Assessment of Bt trait purity in different generations of transgenic cottons.

Adequate expression of Bt (Bacillus thuringiensis) toxins and purity of seeds of Bt-transgenic cottons are important for controlling bollworms, and thereby increasing the cotton productivity. Therefore, we examined the variability in expression of Bt toxin proteins in the seeds and in leaves of different cotton (Gossypium hirsutum (L.) hybrids (JKCH 226, JKCH 1947, JKCH Durga, JKCH Ishwar, JKCH Varun KDCHH 441 and KDCHH 621) expressing Bt toxins in F1 and F2 generations, using bioassays against the cotton bollworm, Helicoverpa armigera (Hübner), and the lateral flow strip (LFS) test. Toxicity of Bt toxin proteins in the seeds of Bt-transgenic cottons to H. armigera correlated with their toxicity in the leaves in one- toxin Bt cotton hybrids. The Bt-F1 and Bt-F2 seeds of JKCH 1947 were more toxic to H. armigera than those of JKCH Varun seeds. The seeds and leaves of F1s showed greater toxicity than the F2 seeds or leaves of one-toxin (cry1Ac) Bt cotton hybrids. However, no significant differences were observed for the two-toxin (cry1Ac and cry2Ab) hybrid, KDCHH 621. Toxicity of leaves to H. armigera increased with crop age, until 112 days after seedling emergence. The Bt trait purity in F1 seeds of four two-toxin Bt cotton hybrids ranged from 86.7 to 100%. The present study emphasizes the necessity of 95% Bt trait purity in seeds of transgenic cotton for sustainable crop production.

Identification and characterization of the Sudanese Bacillus thuringiensis and related bacterial strains for their efficacy against Helicoverpa armigera and Tribolium castaneum.

Forty-four isolates of Bacillus thuringiensis like bacteria from various sources in different locations from Sudan were tested for their insecticidal activity. The toxicity of these isolates ranged from 6.6 to 70% to the neonates of cotton bollworm, Helicoverpa armigera at 10 ppm concentration. The most effective ones are Kb-29, St-6 and Wh-1 comparable with HD-1. Toxicity of isolates to larvae of the red flour beetle, Tribolium castaneum ranged from 20 to 100%. Isolates St-2 and St-23 gave 100% larval mortality within 15 days of exposure and were at par with Ab-8, Ab-12, Kb-26, Kb-30, Om-4, Po-2, Po-5, Po-7, Sa-8 and Wh-5 and were also comparable with E. coli clone expressing Cry3 toxin. The most effective five isolates viz., Kb-29, St-2, St-6, St-23 and Wh-1 belonged to B. thuringiensis. The St-6 isolate, which also showed high toxicity to T. castaneum larvae, had cry1 genes along with coleopteran active cry28 genes, but not cry3 genes. Of the 25 isolates characterized with 16s DNA sequencing, seven belonged to Paenibacillus spp., one Lysinibacillus sphaericus, one Bacillus pumilus, four Bacillus spp., and rest 12 belonged to B. thuringiensis. Biochemical characterization in each species showed variation. The present study shows potential of some isolates like Kb-29, St-2, St-6, St-23 and Wh-1 as promising bioinsecticides.

Potential of Bacillus sp. to produce polyhydroxybutyrate from biowaste.

AIM: To test the Bacillus strains for their abilities to produce polyhydroxybutyrate (PHB) from different sugars and biowaste (Pea-shells). METHODS AND RESULTS: Six Bacillus strains were checked for their ability to produce PHB from GM2 medium supplemented with different sugars at the rate of 1% (w/v) and from biowaste and GM2 (BW : M) combinations (3 : 7, 1 : 1, 7 : 3). Glucose supplemented GM2 medium resulted in maximum PHB production of 435 mg l(-1) constituting 31-62% w/w of the total cell dry mass. Substituting GM2 medium to the extent of 50% with biowaste (pea-shell slurry) resulted in 945-1205 mg l(-1) PHB (55-65% w/w). Optimization for additional nitrogen supplementation, inoculum size resulted in a final PHB production of 3010-3370 mg l(-1) equivalent to 300 g kg(-1) biowaste (dry wt). CONCLUSION: The Bacillus strains were able to produce PHB from biowaste (Pea-shells) as cheap source of substrate. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on usage of pea-shells as feed for PHB production, opening new possibilities for its use for production of PHB and Bacillus as potential candidate for the purpose.

Assessing the susceptibility of cruciferous Lepidoptera to Cry1Ba2 and Cry1Ca4 for future transgenic cruciferous vegetables.

Advances in transgenic plants expressing Bacillus thuringiensis (Bt) insecticidal gene(s) offer a promising alternative to traditional insecticides for control of lepidopteran pests on important cruciferous vegetable crops such as cabbage and cauliflower. A public-private partnership, the Collaboration on Insect Management for Brassicas in Asia and Africa (CIMBAA), was formed in 2005 with the goal of developing dual-gene Bt cauliflower and cabbage, initially for India, to replace the use of broad spectrum, traditional insecticides. As a first step in this effort, the major lepidopteran pests of cruciferous vegetable crops [Plutella xylostella (L.), Pieris rapae (L.), Pieris brassicae (L.), Crocidolomia binotalis (L.), Hellula undalis (F.), Diacrisia obliqua Walker, Spodoptera litura F., and Helicoverpa armigera (Hübner)] were collected over a large geographic area (India, Indonesia, Taiwan, China, Australia, and the United States) and tested against purified Cry1Ba2 and Cry1Ca4 toxins, the toxins proposed to be expressed in the CIMBAA plants. Our results demonstrate that Cry1Ba2 and Cry1Ca4 were effective against the primary target of the CIMBAA plants, P. xylostella, regardless of geographic location, and had LC50 values <1.3 ppm. Furthermore, one or both toxins were effective against the other major pest Lepidoptera, except for S. litura or H. armigera which were less susceptible. No cross-resistance has been found between Cry1Ba2 and Cry1Ca4, suggesting cry1Ba2+cry1Ca4 cauliflower and cabbage could be an effective and sustainable tool to control, P. xylostella, the key lepidopteran pest on cruciferous vegetable crops, as well as most other Lepidoptera. As the CIMBAA plants are being developed, further tests are needed to determine whether they will express these proteins at sufficient levels to control all the Lepidoptera. Sustainable use of the dual-gene plants also is discussed.

Optimizing radiotherapy of brain tumours by a combination of temozolomide & lonidamine.

BACKGROUND & OBJECTIVE: Temozolomide (TMZ), a second generation alkylating drug, an effective cytotoxic agent as well as radiosensitizer for malignant brain tumours, has side effects like myelosuppression. Lonidamine (LND) increases the effectiveness of several experimental multiple chemotherapy protocols, without increasing bone marrow toxicities and is effective in brain tumour patients. The objective of the present studies was to investigate whether combining clinically relevant doses of LND and TMZ could increase the proliferation and radiation response of malignant human brain tumour cells in vitro. METHODS: A malignant human glioma (U373MG) cell line was used in these studies. TMZ (20, 40 or 60 microM) or LND (100, 150 or 200 microM), or the combination of both (20 and 100 microM, respectively) in 0.1 per cent dimethyl sulphoxide (DMSO) were added three days after setting up cultures, in six well plates (5 x 10(4) cells/ well). The effects of continuous treatment for two days on proliferation response and cytotoxicity were studied after trypsinization; by cell counts and the uptake of trypan blue dye (0.5%). For the study of radiation (60Co-Gamma-rays, 2 Gy) response, drugs were removed 4 h after irradiation and cultures were grown further in drug free, normal growth medium for another 20 h or 44 h. RESULTS: Continuous presence of TMZ or LND for two days significantly inhibited cell proliferation in a concentration dependent manner. The frequencies of non viable cells increased significantly only at higher concentrations of LND. Combination of 20 microM TMZ with 100 microM LND had additive effects on proliferation response, without affecting cell viability. Short-term drug treatments without irradiation did not induce micronuclei formation. Cell proliferation and viability were also not affected. However, post-irradiation presence of either of these drugs for 4 h significantly reduced the proliferation response, 24 and 48 h after treatments. It was further inhibited by the combination treatment. On the contrary, radiation induced micronuclei formation was enhanced by either of the drugs; which was significantly increased by the combined treatment, 24 h as well as 48 h after irradiation. No effects on cell viability were observed, immediately after these treatments as well as at later time points. INTERPRETATION & CONCLUSION: Our findings showed that combination of TMZ and LND at clinically achievable, low plasma concentrations could inhibit tumour growth, and lonidamine could reduce the dose of temozolomide required for radiosensitization of brain tumours.

Greenstick fracture of the mandible: a case report.

This case report is an insight in to pediatric traumatology whereby bilateral greenstick fracture of condyle is used as a means to discuss the incidence and anatomic considerations for the management of the same, highlighting the fact that dental surgeons require a unique understanding of the anatomy, growth considerations, healing pattern and operative management involving minimal manipulation while managing pediatric facial fractures.

Effects of 2-deoxy-D-glucose on glycolysis, proliferation kinetics and radiation response of human cancer cells.

The effects of 2-deoxy-D-glucose (2-DG) on energy metabolism, cell proliferation kinetics, radiation-induced DNA repair, and micronuclei formation in HeLa cells have been studied. Results show that the 2-DG induced modifications of the radiation effects are biphasic: at high 2-DG concentrations (greater than 2.5 mM), DNA repair is inhibited and manifestation of radiation damage is enhanced as observed by an increase in the radiation (X ray) induced micronuclei formation; lower concentrations of 2-DG (less than 2.5 mM) do not inhibit DNA repair and a decrease in the frequency of micronuclei formation is observed. These data, in correlation with the effects of 2-DG on glycolysis and cell proliferation kinetics, can be explained by the hypothesis that 2-DG induced modifications of radiation effects arise as a result of energy linked differential inhibitions of pathways of repair and fixation of DNA damage. Implications for cancer therapy are discussed.

Improving cancer radiotherapy with 2-deoxy-D-glucose: phase I/II clinical trials on human cerebral gliomas.

PURPOSE: Evaluation of tolerance, toxicity, and feasibility of combining large fraction (5 Gy) radiotherapy with 2-deoxy-D-glucose (2DG), an inhibitor of glucose transport and glycolysis, which has been shown to differentially inhibit repair of radiation damage in cancer cells. METHODS AND MATERIALS: Twenty patients with supratentorial glioma (Grade 3/4), following surgery were treated with four weekly fractions of oral 2DG (200 mg/kg body weight) followed by whole brain irradiation (5 Gy). Two weeks later, supplement focal radiation to the tumor (14 Gy/7 fractions) was given. Routine clinical evaluation, x-ray computerized tomography (CT), and magnetic resonance (MR) imaging were carried out to study the acute and late radiation effects. RESULTS: All the 20 patients completed the treatment without any interruption. The vital parameters were within normal limits during the treatment. None reported headache during the treatment. Mild to moderate nausea and vomiting were observed during the days of combined therapy (2DG + RT) in 10 patients. No significant deterioration of the neurological status was observed during the treatment period. Seven patients were alive at 63, 43, 36, 28, 27, 19, and 18 months of follow-up. In these patients, the clinical and MR imaging studies did not reveal any late radiation effects. CONCLUSIONS: Feasibility of administering the treatment (2DG + 5 Gy) is demonstrated by the excellent tolerance observed in all 20 patients. Further, the clinical and MR studies also show the absence of any brain parenchymal damage.

HIV gp120 V(1)/V(2) and C(2)-V(3) domains glycoprotein compatibility is required for viral replication.

The envelope gene, especially the V(3) region, of HIV-1 has been shown to be a principal determinant of cell tropism, replication and cytopathogenicity of the virus. In addition, the V(1)/V(2) region of the envelope gene has been found to be an important factor in cell tropism. We examined the compatibility between the V(1)/V(2) and C(2)-V(3) domains of HIV-1 gp120 in different combinations on viral replication by using envelope recombinants between ME1 and ME46, two infectious molecular clones with diverse biologic activity longitudinally isolated from one seropositive subject. Our data demonstrate that a proper interaction between the regions of V(1)/V(2)and C(2) is essential for viral infection and hence replication. Sequence analysis and subsequent site directed mutagenesis study indicate that the pattern of potential envelope N-glycosylation in the V(1)/V(2) and C(2)-V(3) regions may be the determining factor in such interaction between these two regions. It is possible that improper N-glycosylation sites while not affecting virus assembly, can influence through steric hindrance the conformational change of the V(3) region that is required for the co-receptor attachment and hence the viral infectivity.

Radiation-induced DNA damage in tumors and normal tissues. VI. Estimation of the hypoxic fraction of experimental tumors.

For several years, we have concentrated our efforts on validating the use of radiation-induced DNA strand breaks and DNA-protein crosslinks to assess the oxygenation status of tumors and normal tissues. We have demonstrated that (1) the oxygen dependence of strand break formation is identical to that of radiation-induced cell killing; (2) the oxygen dependence of DNA-protein crosslink formation is the mirror image of that of radiation-induced cell killing; and (3) the formation of these radiation-induced DNA lesions is predominantly dependent on the oxygen concentration near the DNA and is independent of the cell type, metabolic status, proliferative status, pH of the surrounding environment, and composition or properties of the proteins tightly associated with the DNA. In the present study, the hypoxic fraction of three experimental tumors was estimated using our assay of radiation-induced DNA damage. The average hypoxic fraction of a large number of tumors estimated with this assay of radiation-induced DNA damage for (1) WiDR human colorectal carcinoma xenografts (40.8 +/- 4.2%), (2) 66 mouse mammary adenocarcinoma tumors (41.8 +/- 3.1%), and (3) subcutaneous tumors grown from 9L rat brain tumor cells (95% CI =-8.2-4.2%) was not statistically different from that of a large number of tumors measured for each of these tumor models by the paired survival curve method (38.3 +/- 6. 3%, 28.9 +/- 5.5%, 95% CI = 2.2-4.4%, respectively). When the hypoxic fraction measured by the alkaline elution method on one half of an individual tumor was compared to that measured by the paired survival curve method on the other half of the same tumor, no statistical correlation was found for either 66 or WiDR tumors. Although this assay of radiation-induced DNA damage can be used effectively in the laboratory to answer a number of important questions about the oxygenation status of animal tumors and normal tissues, failure to reliably estimate the hypoxic fraction of individual tumors and technical considerations make it unlikely that the assay can be used in the clinic to estimate the hypoxic fraction of human tumors.

Antenatal prediction of postpartum urinary and fecal incontinence.

OBJECTIVE: To investigate the effect of pregnancy and delivery on continence and to assess whether physical markers of collagen weakness can predict postpartum urinary and fecal incontinence (including incontinence of flatus). METHODS: In a prospective, longitudinal study in a London teaching hospital, 549 nulliparas were interviewed after 34 weeks' gestation and again 3 months postpartum regarding urinary and fecal symptoms before and during pregnancy and after delivery. Family histories of incontinence, prolapse, and collagen abnormalities were recorded also. Physical examination was done to assess markers of collagen weakness such as striae, hernia, varicose veins, and joint mobility. RESULTS: The prevalence of urinary incontinence before, during, and after pregnancy was 3.6%, 43.7%, and 14.6%, and rates of fecal incontinence were 0.7%, 6.0%, and 5.5%, respectively. Fecal urgency was more common in women who had spontaneous and instrument-assisted vaginal deliveries (n = 413) compared with cesareans (n = 131) (7.3% versus 3.1%; P = .046). Postnatal urinary or fecal dysfunction was not related to antenatal body mass index, smoking, race, striae, varicose veins, hemorrhoids, or family history of incontinence. Higher joint-mobility scores were associated with incontinence of flatus (P = .021) but not with other urinary or fecal symptoms. CONCLUSION: Although collagen weakness was previously implicated in the pathogenesis of incontinence, physical markers in this study could not predict postpartum urinary and fecal incontinence. Either those markers were not representative of collagen weakness, or a larger study with longer follow-up is necessary.

alpha-Melanocyte-stimulating hormone (alpha-MSH) and melanin-concentrating hormone (MCH) modify monoaminergic levels in the preoptic area of the rat.

The effect of perfusion of melanin-concentrating hormone (MCH) or alpha-melanocyte-stimulating hormone (alpha-MSH) (100 ng/microliter) in the ventromedial nucleus (VMN) or medial preoptic area (MPOA) on monoaminergic levels of female rats was measured using microdialysis and HPLC-electrochemical detection. In the MPOA, alpha-MSH raised 5-HIAA concentration, whereas MCH reduced both 5-HT and 5-HIAA. Neither peptide had any effect in the VMN. The opposite effects of the peptides on the serotonergic system might be responsible for their antagonistic or opposite actions previously reported on several CNS functions. Dopamine may mediate the similar effects of the two peptides, because alpha-MSH inhibits dopaminergic release in the MPOA (but not VMN) and MCH tends to follow the same pattern.