Adventitious root induction in Ophiorrhiza prostrata: a tool for the production of camptothecin (an anticancer drug) and rapid propagation

Roots of Ophiorrhiza prostrata D. Don serve as a rich source of camptothecin (CPT), an anticancer drug. Because of the large-scale collection of its roots, the plant has become a threatened species. The present study accomplishes the induction of adventitious roots as a means for the production of CPT as well as for the large-scale propagation of this anticancer drug plant using leaf and internode explants. The biomass yield and CPT content of adventitious roots induced from different explants were compared to roots developed on ex vitro rooted stem cuttings. Adventitious roots were produced on half-strength Murashige and Skoog (MS) medium supplemented with 10.74 μM α-naphthaleneacetic acid and 2.32 μM kinetin at mean fresh weights of 0.753, 0.739 and 0.748 g roots from leaf, internode and shoot, respectively. CPT yield from in vitro derived roots after 50, 80 and 120 days of incubation (0.028, 0.06 and 0.1% dry weight, respectively) was not significantly different from those harvested at the same age from ex vitro rooted (0.03, 0.06 and 0.13%, respectively) stem cuttings. CPT from subcultured roots derived from solid (0.08%) medium was lower than from suspension culture medium (0.12%). Subsequent cultures of the adventitious roots showed a stable production of CPT (0.16%). The yield of CPT from 360-day-old plant-derived roots was 0.19%. Elicitation using methyl jasmonate and acetyl salicylic acid exhibited no enhancement in CPT yield. In vitro propagation through direct shoot regeneration was achieved from the adventitious roots upon transfer to MS medium with 8.87 μM N ⁶-benzyladenine (BA) and 2.46 μM indole-3-butyric acid (IBA) with a mean of 21.2 shoots per culture in 50 days. The shoots upon subculture on medium having the same level of BA and IBA underwent rapid proliferation. The shoots transferred to field conditions after in vitro rooting exhibited 95% survival. Adventitious root induction, from leaf and internode explants, enables the feasible production of CPT as well as the large-scale rapid propagation of this species which can safeguard it from extinction.     

Negative selection of Plasmodium falciparum reveals targeted gene deletion by double crossover recombination.

The genome sequence of Plasmodium falciparum, the causative agent of the most severe form of malaria in humans, rapidly approaches completion, but our ability to genetically manipulate this organism remains limited. Chromosomal integration has only been achieved following the prolonged maintenance of circularised episomal plasmids which selects for single crossover recombinants. It has not been possible to construct genetic deletions via double crossover recombination, presumably due to the low frequency of this event. We have used the Herpes simplex virus thymidine kinase gene and the Escherichia coli cytosine deaminase gene for negative selection of P. falciparum. Parasites were transformed with plasmids expressing the thymidine kinase and cytosine deaminase genes by positive selection for the human dihydrofolate reductase gene. Parasites expressing thymidine kinase are susceptible to the pro-drug ganciclovir while those expressing cytosine deaminase are sensitive to 5-fluorocytosine. Parental parasites were inherently resistant to these drugs. A significant 'bystander effect' was evident in cultures with either ganciclovir or 5-fluorocytosine. Positive and negative selection of the thymidine kinase transformants with both ganciclovir and WR99210 resulted in the selection of parasites containing a genetic deletion of the Pfrh3 gene, the first targeted double crossover deletions in P. falciparum. The use of negative selection for gene disruptions via double crossover recombination will dramatically improve our ability to analyse protein function and opens the possibility of using this strategy for a variety of gene deletion and modification experiments in the analysis of this important infectious agent.     

Membrane insertion of the Bacillus thuringiensis Cry1Ab toxin: single mutation in domain II block partitioning of the toxin into the brush border membrane

The umbrella and penknife models hypothesize that insecticidal Bacillus thuringiensis Cry toxins partition into the apical membrane of the insect midgut by insertion of only two alpha-helices from domain I of the protein, alpha-helices 4 and 5 in the case of the umbrella model and alpha-helices 5 and 6 in the case of the penknife model. Neither model envisages membrane partitioning by domains II and III. In this study, we present data suggesting that mutations in the domain II residue, F371, affect insertion of the whole toxin into Manduca sexta brush border membrane vesicles (BBMVs). Using steady state fluorescence measurements combined with a proteinase K protection assay, we show that mutants of F371 have lost their ability to insert into the BBMV, even though binding to cadherin is almost unaffected. The study also identifies a difference in partitioning of toxins into artificial lipid vesicles (SUVs) as opposed to native BBMVs. While the F371 mutations block insertion of domains I and II into BBMVs, they only block domain II insertion into SUVs. Bioassay and voltage clamping of midguts also confirm the fluorescence data that the noninserting mutants are nontoxic. Our study leads us to propose that, in contrast to previous models of individual free helices inserting into the membrane, the toxin enters into the membrane as a whole molecule or oligomers of the molecule, wherein the domain II residue F371 has a vital role to play in membrane insertion.     

Ascorbate sustains neutrophil NOS expression, catalysis, and oxidative burst

Previous studies from this lab have demonstrated that in vitro ascorbate augments neutrophil nitric oxide (NO) generation and oxidative burst. The present study was therefore undertaken in guinea pigs to further assess the implication of ascorbate deficiency in vivo on neutrophil ascorbate and tetrahydrobiopterin content, NOS expression/activity, phagocytosis, and respiratory burst. Ascorbate deficiency significantly reduced ascorbate and tetrahydrobiopterin amounts, NOS expression/activity, and NO as well as free radical generation in neutrophils from scorbutics. Ascorbate and tetrahydrobiopterin supplementation in vitro, though, significantly enhanced NOS catalysis in neutrophil lysates and NO generation in live cells, but could not restore them to control levels. Although phagocytic activity remained unaffected, scorbutic neutrophils were compromised in free radical generation. Ascorbate-induced free radical generation was NO dependent and prevented by NOS and NADPH oxidase inhibitors. Augmentation of oxidative burst with dehydroascorbate (DHA) was counteracted in the presence of glucose (DHA uptake inhibitor) and iodoacetamide (glutaredoxin inhibitor), suggesting the importance of ascorbate recycling in neutrophils. Ascorbate uptake was, however, unaffected among scorbutic neutrophils. These observations thus convincingly demonstrate a novel role for ascorbate in augmenting both NOS expression and activity in vivo, thereby reinforcing oxidative microbicidal actions of neutrophils.     

Mapping and validation of a major QTL for yellow pigment content on 7AL in durum wheat (<Emphasis Type="Italic">Triticum turgidum</Emphasis> L. ssp. <Emphasis Type="Italic">durum</Emphasis>)

Yellow pigment content in durum wheat (Triticum turgidum L. ssp. durum) is an important criterion for both pasta bright yellow color and human health because of antioxidant properties of carotenoids involved in this pigmentation. In the present study, QTLs for yellow pigment content in durum wheat were mapped in a population of 140 RILs developed from a intraspecific cross between a released variety (PDW 233) and a landrace (Bhalegaon 4). This trait was evaluated in one location for 3 years and in two more locations for one additional year (five different year × location combinations further called “environments”). Yellow pigment content was highly heritable across the five different environments. Analysis of variance showed the significant effect of genotype, environment and genotype × environment interaction on the trait. Five different QTLs linked to yellow pigment content were identified on chromosome 1A, 3B, 5B, 7A and 7B across five different environments. The strongest one located on the distal part of the long arm of chromosome 7A, QYp.macs-7A, explained 55.22% of the variation in the trait, while, remaining four QTLs explained 5–8.75% of phenotypic variation in yellow pigment content. Marker analysis revealed significant association of one ISSR and one AFLP fragment with the trait. These two markers were linked to the major QTL QYp.macs-7A and were converted into SCAR markers. These SCAR markers were further validated on another population as well as 38 diverse genotypes so as to prove their potential in marker assisted selection. These markers will be very useful for the marker assisted breeding of durum wheat for higher yellow pigment content.     

Blastocystis ratti contains cysteine proteases that mediate interleukin-8 response from human intestinal epithelial cells in an NF-kappaB-dependent manner

Blastocystis is a ubiquitous enteric protozoan found in the intestinal tracts of humans and a wide range of animals. Evidence accumulated over the last decade suggests association of Blastocystis with gastrointestinal disorders involving diarrhea, abdominal pain, constipation, nausea, and fatigue. Clinical and experimental studies have associated Blastocystis with intestinal inflammation, and it has been shown that Blastocystis has potential to modulate the host immune response. Blastocystis is also reported to be an opportunistic pathogen in immunosuppressed patients, especially those suffering from AIDS. However, nothing is known about the parasitic virulence factors and early events following host-parasite interactions. In the present study, we investigated the molecular mechanism by which Blastocystis activates interleukin-8 (IL-8) gene expression in human colonic epithelial T84 cells. We demonstrate for the first time that cysteine proteases of Blastocystis ratti WR1, a zoonotic isolate, can activate IL-8 gene expression in human colonic epithelial cells. Furthermore, we show that NF-κB activation is involved in the production of IL-8. In addition, our findings show that treatment with the antiprotozoal drug metronidazole can avert IL-8 production induced by B. ratti WR1. We also show for the first time that the central vacuole of Blastocystis may function as a reservoir for cysteine proteases. Our findings will contribute to an understanding of the pathobiology of a poorly studied parasite whose public health importance is increasingly recognized.