Homology-based analysis of the GRAS gene family in tobacco.

Members of the GRAS gene family are important transcriptional regulators. In this study, 21 GRAS genes were identified from tobacco, and were classified into eight subgroups according to the classification of Arabidopsis thaliana. Here, we provide a preliminary overview of this gene family in tobacco, describing the gene structure, gene expression, protein motif organization, phylogenetic analysis, and comparative analysis in tobacco, Arabidopsis, and rice. Using the sequences of 21 GRAS genes in Arabidopsis to search against the American tobacco genome database, 21 homologous GRAS genes in tobacco were identified. Sequence analysis indicates that these GRAS proteins have five conserved domains, which is consistent with their counterparts in other plants. Phylogenetic analyses divided the GRAS gene family into eight subgroups, each of which has distinct conserved domains and biological functions. Furthermore, the expression pattern of these 21 GRAS genes reveals that most are expressed in all six tissues studied; however, some have tissue specificity. Taken together, this comprehensive analysis will provide a rich resource to assist in the study of GRAS protein functions in tobacco.

The ORION study: comparison of two sirolimus-based regimens versus tacrolimus and mycophenolate mofetil in renal allograft recipients.

Safety and efficacy of two sirolimus (SRL)-based regimens were compared with tacrolimus (TAC) and mycophenolate mofetil (MMF). Renal transplantation recipients were randomized to Group 1 (SRL+TAC; week 13 TAC elimination [n = 152]), Group 2 (SRL + MMF [n = 152]) or Group 3 (TAC + MMF [n = 139]). Group 2, with higher-than-expected biopsy-confirmed acute rejections (BCARs), was sponsor-terminated; therefore, Group 2 two-year data were limited. At 1 and 2 years, respectively, graft (Group 1: 92.8%, 88.5%; Group 2: 90.6%, 89.9%; Group 3: 96.2%, 95.4%) and patient (Group 1: 97.3%, 94.4%; Group 2: 95.2%, 94.5%; Group 3: 97.0%, 97.0%) survival rates were similar. One- and 2-year BCAR incidence was: Group 1, 15.2%, 17.4%; Group 2, 31.3%, 32.8%; Group 3, 8.2%, 12.3% (Group 2 vs. 3, p < 0.001). Mean 1- and 2-year modified intent-to-treat glomerular filtration rates (mL/min) were similar. Primary reason for discontinuation was adverse events (Group 1, 34.2%; Group 2, 33.6%; Group 3, 22.3%; p < 0.05). In Groups 1 and 2, delayed wound healing and hyperlipidemia were more frequent. One-year post hoc analysis of new-onset diabetes posttransplantation was greater in TAC recipients (Groups 1 and 3 vs. 2, 17% vs. 6%; p = 0.004). Between-group malignancy rates were similar. The SRL-based regimens were not associated with improved outcomes for kidney transplantation patients.

Comparison of the safety and efficacy of cyclosporine minimization versus cyclosporine elimination in de novo renal allograft patients receiving sirolimus.

The safety and efficacy of concentration-controlled use of sirolimus (SRL) and cyclosporine (CsA) followed by CsA minimization (CsAm) or elimination (CsAe) beginning at week 13 was compared in a phase 4, open-label, randomized (1:1) trial of renal transplant recipients enrolled between March 2004 and November 2005. The primary endpoint was renal function, measured at 12 months using the Nankivell formula, in patients remaining on therapy. Though a total enrollment of 140 patients in each group was planned to provide an 80% power to detect a difference in means, only 207 subjects were enrolled in this study. Demographic characteristics were similar between groups, with 98.1% recipients of first grafts, 69.1% from living donors, and 7.2% diabetics. At 12 months, there were no differences in renal function (61.08 vs 65.24 mL/min, P = .132); incidence of biopsy-confirmed acute rejection (14.3% vs 22.5%, P = .152); and patient (89.5% vs 92.2%, P = .632), graft (87.6% vs 88.2%, P = .999), and death-censored graft (98.1% vs 94.1%, P = .166) survivals between CsAm and CsAe groups, respectively. There were no differences in the overall rate of study-drug discontinuation (32.4% vs 36.3%, P = .562) but more patients discontinued because of lack of efficacy/graft loss in the CsAe group (4.8% vs 14.7%, P = .018). This study was underpowered to demonstrate the superiority of one regimen over the other. In summary, SRL immunotherapy combined with CsA minimization or elimination showed comparative safety and efficacy. Both regimens offer potential treatment options for de novo renal allograft recipients.

Manipulating biological samples for environmental scanning electron microscopy observation.

Biological samples having different characteristics were observed by environmental scanning electron microscopy (ESEM). The environmental conditions for untreated biological samples was determined by optimizing sample temperature and chamber pressure. When the temperature was at 4 degrees - 6 degrees C and chamber pressure was 5.2-5.9 Torr, the relative humidity in the specimen chamber was about 85%. Under these conditions, the surface features of the sample were completely exposed and did not exhibit charging. The images obtained from the untreated samples at different ESEM conditions were also compared with fixed and coated samples observed under high vacuum.

Randomised controlled trial of an interactive multimedia decision aid on benign prostatic hypertrophy in primary care.

OBJECTIVE: To determine whether a decision aid on benign prostatic hypertrophy influences decision making, health outcomes, and resource use. DESIGN: Randomised controlled trial. SETTING: 33 general practices in the United Kingdom. PARTICIPANTS: 112 men with benign prostatic hypertrophy. INTERVENTION: Patients' decision aid consisting of an interactive multimedia programme with booklet and printed summary. OUTCOME MEASURES: Patients' and general practitioners' perceptions of who made the decision, decisional conflict scores, treatment choice and prostatectomy rate, American Urological Association symptom scale, costs, anxiety, utility, and general health status. RESULTS: Both patients and general practitioners found the decision aid acceptable. A higher proportion of patients (32% v 4%; mean difference 28%, 95% confidence interval 14% to 41%) and their general practitioners (46% v 25%; 21%, 3% to 40%) perceived that treatment decisions had been made mainly or only by patients in the intervention group compared with the control group. Patients in the intervention group had significantly lower decisional conflict scores than those in the control group at three months (2.3 v 2.6; -0.3, -0.5 to -0.1, P<0.01) and this was maintained at nine months. No differences were found between the groups for anxiety, general health status, prostatic symptoms, utility, or costs (excluding costs associated with the video disc equipment). CONCLUSIONS: The decision aid reduced decisional conflict in men with benign prostatic hypertrophy, and the patients played a more active part in decision making. Such programmes could be delivered cheaply by the internet, and there are good arguments for coordinated investment in them, particularly for conditions in which patient utilities are important.

Molecular cloning of 11S globulin and 2S albumin, the two major seed storage proteins in sesame.

Insoluble 11S globulin and soluble 2S albumin, conventionally termed alpha-globulin and beta-globulin, are the two major storage proteins and constitute 80-90% of total seed proteins in sesame. Two full-length cDNA clones were sequenced and deduced to encode sesame 11S globulin and 2S albumin precursors, respectively. Deduced amino acid composition reveals that 2S albumin, but not 11S globulin, is a sulfur-rich protein. Three abundant polypeptides of 50-60 kDa were resolved on SDS-PAGE when seed-purified 11S globulin was prepared in nonreducing conditions. Immunological analysis suggests that these three polypeptides are encoded by homologous genes. Immunodetection on the overexpressed protein of the 11S globulin clone in Escherichia coli indicates that this clone encodes the precursor protein of one of the three purified 11S globulin polypeptides.

Identification of three novel unique proteins in seed oil bodies of sesame.

Plant seeds store triacylglycerols in discrete organelles called oil bodies. An oil body preserves a matrix of triacylglycerols surrounded by a monolayer of phospholipids embedded with abundant structural proteins termed oleosins and probably some uninvestigated minor proteins of higher molecular mass. Three polypeptides of 27, 37, and 39 kDa (temporarily denominated as Sop1, Sop2, and Sop3) were regularly co-purified with seed oil bodies of sesame. Comparison of amino acid composition indicated that they were substantially less hydrophobic than the known oleosins, and thus should not be aggregated multimers of oleosins. The results of immuno-recognition to sesame proteins extracted from subcellular fractions of mature seeds, various tissues, and oil bodies purified from different stages of seed formation revealed that these three polypeptides were unique proteins gathered in oil bodies, accompanying oleosins and triacylglycerols, during the active assembly of the organelles in maturing seeds. Both in vivo and in intro, immunofluorescence labeling using secondary antibodies conjugated with FITC (fluorescein isothiocyanate) confirmed the localization of these three polypeptides in oil bodies.

Characterization of hemin binding activity of Streptococcus pneumoniae.

Streptococcus pneumoniae is a causative agent of bacterial pneumonia, otitis media, meningitis, and bacteremia. It causes considerable morbidity and mortality throughout the world, especially among children, the elderly, and immunocompromised individuals. We have demonstrated previously that the growth of S. pneumoniae is limited under iron-depleted conditions and can be restored by the addition of either hemin or hemoglobin. In the present study, we showed that S. pneumoniae had the ability to bind hemin and that the level of hemin binding activity was not affected by supplementation of the growth medium with iron. Approximately 70 to 80% of the hemin binding activity was mediated by proteinase-resistant components, and the remainder was mediated by proteins. Hemin binding proteins were located in both soluble extract and envelope fractions of pneumococcal cells. By batch affinity chromatography, a major hemin binding polypeptide with an apparent molecular mass of 43 kDa was identified in the cell lysate of S. pneumoniae. Polyclonal antibodies against this polypeptide were raised. By immunoblot analysis, this hemin binding polypeptide was localized in the envelope and did not exhibit any variation in molecular weight among all serotypes tested. The subcellular distribution of hemin binding activity may have functional implications.

Cloning of a Corynebacterium diphtheriae iron-repressible gene that shares sequence homology with the AhpC subunit of alkyl hydroperoxide reductase of Salmonella typhimurium.

To understand how Corynebacterium diphtheriae responds to iron limitation, we compared the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) protein profiles of both wild-type cells and iron uptake mutants grown in either high- or low-iron medium. The removal of iron by ethylene diamine di-(o-hydroxy-phenyl acetic acid) from the growth medium of wild-type cells resulted in induction of at least 14 polypeptides. DirA, a major iron-repressible polypeptide, was purified from wild-type cells by preparative SDS-PAGE, and the dirA structural gene was isolated from a genomic library of nontoxigenic C. diphtheriae. The nucleotide sequence of dirA was determined, and the deduced amino acid sequence of DirA revealed strong homologies with the AhpC subunit of Salmonella typhimurium alkyl hydroperoxide reductase and polypeptides of other microorganisms associated with oxidation reduction activity. Like AhpC, cloned DirA reduced the susceptibility of an Escherichia coli ahp mutant to cumene hydroperoxide, suggesting that DirA has alkyl hydroperoxide reductase activity.

Determination of pyrantel in swine liver by flame ionization gas chromatography and confirmation by gas chromatography/mass spectrometry.

During an evaluation of the gas chromatography/mass spectrometry (GC/MS) confirmatory procedure of Lynch and Bartolucci for pyrantel residues in swine tissues, we developed a GC flame ionization method for quantitating pyrantel residues in extracts of swine liver. The method was subjected to trial principally in the laboratories of Biospherics, Inc., using control liver, fortified control liver, and incurred liver tissue samples. Although the method does not meet all of the current Food and Drug Administration criteria, it compares favorably to the official determinative method. Portions of the same extract can be used for quantitation and for GC/MS confirmation, true recoveries appear to be slightly higher, and an internal standard is not required. The precision of this method equals or exceeds that of the official determinative method.