Comparative analysis of the differential regeneration response of various genotypes of <Emphasis Type="Italic">Triticum aestivum</Emphasis>, <Emphasis Type="Italic">Triticum durum</Emphasis> and <Emphasis Type="Italic">Triticum dicoccum</Emphasis>

An efficient genotype independent, in vitro regeneration system was developed for nine popular Indian wheat cultivars, three each of Triticum aestivum L. viz., CPAN1676, HD2329 and PBW343, Triticum durum Desf. viz., PDW215, PDW233 and WH896, and Triticum dicoccum Schrank. Schubl. viz., DDK1001, DDK1025 and DDK1029, by manipulating the concentration and time of exposure to the growth regulator, thidiazuron (TDZ). A total of 18 (for immature inflorescence and embryo explant) and six (for mature embryo explant) different combinations of growth regulators were tried for callusing and regeneration, respectively. Media combination with low concentration of TDZ (2.2 μM) in combination to auxin and/or cytokinin (depending upon culture stage), was found to be effective for immature and mature explants. Compact, nodular and highly embryogenic calli were obtained by using immature embryo, immature inflorescence and mature embryo explants, and regeneration frequency up to 25 shoots/explant with an overall 80% regeneration was achieved. Comparable regeneration frequency was achieved for mature embryo explants. No separate hormone combination for rooting was required and plantlets ready to transfer to soil could be obtained in a short period of 8–10 weeks. This protocol can be used for raising transgenic plants for functional genomics analysis of agronomically important traits in the three species of wheat.     

The influence of larval diet on adult feeding behaviour in the tobacco hornworm moth, Manduca sexta

Lab-reared sphingid and noctuid moths appear to feed less than wild moths, and often are starved to enhance responsiveness in feeding assays. To measure the impact of larval nutrition on adult feeding, we raised a model sphingid species, Manduca sexta, on control or modified diets (reduced sugar, protein or water, supplemented beta-carotene) or cut tobacco leaves, then conducted feeding assays with artificial flowers. Behaviour was scored and analysed in a double-blind manner. Larval diet affected adult eclosion time, size and fat content, the latter of which was inversely proportional to moth approaches to the floral array in a flight cage. In contrast, behaviours refractory to feeding (sitting, escaping) were associated with sex and barometric pressure, but not with diet or fat content. Frequency of floral approaches and probing was not associated with any variable. However, moths reared on beta-carotene-supplemented diet were 2-3 times more likely to feed, and significantly less likely to sit or show "escape" behaviour than were moths from most other treatments. Our results suggest that decreased visual sensitivity, rather than increased fat content, accounts for reduced adult feeding by lab-reared M. sexta.     

A role for Rab5 in structuring the endoplasmic reticulum

The endoplasmic reticulum (ER) is a contiguous network of interconnected membrane sheets and tubules. The ER is differentiated into distinct domains, including the peripheral ER and nuclear envelope. Inhibition of two ER proteins, Rtn4a and DP1/NogoA, was previously shown to inhibit the formation of ER tubules in vitro. We show that the formation of ER tubules in vitro also requires a Rab family GTPase. Characterization of the 29 Caenorhabditis elegans Rab GTPases reveals that depletion of RAB-5 phenocopies the defects in peripheral ER structure that result from depletion of RET-1 and YOP-1, the C. elegans homologues of Rtn4a and DP1/NogoA. Perturbation of endocytosis by other means did not affect ER structure; the role of RAB-5 in ER morphology is thus independent of its well-studied requirement for endocytosis. RAB-5 and YOP-1/RET-1 also control the kinetics of nuclear envelope disassembly, which suggests an important role for the morphology of the peripheral ER in this process.     

Functional expression of Escherichia coli fhuA gene in Rhizobium spp. of Cajanus cajan provides growth advantage in presence of Fe3+: ferrichrome as iron source

Cajanus cajan rhizobial isolates were found to be unable to utilize iron bound to ferrichrome, desferrioxamine B or rhodotorulic acid, all being hydroxamate type siderophores. A broad host range expression vector containing the Escherichia coli fhuA gene, encoding the outer membrane receptor for Fe-ferrichrome, was constructed. The plasmid construct (pGR1), designed to express fhuA under the lac promoter of E. coli, complemented E. coli MB97 ΔfhuA mutant for ferri-ferrichrome utilization and also allowed Rhizobium spp. ST1 and Rhizobium spp. IC3123 to grow using iron bound to ferrichrome. Sensitivity to the antibiotic albomycin, transported via the FhuA receptor, was found in case of MB97 as well as rhizobial transformants harboring pGR1. The rhizobial transformants expressing fhuA showed growth stimulation when co-inoculated with Ustilago maydis, a fungal species known to produce ferrichrome under iron starved conditions. Growth stimulation was also observed in the presence of externally supplied ferrichrome. The significance of these findings in terms of the potential for improving the survivability of rhizobial bioinoculant strains in natural soils is discussed.     

O-2Preliminary results of the ARTISTIC study: a randomised trial in screening to improve cytology

Annually in the UK around 250 000 cervical smears show low-grade abnormalities. Alternative management policies following a low-grade smear are cytological surveillance or referral for colposcopy. Their effectiveness and cost-effectiveness, and the potential for human papillomavirus (HPV) testing to triage women to either management, has been debated. Trial of management of borderline and other low-grade abnormal smears (TOMBOLA) (a large RCT) addresses these uncertainties, considering clinical, psychosocial and economic outcomes. 4439 women aged 20–59, resident in Grampian, Tayside or Nottingham with a low-grade smear were randomised to cytological surveillance (six-monthly smears in primary care) or hospital-based colposcopy. At colposcopy, women with visible abnormality were randomised to immediate treatment or biopsy and recall for treatment if necessary. Recruitment HPV status was assessed using PCR techniques. Women were followed for three years to an exit colposcopy. Cumulative incidence of CIN2 or more severe disease (CIN2+) in the colposcopy arm was 7.9% per year, higher than in cytological surveillance (5.8%; OR = 1.43, 95% CI 1.23–1.67). This difference was less marked for CIN3+ (OR = 1.27, 1.04–1.55), suggesting spontaneous regression of some CIN2, and that initial colposcopy can lead to over-treatment. There was little difference in psychosocial outcomes between arms. In comparison of biopsy and recall versus immediate treatment, there was no difference in cumulative incidence of CIN2+ or psychosocial outcomes. There was over-treatment and increased frequency/duration of bleeding with immediate treatment. There was no compelling economic reason to favour any one management method. Testing for HPV does not appear to be effective in triage. Based on these findings, we make management recommendations for women with low-grade smears.     

Application of response surface methodology to cell immobilization for the production of palatinose

Response surface methodology (RSM), based on multivariate non-linear model, was applied to study the interactions and optimization of the immobilization parameters for cell entrapment, namely alginate concentration, cell loading and bead diameter using Erwinia rhapontici NCPPB 1578 that produced palatinose. ANOVA analysis and statistical parameters calculations showed that RSM could be used effectively to model and improve a complex system like cell immobilization. Palatinose yield was increased by 40%. The maximum yield of 140 mg/ml was achieved in a batch of 1h at alginate concentration of 5% w/v, cell loading of 5 g l(-1) and 2.25 mm bead diameter. Thus, the E. rhapontici NCPPB 1578 immobilization in alginate bead and subsequent palatinose yield was successfully improved by application of RSM technique.     

Extraction of inhibitor-free metagenomic DNA from polluted sediments, compatible with molecular diversity analysis using adsorption and ion-exchange treatments

PCR inhibitor-free metagenomic DNA of high quality and high yield was extracted from highly polluted sediments using a simple remediation strategy of adsorption and ion-exchange chromatography. Extraction procedure was optimized with series of steps, which involved gentle mechanical lysis, treatment with powdered activated charcoal (PAC) and ion-exchange chromatography with amberlite resin. Quality of the extracted DNA for molecular diversity analysis was tested by amplifying bacterial 16S rDNA (16S rRNA gene) with eubacterial specific universal primers (8f and 1492r), cloning of the amplified 16S rDNA and ARDRA (amplified rDNA restriction analysis) of the 16S rDNA clones. The presence of discrete differences in ARDRA banding profiles provided evidence for expediency of the DNA extraction protocol in molecular diversity studies. A comparison of the optimized protocol with commercial Ultraclean Soil DNA isolation kit suggested that method described in this report would be more efficient in removing metallic and organic inhibitors, from polluted sediment samples.     

Plc1p is required for SAGA recruitment and derepression of Sko1p-regulated genes

In Saccharomyces cerevisiae, many osmotically inducible genes are regulated by the Sko1p-Ssn6p-Tup1p complex. On osmotic shock, the MAP kinase Hog1p associates with this complex, phosphorylates Sko1p, and converts it into an activator that subsequently recruits Swi/Snf and SAGA complexes. We have found that phospholipase C (Plc1p encoded by PLC1) is required for derepression of Sko1p-Ssn6p-Tup1p-controlled osmoinducible genes upon osmotic shock. Although plc1Delta mutation affects the assembly of the preinitiation complex after osmotic shock, it does not affect the recruitment of Hog1p and Swi/Snf complex at these promoters. However, Plc1p facilitates osmotic shock-induced recruitment of the SAGA complex. Like plc1Delta cells, SAGA mutants are osmosensitive and display compromised expression of osmotically inducible genes. The reduced binding of SAGA to Sko1p-Ssn6p-Tup1p-repressed promoters in plc1Delta cells does not correlate with reduced histone acetylation. However, SAGA functions at these promoters to facilitate recruitment of the TATA-binding protein. The results thus provide evidence that Plc1p and inositol polyphosphates affect derepression of Sko1p-Ssn6p-Tup1p-controlled genes by a mechanism that involves recruitment of the SAGA complex and TATA-binding protein.     

Cytoplasmic localization of BAG-1 in leukoplakia and carcinoma of the tongue: correlation with p53 and c-erbB2 in carcinoma

BACKGROUND: The present study evaluated the clinical significance of BAG-1, an antiapoptotic protein, in leukoplakia and carcinoma of the tongue. METHODS: BAG-1 expression was evaluated by immunohistochemistry in paraffin-embedded tissues of leukoplakia (n=25) and carcinoma of the tongue (n=61). RESULTS: Cytoplasmic expression was predominantly seen in 80% and 70% of patients with leukoplakia and carcinoma, respectively. BAG-1 expression was found to be significantly lower in tobacco users than in non-tobacco users. BAG-1 expression in tobacco-using leukoplakia and carcinoma patients was compared by grouping the carcinoma patients according to lymph node status and disease stage. Carcinoma patients with tumor-positive lymph nodes had significantly lower BAG-1 expression than patients with negative lymph nodes and leukoplakia. Further, a trend towards an inverse correlation was observed with p53 and c-erbB2. In univariate and multivariate survival analysis, patient subgroups with 2+ or 3+ marker positivity (BAG-1 negativity, p53 and c-erbB2 positivity) had a reduced overall survival compared with patient subgroups with 1+ marker positivity or negativity. CONCLUSION: BAG-1 negativity in association with p53 and c-erbB2 positivity identified a subgroup of tongue cancer patients with an aggressive phenotype. Hence, an antiapoptotic protein, BAG-1, was found to be down-regulated in chewing-tobacco-mediated tongue carcinogenesis.     

Anillin and the septins promote asymmetric ingression of the cytokinetic furrow

During cytokinesis, constriction of a cortical contractile ring generates a furrow that partitions one cell into two. The contractile ring contains three filament systems: actin, bipolar myosin II filaments, and septins, GTP-binding hetero-oligomers that polymerize to form a membrane-associated lattice. The contractile ring also contains a potential filament crosslinker, Anillin, that binds all three filament types. Here, we show that the contractile ring possesses an intrinsic symmetry-breaking mechanism that promotes asymmetric furrowing. Asymmetric ingression requires Anillin and the septins, which promote the coalescence of components on one side of the contractile ring, but is insensitive to a 10-fold reduction in myosin II levels. When asymmetry is disrupted, cytokinesis becomes sensitive to partial inhibition of contractility. Thus, asymmetric furrow ingression, a prevalent but previously unexplored feature of cell division in metazoans, is generated by the action of two conserved furrow components and serves a mechanical function that makes cytokinesis robust.