Transformation of Lotus japonicus using the herbicide resistance bar gene as a selectable marker

Transgenic plants of the model legume Lotus japonicus were regenerated by hypocotyl transformation using a bar gene as a selectable marker. The bar encodes for Phosphinothricin Acetyl Transferase that detoxifies phosphinothricin (PPT), the active ingredient of herbicides such as Ignite (AgrEvo) and Basta (Hoechst). Transgenic L. japonicus plants resistant to PPT were positive upon PCR by bar gene-specific primers. In 5 out of 7 independent lines tested, PPT resistance segregated as a single dominant allele indicating a single T-DNA insertion into the plant genome. All regenerated plants were fertile and void of visible somaclonal abnormalities contrary to 14% infertility when antibiotic selectable markers were used. The lack of somaclonal variation, ease of PPT application and low cost of PPT makes this protocol an attractive alternative for the regeneration of transgenic L. japonicus. The production of PPT herbicide-resistant L. japonicus plants may have significant commercial applications in crop production.

Horizontal transmission of entomopathogenic fungi by the diamondback moth

The relative potential of the pathogenic fungi Beauveria bassiana and Zoophthora radicans for use as autodisseminated biological control agents of the diamondback moth (Plutella xylostella) was compared. The LC50 of B. bassiana conidia to third instar larvae was 499 conidia/mm(2) of leaf surface and individual cadavers of mycosed fourth instar larvae yielded a mean of 67.5 X 10(6) (+/- 7.5 x 10(6)) conidia. All concentrations of B. bassiana tested in inoculation chambers (0.24, 2.4, and 6.2 mug/mm(2)) induced 100% mortality in adult male moths within 7 days. The times to death and sporulation were concentration and exposure duration dependent. A standard procedure for inoculating male moths resulted in > 85% mortality from Z. radicans and > 93% mortality from B. bassiana. Pairing of inoculated males with clean moths of both sexes yielded higher rates of passive transmission of B. bassiana than Z. radicans, but there was no evidence for sexual transmission of either pathogen. Similarly, B. bassiana was more effectively transmitted from inoculated male moths to larvae foraging on whole plants. Single sporulating cadavers producing B. bassiana or Z. radicans conidia placed on plants infested with larvae resulted in a similar rate of transmission for both pathogens. However, an increase of the density of sporulating cadavers from one to three/plant increased Z. radicans transmission (greater than fourfold) but had no effect on B. bassiana transmission. Simultaneous inoculations of larvae with conidia of both fungi reduced the mortality induced by each pathogen, the reduction being most acute for B. bassiana-induced mortality. Inoculation of adults with both fungi showed that, at concentrations required for effective passive transmission to larvae, B. bassiana severely inhibited Z. radicans mycosis in adults. (C) 2001 Academic Press.

Post-antibiotic growth suppression of linezolid against Gram-positive bacteria

The in vitro post-antibiotic effects (PAEs) of eight different concentrations of linezolid against Gram-positive cocci were investigated and the results analysed using the sigmoid E-max model for mathematically modelling the PAE. Mean maximal linezolid PAEs against strains of Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Enterococcus faecium and Streptococcus pneumoniae were 2.2, 1.8, 2.8, 2.0 and 3.0 h, respectively. Resistance to methicillin (for the staphylococci), vancomycin (for the enterococci) and penicillin (for the pneumococci) had no effect on the duration of the PAE. Results of PAE testing support twice-daily dosing of linezolid in humans.

Protein targeting to the plasma membrane of adult skeletal muscle fiber: An organized mosaic of functional domains

The plasma membrane of differentiated skeletal muscle fibers comprises the sarcolemma, the transverse (T) tubule network, and the neuromuscular and muscle-tendon junctions. We analyzed the organization of these domains in relation to defined surface markers, beta -dystroglycan, dystrophin, and caveolin-3, These markers were shown to exhibit highly organized arrays along the length of the fiber. Caveolin-3 and beta -dystroglycan/dystrophin showed distinct, but to some extent overlapping, labeling patterns and both markers left transverse tubule openings clear. This labeling pattern revealed microdomains over the entire plasma membrane with the exception of the neuromuscular and muscle-tendon junctions which formed distinct demarcated macrodomains. Our results suggest that the entire plasma membrane of mature muscle comprises a mosaic of T tubule domains together with sareolemmal caveolae and beta -dystroglycan domains. The domains identified with these markers were examined with respect to targeting of viral proteins and other expressed domain-specific markers, We found that each marker protein was targeted to distinct microdomains, The macrodomains were intensely labeled with all our markers. Replacing the cytoplasmic tail of the vesicular stomatitis virus glycoprotein with that of CD4 resulted in retargeting from one domain to another. The domain-specific protein distribution at the muscle cell surface may be generated by targeting pathways requiring specific sorting information but this trafficking is different from the conventional apical-basolateral division. (C) 2001 Academic Press.

Novel MUCI splice variants are expressed in cervical carcinoma

Objectives. The MUC1 antigen can be used to identify epithelial cells from the background of hemopoietic cells. The present investigation describes patterns of overexpression of two novel MUC1 splice variants in human cervical carcinoma cell lines. Methods. RT-PCR was carried out to determine MUC1 splice variants in the cervical cancer cell lines C-4 II, C-33A, DoTc 2 4510, C-4 I, SiHa, HT3, Hs 636 T (C4-I), and HeLa. Results. The novel MUC1 splice variant D was expressed in all cell lines and the novel MUC1 splice variant C was expressed in all cell lines but C-33A. Variants A and B were expressed in all (variant A) and all but one (variant B) cell line. MUC1/REP was expressed in all cell lines and MUC1/SEC was positive in all but two cell lines (C-33 A, DoTc 2 4510). All but one cell line (C-33A) expressed MUC1/X and MUC1/Y, and two cell lines (C-33 A, DoTc 2 4510) did not express MUC1/Z, respectively. MUC1 variants A, D, and REP could be demonstrated consistently among all eight cervical carcinoma cell lines we have examined. Conclusions. The present study describes the feasibility of detecting a large number of MUC1 variants, including MUC1 variants C and D which are described for cervical carcinoma cells for the first time. Further studies will examine the presence of MUC1 splice variants' expression in human cervical carcinoma tissue.

Safety and efficacy of low anterior en bloc resection as part of cytoreductive surgery for patients with ovarian cancer

Objective. To examine the feasibility and safety of a low anterior resection of the rectosigmoid plus adjacent pelvic tumour as part of primary cytoreduction for ovarian cancer. Methods. This study included 65 consecutive patients with primary ovarian cancer who had debulking surgery from 1996 through 2000. All patients underwent an en bloc resection of ovarian cancer and a rectosigmoid resection followed by an end-to-end anastomosis. Parameters for safety and efficacy were considered as primary statistical endpoints for the aim of this analysis. Results. Postoperative residual tumour was nil, 1 cm in 14, 34, and 14 patients, respectively. The median postoperative hospital stay was 11 days (range, 6 to 50 days). Intraoperative complications included an injury to the urinary bladder in one patient. Postoperative complications included wound complications (n=14, 21.5%), septicemia (n=9, 13.8%), cardiac complications (n=7, 10.8%), thromboembolic complications (n=5, 7.7%) ileus (n=2, 3.1%) anastomotic leak (n=2, 3.1%) and fistula (n=1, 1.5%). Reasons for a reoperation during the same admission included repair of an anastomotic leak (n=1), postoperative hemorrhage (n=1), and wound debridement (n=1). Wound complications, septicemia, and anastomotic leak formation were more frequent in patients who had a serum albumin level of less than or equal to 30 g/L preoperatively. There was one surgically related mortality in a patient who died from a cerebral vascular accident 2 days postoperatively. Conclusions. An en bloc resection as part of primary cytoreductive surgery for ovarian cancer is effective and its morbidity is acceptably low. (C) 2001 Academic Press.

Postoperative vaginal vault brachytherapy for node-negative Stage II (occult) endometrial carcinoma

Objective. The aim of this study is to look at the efficacy of extended surgical staging and postoperative vaginal vault brachytherapy in patients with Stage II (occult) endometrial carcinoma. Methods. Between January 1989 and December 1997, there were 30 patients with Stage II (occult) endometrial carcinoma who received postoperative vaginal vault brachytherapy as the only adjuvant treatment. The study group consisted of 15 of these patients who had extended surgical staging (including lymphadenectomy). Results. At a median follow-up of 36 months (range 17 to 113 months), there has been no recurrence. There were no major complications from surgery. Only 1 patient had mild rectal bleeding following vaginal vault brachytherapy and there were no grade 3 or 4 bowel toxicities. Conclusions. Extended surgical staging and postoperative vaginal vault brachytherapy for Stage II (occult) endometrial carcinoma is associated with minimal morbidity and excellent survival. (C) 2001 Academic Press.

Quality Assurance Through a Continuous Curriculum Review (CCR) Strategy: Reflections on a Pilot Project

Institutional research can be defined as "the activity in which the research effort of an academic institution is directed at the solution of its own problems and to the enhancement of its own performance" (Woodward, 1993, p. 113). This paper describes and reflects on an attempt at the University of Queensland to address the need for course quality appraisal for improvement. The strategy, Continuous Curriculum Review (CCR) is simply an attempt to trial and promote regular comprehensive data collection for developing 'snapshot' views of whole curricula so that decisions about what to change and what to change first can be made in an empirically defensible and timely manner. The strategy and reporting protocols that were developed are described, and the costs and benefits of engaging in this kind of data gathering exercise for quality assurance and quality enhancement purposes are discussed.

Integrating technology to improve the efficiency of qualitative data analysis - A note on methods

Qualitative data analysis (QDA) is often a time-consuming and laborious process usually involving the management of large quantities of textual data. Recently developed computer programs offer great advances in the efficiency of the processes of QDA. In this paper we report on an innovative use of a combination of extant computer software technologies to further enhance and simplify QDA. Used in appropriate circumstances, we believe that this innovation greatly enhances the speed with which theoretical and descriptive ideas can be abstracted from rich, complex, and chaotic qualitative data. © 2001 Human Sciences Press, Inc.

Characterization of the EphA1 receptor tyrosine kinase: Expression in epithelial tissues

The Eph family (of receptor tyrosine kinases plays a crucial role during development and is implicated in oncogenesis. Using a partial cDNA clone of an Eph-related kinase (Esk) we isolated the complete coding region of a gene which we show to be murine EphA1 by both structural and functional criteria. The chromosomal localization is shown to be syntenic to hEphA1 and the genomic organization also shows distinct features found in the hEphA1 gene. Functionally, in keeping with findings for the human homologue, both soluble recombinant and native mEphA1 show preferential binding to ephrin A1. However, we also observed significant binding to other A-type ligands as has been observed for other Eph receptors. We analysed the expression of mEphA1 mRNA by in situ hybridization on tissue sections. mEphA1 was expressed in epithelial elements of skin, adult thymus, kidney and adrenal cortex. Taken together with previous Northern blotting data these results suggest that mEphA1 is expressed widely in differentiated epithelial cells.

SOX18 directly interacts with MEF2C in endothelial cells

Recently, we demonstrated that mutations in the Sry-related HMG box gene Sox18 underlie vascular and hair follicle defects in the mouse allelic mutants ragged (Ra) and RaJ. Ra mice display numerous anomalies in the homozygote including, oedema, peritoneal secretions, and are almost completely naked. Sox18 and the MADS box transcription factor, Mef2C, are expressed in developing endothelial cells. Null mutants in Sox18 and Mef2c display overlapping phenotypic abnormalities, hence, we investigated the relationship between these two DNA binding proteins. We report here the direct interaction between MEF2C and SOX18 proteins, and establish that these proteins are coexpressed in vivo in endothelial cell nuclei. MEF2C expression potentiates SOX18-mediated transcription in vivo and regulates the function of the SOX18 activation domain. Interestingly, MEF2C fails to interact or co-activate transcription with the Ra or RaJ mutant SOX18 proteins. These results suggest that MEF2C and SOX18 may be important partners directing the transcriptional regulation of vascular development. (C) 2001 Academic Press.

Mathematical methods for spatially cohesive reserve design

The problem of designing spatially cohesive nature reserve systems that meet biodiversity objectives is formulated as a nonlinear integer programming problem. The multiobjective function minimises a combination of boundary length, area and failed representation of the biological attributes we are trying to conserve. The task is to reserve a subset of sites that best meet this objective. We use data on the distribution of habitats in the Northern Territory, Australia, to show how simulated annealing and a greedy heuristic algorithm can be used to generate good solutions to such large reserve design problems, and to compare the effectiveness of these methods.