procedures that can be performed using different applications and in different specialties, including plastic surgery. The endoscope is a popular instrument used in many fields, including plastic surgery. Although the operating microscope is still a must for microsurgical performance, microsurgery could be performed, depending on the experiences and facilities, by using other visual-assisting equipment. From this point of view, the authors tried to find less costly and more widespread equipment suitable for performing microsurgery that can, furthermore, be applied in special situations and indications, such as operating in an optical cavity. The authors investigated this issue with the endoscope. In this experimental project, the authors performed vascular microsurgical anastomoses of the rats' femoral vessels to create an optical cavity in a prefabricated skin retraction model in the groin area of 10 Sprague-Dawley male rats. The microsurgical anastomoses of the femoral vessels and nerves were performed easily in a reasonable time, without recorded difficulties, and with maximum physical and visual comfort for the surgeon. The authors spent a mean time of 28.1, 27.3, and 19.2 minutes for the arterial, venous, and neural anastomoses, respectively. In this group of animals, 90 percent vascular patency and 100 percent accurate neural anastomoses were recorded. The advantage the authors noted was that this new technique of operating in the field of microsurgery, with its feasibility and difficulties, would be a point of research and application for the young generations of microsurgeons.     

Ultrastructure of Hendra virus and Nipah virus within cultured cells and host animals

The ultrastructure of Hendra and Nipah viruses is described in cultured cells, pigs, horses and humans. Differences in ultrastructure between the viruses are evident within infected cell cultures and lungs from infected amplifier hosts. These differences are important in viral identification and differentiation and understanding the pathogenesis of disease.     

Molecular characterization of phenol-degrading bacteria isolated from different Egyptian ecosystems

Twelve selected phenol-degrading bacterial isolates were obtained on phenol agar plates using culture enrichment technique. Molecular identification of the isolates was performed using eubacterial 16S rRNA PCR specific primers. Based on 16S rDNA sequence analysis, the results revealed that the majority of the isolates (8 out of 12) are affiliated to the g-subdivision of Proteobacteria. Four out of the eight isolates are closely related to the genus Acinetobacter. Molecular heterogeneity among the phenol-degrading isolates was further investigated by using rep-PCR chromosomal fingerprinting and correlated with plasmid and antibiotic profile analysis. Rep-PCR results strongly confirmed that the bacterial isolates from different environmental sites produced different fingerprinting patterns. The mineralization of phenol by all isolates was evaluated using 14C-labeled phenol assay. Phenol mineralization ranged from 55% (W-17) to 0.4% (Sea-9). This was further confirmed by the detection of several monoaromatic and polyaromatic degrading genes, e.g., pheA, MopR, XylE, and NahA. In addition, catalytic enzymes such as catalase and dioxygenase were also monitored.     

Successful immunotherapy with matrix metalloproteinase-derived peptides in adjuvant arthritis depends on the timing of peptide administration

We have recently found that matrix metalloproteinases (MMPs) are targets for T-cell and B-cell reactivity in experimental arthritis. In the present article, we investigate whether modulation of MMP-specific T-cell responses could influence the course of adjuvant arthritis (AA). Lewis rats were treated nasally with MMP peptides prior to or after AA induction. Administration of the MMP-10 or the MMP-16 peptide prior to AA induction reduced the arthritic symptoms. In contrast, administration of the MMP-10 peptide after AA induction aggravated the arthritic symptoms. The present study shows the possible usefulness of MMP peptides for immunotherapy. However, a clear understanding of proper timing of peptide administration is crucial for the development of such therapies.     

Anti-sperm antibodies and seminal characteristics after testicular biopsy or epididymal aspiration in dogs

A study was performed to determine if performing testicular biopsies or epididymal aspirates in dogs would induce sperm-bound anti-sperm antibodies (ASA), affect long-term sperm production or semen quality. Semen was collected from 8 mature dogs 3 times a week before and after hemicastration and then 3 times a week after testicular biopsy (n=3 and 1 control) or epididymal aspiration (n=3 and 1 control). Detection of anti-sperm IgG (ASA) on sperm cells was performed by flow cytometry analysis using a flow cytometer. Two dogs with testicular biopsies became positive for ASA 16 d after testicular biopsy and remained positive for 7 and 9 d, respectively. One dog that had an epididymal aspirate became positive 13 d after epididymal aspiration and remained positive for 35 d. One dog became positive 21 d after hemicastration and remained positive for 28 d. Sperm output declined significantly in 7 of 8 dogs after hemicastration. A control epididymal aspirate treatment dog had decreased sperm output, and a testicular biopsy treatment dog had increased sperm output. None of the dogs with ASA had significant changes in sperm output after treatment. Sperm motility declined significantly in 3 dogs after hemicastration. An epididymal aspiration treatment dog had a decrease in sperm motility, a control epididymal aspirate treatment dog and a control testicular biopsy treatment dog each had increases in sperm motility. None of the dogs with ASA had significant changes in motility. The percentage of normal spermatozoa significantly decreased in 3 dogs and significantly increased in 1 dog after hemicastration. Two dogs that had testicular biopsies and 1 dog that had an epididymal aspiration had decreases in percent normal sperm. Two of 3 dogs with decreases in percent normal sperm after treatment had ASA, but 2 dogs with ASA had no change in motility. Hemicastration, epididymal aspiration, and testicular biopsy can induce ASA production within 2 wk of the procedure, but ASA are transient and do not have a predictably negative effect on total sperm output or motility. Testicular biopsy and epididymal aspiration are safe diagnostic procedures, but further work investigating post-treatment fertility must be done before final conclusions can be made.     

Effects of medium viscosity on kinetics of the enzymatic reaction catalyzed by bacterial RNase

Effects of medium viscosity on kinetic parameters of poly(U) hydrolysis catalyzed by RNase from Bac. intermedius 7P (binase) were studied in solutions of sucrose (4-50 wt. %) and glycerol (35-62 wt. %) in Tris--sodium acetate buffer (pH 7.5) at 25 degreesC. The rate constant of reaction kcat was practically unchanged over a wide range of viscosities (1-15 cP for sucrose and 2.5-3 cP for glycerol). In glycerol solutions, kcat slightly increased with viscosity increase from 4 to 10 cP. Addition of NaCl to the buffer medium resulted in an inhibitory effect of Na+ on kcat, prevented by 50% sucrose or 60% glycerol. It is concluded that binase-catalyzed poly(U) cleavage occurs through a "tense"-substrate mechanism, similarly to reactions catalyzed by alpha-chymotrypsin, trypsin, and laccase.