The Arabidopsis cinnamoyl CoA reductase irx4 mutant has a delayed but coherent (normal) program of lignification

Previous studies have indicated that the Arabidopsis thalianairregular xylem 4 (irx4) mutant is severely lignin-deficient, forming abnormal lignin from aberrant monomers. Studies of lignin structure in dwarfed cinnamoyl CoA reductase (CCR)-downregulated tobacco were also previously reported to incorporate feruloyl tyramine derivatives. The lignin in the Arabidopsis irx4 mutant was re-investigated at 6 weeks and at maturation (9 weeks). Application of (1)H, (13)C, 2D Heteronuclear Multiple Quantum Coherence and 2D Heteronuclear Multiple Bond Coherence spectroscopic analyses to the lignin-enriched isolates from both Arabidopsis wild-type (Ler) and the CCR-irx4 mutant at both developmental stages revealed that only typical guaiacyl/syringyl lignins were formed. For the irx4 mutant, the syringyl content at 6 weeks growth was lower, in accordance with a delayed but coherent program of lignification. At maturation, however, the syringyl/guaiacyl ratio of the irx4 mutant approached that of wild-type. There was no evidence for feruloyl tyramines, or homologues thereof, accumulating as a chemical signature in lignins resulting from CCR mutation. Nor were there any noticeable increases in other phenolic components, such as hydroxycinnamic acids. These findings were further confirmed by application of thioacidolysis, alkaline nitrobenzene oxidation and acetyl bromide analyses. Moreover, in the case of CCR downregulation in tobacco, there were no NMR spectroscopic correlations that demonstrated feruloyl tyramines being incorporated into the lignin biopolymers. This study thus found no evidence that abnormal lignin formation occurs when CCR activity is modulated.     

The determination of n-alkanes in the cuticular wax of leaves of Ludwigia adscendens L

An n-hexane extract of fresh, mature leaves of Ludwigia adscendens, containing a thin layer of epicuticular waxes, has been analysed for the first time by TLC, IR and GC using standard hydrocarbons. The leaves contained 22 identified long chain (C15-C36) n-alkanes, accounting for 74.27% of the hydrocarbons present, and an unknown number of unidentified branched chain alkanes. The predominant n-alkane was C25 (11.02%), whilst C18 (7.62%), C20 (6.14%), C29 (5.36%) and C27 (5.29%) n-alkanes were moderately abundant: the C35 homologue was present only in minor amounts (0.22%).     

A modified spray reagent for the detection of amino acids on thin layer chromatography plates

Summary. Identification of amino acids is extremely important for the evaluation of protein structure. Thin layer chromatography is an important tool for detecting amino acids by variety of spray reagents. Among these ninhydrin is the most popular due to its high sensitivity. However, ninhydrin produces the same purple/violet color with most amino acids. A spray reagent with high sensitivity for easy and rapid identification of amino acids on thin-layer plates has been introduced. Received March 14, 2000 Accepted August 31, 2000     

Exploring the structure of opioid receptors with homology modeling based on single and multiple templates and subsequent docking: A comparative study

Opioid receptors are the principal targets for opioids, which have been used as analgesics for centuries. Opioid receptors belong to the rhodopsin family of G-protein coupled receptors (GPCRs). In the absence of crystal structures of opioid receptors, 3D homology models have been reported with bovine rhodopsin as a template, though the sequence homology is low. Recently, it has been reported that use of multiple templates results in a better model for a target having low sequence identity with a single template. With the objective of carrying out a comparative study on the structural quality of the 3D models based on single and multiple templates, the homology models for opioid receptors (mu, delta and kappa) were generated using bovine rhodopsin as single template and the recently deposited crystal structures of squid rhodopsin, turkey β-1 and human β-2 adrenoreceptors along with bovine rhodopsin as multiple templates. In this paper we report the results of comparison between the refined 3D models based on multiple sequence alignment (MSA) and models built with bovine rhodopsin as template, using validation programs PROCHECK, PROSA, Verify 3D, Molprobity and docking studies. The results indicate that homology models of mu and kappa with multiple templates are better than those built with only bovine rhodopsin as template, whereas, in many aspects, the homology model of delta opioid receptor with single template is better with respect to the model based on multiple templates. Three nonselective ligands were docked to both the models of mu, delta and kappa opioid receptors using GOLD 3.1. The results of docking complied well with the pharamacophore, reported for nonspecific opioid ligands. The comparison of docking results for models with multiple templates and those with single template have been discussed in detail. Three selective ligands for each receptor were also docked. As the crystallographic structures are not yet known, this comparison will help in choosing better homology models of opioid receptors for studying ligand receptor interactions to design new potent opioid antagonists.     

HSP90 controls SIR2 mediated gene silencing

In recent years, Hsp90 is found to interact with several telomeric proteins at various phases of cell cycle. The Hsp90 chaperone system controls assembly and disassembly of telomere structures and thus maintains the dynamic state of telomere. Here, for the first time we report that the activity of another telomeric protein Sir2p is modulated by Hsp82, the ortholog of Hsp90 from budding yeast (Saccharomyces cerevisiae). In a temperature sensitive Hsp90 deficient yeast strain (iG170Dhsp82), less abundant Sir2p is observed, resulting in de-repression of telomere silencing and a complete loss of mating type silencing. Intriguingly, over expression of Hsp90, either by exposing cells to heat shock or by introducing HSP82 overexpression plasmid also yields reduced level of Sir2p, with a consequential loss of telomere silencing. Thus, Hsp90 homeostasis maintains the cellular pool of Sir2p and thereby controls the reversible nature of telomere silencing. Interestingly, such regulation is independent of one of its major co-chaperones Sba1 (human ortholog of p23).     

Epigenetic,Genetic and Environmental Interactions in Esophageal Squamous Cell Carcinoma from Northeast India

Background

Esophageal squamous cell carcinoma (ESCC) develops as a result of complex epigenetic, genetic and environmental interactions. Epigenetic changes like, promoter hypermethylation of multiple tumour suppressor genes are frequent events in cancer, and certain habit-related carcinogens are thought to be capable of inducing aberrant methylation. However, the effects of environmental carcinogens depend upon the level of metabolism by carcinogen metabolizing enzymes. As such key interactions between habits related factors and carcinogen metabolizing gene polymorphisms towards modulating promoter methylation of genes are likely. However, this remains largely unexplored in ESCC. Here, we studied the interaction of various habits related factors and polymorphism of GSTM1/GSTT1 genes towards inducing promoter hypermethylation of multiple tumour suppressor genes.

Methodology/Principal Findings

The study included 112 ESCC cases and 130 age and gender matched controls. Conditional logistic regression was used to calculate odds ratios (OR) and multifactor dimensionality reduction (MDR) was used to explore high order interactions. Tobacco chewing and smoking were the major individual risk factors of ESCC after adjusting for all potential confounding factors. With regards to methylation status, significantly higher methylation frequencies were observed in tobacco chewers than non chewers for all the four genes under study (p<0.01). In logistic regression analysis, betel quid chewing, alcohol consumption and null GSTT1 genotypes imparted maximum risk for ESCC without promoter hypermethylation. Whereas, tobacco chewing, smoking and GSTT1 null variants were the most important risk factors for ESCC with promoter hypermethylation. MDR analysis revealed two predictor models for ESCC with promoter hypermethylation (Tobacco chewing/Smoking/Betel quid chewing/GSTT1 null) and ESCC without promoter hypermethylation (Betel quid chewing/Alcohol/GSTT1) with TBA of 0.69 and 0.75 respectively and CVC of 10/10 in both models.

Conclusion

Our study identified a possible interaction between tobacco consumption and carcinogen metabolizing gene polymorphisms towards modulating promoter methylation of tumour suppressor genes in ESCC.     

Reassessment of effects on lignification and vascular development in the irx4 Arabidopsis mutant

The Arabidopsis thaliana irregular xylem4 (irx4) cinnamoyl-CoA reductase 1 (CCR1) mutant was reassessed for its purported exclusive rate-limiting or key effects on lignification. Analyses of gross growth characteristics and stem cross-section anatomy, from seedling emergence to senescence, revealed that stunted irx4 mutant lines were developmentally delayed, which in turn indirectly but predictably led to modest reductions (ca. 10-15%) in overall lignin amounts. Such developmental changes are not generally observed in suppression of other monolignol pathway forming enzymes (e.g., 4-coumarate CoA ligase) even when accompanied by significant reductions in lignin amounts. With the greatly arrested development of the irx4 mutant, formation of the lignin-derived syringyl moieties was also predictably delayed (by about 1-2 weeks), although at maturation the final guaiacyl:syringyl ratios were essentially identical to wild-type. No evidence was obtained for so-called abnormal lignin precursors being incorporated into the lignin, as shown by solid-state 13C NMR spectroscopic analysis in contrast to a claim to the contrary [Jones, L., Ennos, A.R., Turner, S.R., 2001. Cloning and characterization of irregular xylem4 (irx4): a severely lignin-deficient mutant of Arabidopsis. Plant J. 26, 205-216]. A previous claim of an "abnormal" lignin present in stunted CCR downregulated tobacco was also not substantiated, with only trace differences being noted in the presumed cell-wall constituent levels. More importantly, a linear correlation between total lignin amounts and lignin-derived fragmentation products was observed at all stages of Arabidopsis growth/development in both wild-type and irx4 mutant lines, regardless of lignin content, i.e., in harmony with an exquisitely controlled and predictable macromolecular assembly process. Recombinant CCR1 displayed fairly broad substrate versatility for all phenylpropanoid CoA substrates, with both feruloyl and 5-hydroxyferuloyl CoA being the best substrates. Taken together, these data indicate that other CCR isoforms are apparently capable of generating monolignol-derived lignified elements in irx4 when CCR1 is impaired, i.e., indicative of a functionally redundant CCR metabolic network operative in Arabidopsis. Other dwarfed phenotypes have also been observed following downregulation/disruption of unrelated metabolic processes but which also involve CoA ester metabolism, i.e., with hydroxymethylglutaryl CoA reductases in Arabidopsis and a bacterial enoyl CoA hydratase/lyase overexpressed in tobacco. Although the reasons for dwarfing in each case are unknown, a common mechanism for the various pleiotropic effects is proposed through perturbation of CoASH pool levels. Finally, this study demonstrates the need for progressive analyses over the lifespan of an organism, rather than at a single time point which cannot reveal the progressive developmental changes occurring.     

Mitochondrial DNA Copy Number and Risk of Oral Cancer: A Report from Northeast India

Background

Oral squamous cell carcinoma (OSCC) is the sixth most common cancer globally. Tobacco consumption and HPV infection, both are the major risk factor for the development of oral cancer and causes mitochondrial dysfunction. Genetic polymorphisms in xenobiotic-metabolizing enzymes modify the effect of environmental exposures, thereby playing a significant role in gene–environment interactions and hence contributing to the individual susceptibility to cancer. Here, we have investigated the association of tobacco - betel quid chewing, HPV infection, GSTM1-GSTT1 null genotypes, and tumour stages with mitochondrial DNA (mtDNA) content variation in oral cancer patients.

Methodology/Principal Findings

The study comprised of 124 cases of OSCC and 140 control subjects to PCR based detection was done for high-risk HPV using a consensus primer and multiplex PCR was done for detection of GSTM1-GSTT1 polymorphism. A comparative ΔCt method was used for determination of mtDNA content. The risk of OSCC increased with the ceased mtDNA copy number (P_trend = 0.003). The association between mtDNA copy number and OSCC risk was evident among tobacco – betel quid chewers rather than tobacco – betel quid non chewers; the interaction between mtDNA copy number and tobacco – betel quid was significant (P = 0.0005). Significant difference was observed between GSTM1 - GSTT1 null genotypes (P = 0.04, P = 0.001 respectively) and HPV infection (P<0.001) with mtDNA content variation in cases and controls. Positive correlation was found with decrease in mtDNA content with the increase in tumour stages (P<0.001). We are reporting for the first time the association of HPV infection and GSTM1-GSTT1 null genotypes with mtDNA content in OSCC.

Conclusion

Our results indicate that the mtDNA content in tumour tissues changes with tumour stage and tobacco-betel quid chewing habits while low levels of mtDNA content suggests invasive thereby serving as a biomarker in detection of OSCC.