Expression of a Chimeric Gene Encoding Insecticidal Crystal Protein Cry1Aabc of Bacillus thuringiensis in Chickpea (Cicer arietinum L.) Confers Resistance to Gram Pod Borer (Helicoverpa armigera Hubner.).

Domain swapping and generation of chimeric insecticidal crystal protein is an emerging area of insect pest management. The lepidopteran insect pest, gram pod borer (Helicoverpa armigera H.) wreaks havoc to chickpea crop affecting production. Lepidopteran insects were reported to be controlled by Bt (cryI) genes. We designed a plant codon optimized chimeric Bt gene (cry1Aabc) using three domains from three different cry1A genes (domains I, II, and III from cry1Aa, cry1Ab, and cry1Ac, respectively) and expressed it under the control of a constitutive promoter in chickpea (cv. DCP92-3) to assess its effect on gram pod borer. A total of six transgenic chickpea shoots were established by grafting into mature fertile plants. The in vitro regenerated (organogenetic) shoots were selected based on antibiotic kanamycin monosulfate (100 mg/L) with transformation efficiency of 0.076%. Three transgenic events were extensively studied based on gene expression pattern and insect mortality across generations. Protein expression in pod walls, immature seeds and leaves (pre- and post-flowering) were estimated and expression in pre-flowering stage was found higher than that of post-flowering. Analysis for the stable integration, expression and insect mortality (detached leaf and whole plant bioassay) led to identification of efficacious transgenic chickpea lines. The chimeric cry1Aabc expressed in chickpea is effective against gram pod borer and generated events can be utilized in transgenic breeding program.

Isolated proximal rupture of flexor digitorum longus tendon in a traumatic open subtalar dislocation.

Tendon ruptures associated with ankle fractures and dislocations or subtalar dislocations are very rare entities with only a few reports of these in the literature. We report a case of an open subtalar dislocation and associated isolated proximal rupture of the flexor digitorum longus tendon at the musculotendinous junction, following a relatively low energy trauma. The finding of the avulsed flexor digitorum longus tendon was intraoperative, and management involved thorough washout of the joint and a primary side-to-side tenodesis with the flexor hallucis longus tendon. The patient has made a good functional recovery from his injuries. This case is reported because of the rarity of this combination of injuries and the associated management dilemma with which it presented us.

Binding of Bacillus thuringiensis Cry1A toxins with brush border membrane vesicles of maize stem borer (Chilo partellus Swinhoe).

Maize stem borer (Chilo partellus) is a major insect pest of maize and sorghum in Asia and Africa. Bacillus thuringiensis (Bt) δ-endotoxins have been found effective against C. partellus, both in diet-overlay assay and in transgenic plants. Gene stacking as one of the resistance management strategies in Bt maize requires an understanding of receptor sharing and binding affinity of δ-endotoxins. In the present study, binding affinity of three fluorescein isothiocyanate labeled Cry1A toxins showed high correlation with the toxicity of respective δ-endotoxins. Competitive binding studies showed that Cry1Ab toxins share some of the binding sites with Cry1Aa and Cry1Ac with low affinity and that Cry1Ab may have additional binding sites that are unavailable to the other two toxins tested.

In vitro alpha-amylase inhibition and in vivo antioxidant potential of Amaranthus spinosus in alloxan-induced oxidative stress in diabetic rats.

Amaranthus spinosus Linn. (Amaranthaceae), commonly known as "Mulluharivesoppu" in Kannada, is used in the Indian traditional system of medicine for the treatment of diabetes. The present study deals with the scientific evaluation of alpha amylase and the antioxidant potential of methanol extract of A. spinosus (MEAS). The aim of this study was to investigate in vitro alpha-amylase enzyme inhibition by CNPG3 (2-chloro-4-nitrophenol α-d-maltotrioside) and in vivo antioxidant potential of malondialdehyde (MDA), glutathione (GSH), catalase (CAT) and total thiols (TT) in alloxan-induced diabetic rats of a methanolic extract of A. spinosus. Blood sugar was also determined in MEAS-treated alloxan-induced diabetic rats. MEAS showed significant inhibition of alpha-amylase activity and IC50 46.02 µg/ml. Oral administration of MEAS (200 and 400 mg/kg) for 15 days showed significant reduction in the elevated blood glucose, MDA and restores GSH, CAT and TT levels as compared with a diabetic control. The present study provides evidence that the methanolic extract of A. spinosus has potent alpha amylase, anti-diabetic and antioxidant activities.

Synergistic activity between Bacillus thuringiensis Cry1Ab and Cry1Ac toxins against maize stem borer (Chilo partellus Swinhoe).

AIM: To select a toxin combination for the management of maize stem borer (Chilo partellus) and to understand possible mechanism of synergism among Bacillus thuringiensis Cry1A toxins tested. METHODS AND RESULTS: Three Cry1A toxins were over expressed in Escherichia coli strain JM105 and used for diet overlay insect bioassay against C. partellus neonate larvae, both alone and in combinations. Probit analysis revealed that the three Cry1A toxins tested have synergistic effect against C. partellus larvae. In vitro binding analysis of fluorescein isothiocyanate (FITC)-labelled Cry1A toxins to midgut brush border membrane vesicle (BBMV) shows that increase in toxicity is directly correlated to an increase in binding of toxin mix. CONCLUSIONS: A high Cry1Ac to Cry1Ab ratio leads to an increase in efficacy of these toxins towards C. partellus larvae and this increase in toxicity comes from an increase in toxin binding. SIGNIFICANCE AND IMPACT OF THE STUDY: Use of Cry1Ab and Cry1Ac combination could be an effective approach to control C. partellus. Furthermore, we show it first time that possible reason behind increase in toxicity of synergistic Cry1A proteins is an increase in toxin binding.

Isolation and characterization of a green tissue-specific promoter from pigeonpea [Cajanus cajan (L.) Millsp.].

Expression of rbcS genes encoding small subunit of rubisco, most abundant protein in green tissue, is regulated by at least three parameters--tissue type, light conditions and stage of development. One of the green tissue-specific promoters of rbcS gene family was isolated from pigeonpea by PCR. Expression of uidA gene encoding beta-glucuronidase in the transgenic tobacco plants under the control of pigeonpea rbcS promoter, clearly showed that this promoter was as strong as pea rbcS3A promoter characterized earlier. Study of the sequence similarity with pea rbcS3A promoter, especially the region (boxes I and III) that is required for rbcS3A expression, showed more than 50% divergence. In contrast, pigeonpea promoter sequence isolated in the present study was more similar to that of spinach and rice rbcS promoters.

Genotoxicity evaluation of polluted ground water in human peripheral blood lymphocytes using the comet assay.

This paper presents the genotoxicity experiments with the ground water collected from an area under the influence of textile dyeing and bleaching industries in Tirupur, Tamilnadu, India. The alkaline single cell gel electrophoresis (SCGE) assay was performed in vitro with human peripheral blood lymphocytes. The cells were exposed to two doses of non-volatile organic agents extracted from ground water samples. Ground water samples were collected from 12 locations distributed in and around Tirupur and extracts were taken at different pHs (without pH adjustment and acidic pH 2.0). The persistence of the DNA damage after exposure to the organic extracts was also studied. All the samples were found to contain substances capable of inducing DNA damage in human lymphocytes. Extracts from acidified waters (pH=2.0) were found to induce more DNA damage than extracts from without pH adjustment (natural pH). The DNA damage was not fully repaired after incubation for 2h at 37 degrees C. The chemical characterization of the sub-fractions revealed the existence of aromatic amines in the extracts, which may be responsible for the DNA damaging activity of the water samples. The results of this investigation demonstrate the application of the comet assay in environmental monitoring studies.

Cross-face nerve graft with free-muscle transfer for reanimation of the paralyzed face: a comparative study of the single-stage and two-stage procedures.

The most accepted method for reanimation of the paralyzed face is the two-stage method that combines cross-face nerve graft with free-muscle transfer. Although the results of reconstruction with this method are satisfactory, there is an excessive delay between stages, which prolongs the period of rehabilitation. In 1995, Kumar overcame this and presented his preliminary results from a single-stage transfer of the gracilis. We compared the long-term results of the single-stage with the two-stage method of reconstruction. Patients selected the method of reconstruction on the basis of the information, photographs of scars, and video recording of results given to them at a special facial palsy clinic. Ten patients selected the single-stage reconstruction and 15 selected the two-stage reconstruction. The mean follow-up period was 3 years. The results of the two methods are comparable, with 90 percent of the patients who underwent the single-stage and 93 percent of those who underwent the two-stage procedures having good and fair results. However, the two-stage method scored good symmetry at rest in 67 percent, compared with 20 percent for the single-stage method. There were fewer complications with the single-stage method, and none of the patients had any problems relating to the donor site. The period of rehabilitation was reduced by 10 months with the single-stage method.

Expression of type I adenylyl cyclase in intrinsic pathways of the hippocampal formation of the macaque (Macaca nemestrina).

The mossy fiber pathway of the hippocampal formation and type 1 adenylyl cyclase (AC1) have been implicated in long-term potentiation and memory function. Using immunohistochemical labeling and light microscopy we demonstrated intense labeling of AC1 in the mossy fibers and less intense labeling in the molecular layers of both the dentate gyrus and fields CA1, CA2 and CA3 of the hippocampus, i.e. in terminal fields of the perforant pathway. These findings indicate that, in the non-human primate, AC1 is found in the mossy fibers and in terminal fields of the perforant pathway where it may play a role in long term potentiation similar to that demonstrated in the rodent.

Genotoxicity of some sulfur dyes on tadpoles (Rana hexadactyla) measured using the comet assay.

This report presents the results of a genotoxicity study to evaluate the DNA damage caused by four sulfur dyes used in the textile and tannery industries. Alkaline single-cell gel electrophoresis assay (SCGE) was performed on erythrocytes from Rana hexadactyla tadpoles following whole-body exposure to increasing concentrations of the dyes. The dyes, along with their active ingredients, were Sandopel Basic Black BHLN, Negrosine, Dermapel Black FNI, and Turquoise Blue. The dye-treated tadpoles showed significant DNA damage, measured as mean DNA length:width ratio, when compared with unexposed control animals. Among the four tested dyes Sandopel Basic Black BHLN appears to be highly genotoxic, Dermapel Black FNI was least genotoxic, and Negrosine and Turquoise Blue were moderately toxic to R. hexadactyla tadpoles. The tadpoles showed a significant reduction in DNA damage when placed in dechlorinated tap water after exposure for a 24-hr period to the dye solutions.

Characterization of genetic diversity of some serovars of Bacillus thuringiensis by RAPD.

RAPD based fingerprinting of 21 serovars of Bacillus thuringiensis (Bt) representing different serotypes was performed using 19 random decamer primers. A total of 172 polymorphic fragments, ranging in size from 161-2789 bp, were amplified from 13 of the 19 primers. Pairwise genetic similarity analysis revealed very low similarity values, ranging from 3-68%, among the serovars of Bt, indicating high genetic divergence. Nineteen serovars of Bt fell in two major clusters and remaining two formed solitary clusters in the dendogram. Clustering of Bt strains established genetic relatedness between serovars and serotypes. It has been suggested that RAPD analysis can be used for genotypic characterization of Bt to complement flagellar serotyping.

Amino acid substitution in alpha-helix 7 of Cry1Ac delta-endotoxin of Bacillus thuringiensis leads to enhanced toxicity to Helicoverpa armigera Hubner.

Insecticidal proteins or delta-endotoxins of Bacillus thuringiensis are highly toxic to a wide range of agronomically important pests. The toxins are formed of three structural domains. The N-terminal domain is a bundle of eight alpha-helices and is implicated in pore formation in insect midgut epithelial membranes. All the delta-endotoxins share a common hydrophobic motif of eight amino acids in alpha-helix 7. A similar motif is also present in fragment B of diphtheria toxin (DT). Site-directed mutagenesis of Cry1Ac delta-endotoxin of B. thuringiensis was carried out to substitute its hydrophobic motif with that of DT fragment B. The mutant toxin was shown to be more toxic to the larvae of Helicoverpa armigera (cotton bollworm) than the wild-type toxin. Voltage clamp analysis with planar lipid bilayers revealed that the mutant toxin opens larger ion channels and induces higher levels of conductance than the wild-type toxin.

Effect of immunostimulation on cytotoxic activity of intestinal intraepithelial lymphocytes of chickens in infectious bursal disease and Eimeria tenella infections.

In chickens, intestinal intraepithelial lymphocytes (iIELs) exhibit spontaneous natural killer (NK) cell like activity, by which they are active in the first line of defence on gut epithelial linings. In the present study, the cytotoxic activity of iIELs was found to be drastically suppressed in chickens experimentally infected with infectious bursal disease (IBD) virus at the age of 5 weeks and also in chickens experimentally infected with Eimeria tenella oocysts at the age of 8 weeks (p < 0.01). As nonspecific immunostimulation is gaining importance, immunostimulants such as immunostimulating Mycobacterium phlei (ISMP) and bone marrow culture supernatant (BMCS) were tested for their influence on the functional activity of iIELs of chickens in IBD and E. tenella infections. In chickens primed with ISMP a week prior to respective experimental infections, it was found that the cytotoxic activity of iIELs was restored (p < 0.01) in both IBD and E. tenella infections. At the same time, in chickens primed with BMCS a week prior to respective experimental infections, the cytotoxic activity of iIELs was restored to a certain extent (p < 0.01) in E. tenella but not at all in IBD infection. These results showed that application of immunostimulation helped potentiate and restore the functional activity of iIELs of chickens in IBD and E. tenella infections.

Zinc status of breastfed and formula-fed infants of different gestational ages.

Zinc status in 186 full term and preterm infants was determined at birth, and 3, 6, 9, and 12 months of age along with determination of zinc levels in breast or formula milk to find out if routine zinc supplements are needed during infancy. The leukocyte and plasma zinc levels in all breastfed infants were high at birth and gradually declined reaching lowest values by 4-6 months of age, and improved to normal levels by 9 months following weaning. The preterm infants however, had significantly (P < 0.05) higher leukocyte zinc (213.6 +/- 46.91 micrograms/10(10) cells) at birth compared to term infants. Colostrum of all the mothers had higher zinc concentrations which declined to significantly lower levels in breastmilk by 4-6 months of lactation, corresponding to the age when the breastfed infants had lower zinc levels. The improvement of the levels to normal after weaning suggests that the fall in zinc status during early infancy could be a transient phenomenon which could be reversed by proper weaning, thus strengthening the plea for timely food supplements rather than the need for single nutrient supplements. Formula-fed full term infants had significantly lower leukocyte zinc levels (49.3 +/- 2.59 micrograms/10(10) cells) at 3 months of age compared to breastfed infants of the same age (92.8 +/- 14.04 micrograms/10(10) cells). Even these infants improved their zinc status after weaning on par with breastfed infants. The functional significance of their transient, but low zinc values during early infancy needs to be investigated.

A colorimetric assay to evaluate the cytotoxic activity of the intestinal intraepithelial lymphocytes of chickens.

After the successful use of 3-[4,5-(dimethylthiazol-2-yl)]-2,5-diphenyltetrazolium bromide (MTT) in cell proliferation assays, its use has been established by different workers in cytotoxicity assays and research on leukaemia. In the present study, a colorimetric assay using MTT was adopted to evaluate the cytotoxic activity of chicken intestinal intraepithelial lymphocytes (iIELs), which constitute an important cellular component of the gut-associated lymphoid tissue (GALT). These iIELs are found to exhibit natural killer (NK) cell-like cytotoxic activity, which is spontaneous, non-MHC-restricted, and does not need to be primed. Hitherto, conventional chromium-release assays have been used to evaluate the cytotoxic activity of iIELs, but these assays have disadvantages such as radiation hazards and loss of the cells in washing steps. The mean percentage cytotoxic activity of chicken iIELs evaluated by the colorimetric assay was 90.37 +/- 2.53 in a group of 5-week-old chickens and 80.2 +/- 3.45 in a group of 8-week-old chickens. These findings established the successful use of a colorimetric assay using MTT for evaluating the cytotoxic activity of chickens iIELs.

Cross-face reanimation of the paralysed face, with a single stage microneurovascular gracilis transfer without nerve graft: a preliminary report.

This paper describes a method of cross-face reanimation of the paralysed face by a single stage transfer of gracilis. In this method, the nerve to the gracilis is kept long and tunnelled across the upper lip and joined directly to branches of the facial nerve on the normal side. The method avoids the need for a nerve graft and shortens the period of rehabilitation by 12 months. The results in 3 cases were good in 2 cases and fair in 1 case.

Integration of a cyanobacterial protein involved in nitrate reduction (narB) into isolated Synechococcus but not into pea thylakoid membranes.

Chimeric genes comprised of Rubisco small subunit transit peptide fused in frame with full-length and truncated sequences of a nitrate reductase (narB) structural gene of Synechococcus were constructed. Fusion proteins were synthesized in a rabbit reticulocyte system. In thylakoido integration of synthetic proteins resulted in the association of the full-length narB-coded protein to the Synechococcus photosynthetic membranes. The membrane-associated protein was sensitive to trypsin treatment but could not be removed by washing in the presence of NaBr. Trypsin pretreatment of thylakoids abolished the capability for association. The association of the narB-coded protein with thylakoids might require another membrane protein whose identity is not known. It is proposed that the Synechococcus narB polypeptide is a peripheral, membrane bound protein anchored to the thylakoids via a short hydrophobic domain while the major part of the protein resides on the outer side of the thylakoid membranes. The chimeric narB proteins were processed and imported by intact pea chloroplasts in vitro; however, the mature proteins were found localized in the stroma and not in the thylakoid membrane fraction. Similarly, the attempt to integrate the protein in vitro into isolated pea thylakoid membranes failed although these membranes incorporate early light-inducible proteins.