In vitro propagation,genetic and phytochemical assessment of <Emphasis Type="Italic">Habenaria edgeworthii</Emphasis>: an important Astavarga plant

An efficient in vitro propagation protocol for Habenaria edgeworthii Hook. f. ex. Collett using seed-derived callus was established. The maximum seed germination was observed in Murashige and Skoog (MS) medium supplemented with 1.0 μM α-naphthalene acetic acid (NAA). Induction of callus was achieved on full and ½-strength MS medium supplemented with 1.0 μM NAA. The highest number of shoot (11.9 shoots/explant) was achieved in MS medium supplemented with 0.1 μM 6-benzyladenine (BA) and 0.01 μM NAA. Further, elongated shoots when transferred to ½-strength MS rooting medium with different auxin concentrations induced roots (41.6–83.3%) and tubers (0–20.8%); however, a maximum of 87.5% rooting was achieved in a plant growth regulator (PGR)-free MS medium. Rooted shoots (plantlets) when transferred to a mixture of soil:sand:perlite (1:1:1 ratio) resulted in 68% survival. Inter-simple sequence repeats (ISSR) markers confirmed the genetic stability among regenerated plants. The phytochemical analysis of tissue culture-raised tubers showed higher phenolic content than wild tuber. The regeneration protocol developed in this study provides a basis for germplasm conservation and harnessing the total phenol and phenolic compounds of H. edgeworthii. Further, the methods can open avenues for application in other Orchidaceous plants of the Indian Himalayan region.     

Whole-Genome Shotgun Sequencing of the Extremophile Alkalibacillus haloalkaliphilus C-5, of Indian Origin

Alkalibacillus haloalkaliphilus C-5 is a haloalkaliphilic bacterium that was isolated from a soil sample from the salty Sambhar Lake, Rajasthan, India. The organism is capable of alkaline protease production under conditions of pH 10 and 10% (wt/vol) salt. We sequenced and have reported the whole genome of Alkalibacillus haloalkaliphilus C-5, of Indian origin, for the first time.     

Phylogenetic analysis, homology modelling, molecular dynamics and docking studies of caffeoyl–CoA-O- methyl transferase (CCoAOMT 1 and 2) isoforms isolated from subabul (Leucaena leucocephala)

Caffeoyl coenzyme A O-methyltransferase (CCoAOMT) is an important enzyme that participates in lignin biosynthesis especially in the formation of cell wall ferulic esters of plants. It plays a pivotal role in the methylation of the 3-hydroxyl group of caffeoyl CoA. Two cDNA clones that code CCoAOMT were isolated earlier from subabul and in the present study; 3D models of CCoAOMT1 and CCoAOMT2 enzymes were built using the MODELLER7v7 software to find out the substrate binding sites. These two proteins differed only in two amino acids and may have little or no functional redundancy. Refined models of the proteins were obtained after energy minimization and molecular dynamics in a solvated water layer. The models were further assessed by PROCHECK, WHATCHECK, Verify_3D and ERRAT programs and the results indicated that these models are reliable for further active site and docking analysis. The refined models showed that the two proteins have 9 and 10 α-helices, 6 and 7 β-sheets respectively. The models were used for docking the substrates CoA, SAM, SAH, caffeoyl CoA, feruloyl CoA, 5-hydroxy feruloyl CoA and sinapyl CoA which showed that CoA and caffeoyl CoA are binding with high affinity with the enzymes in the presence and absence of SAM. It appears therefore that caffeoyl CoA is the substrate for both the isoenzymes. The results also indicated that CoA and caffeoyl CoA are binding with higher affinity to CCoAOMT2 than CCoAOMT1. Therefore, CCoAOMT2 conformation is thought to be the active form that exists in subabul. Docking studies indicated that conserved active site residues Met58, Thr60, Val63, Glu82, Gly84, Ser90, Asp160, Asp162, Thr169, Asn191 and Arg203 in CCoAOMT1 and CCoAOMT2 enzymes create the positive charge to balance the negatively charged caffeoyl CoA and play an important role in maintaining a functional conformation and are directly involved in donor-substrate binding.     

Repetitive somatic embryogenesis and plant regeneration in Garcinia indica choiss

Summary Immature seeds of Garcinia indica Choiss, were exeised from immature fruits and cultured on Lloyd and McCown (1980), woody plant medium (WPM) with different combinations of auxins and cytokinins. Somatic embryos were obtained on the media supplemented with 6-benzy laminopurine (BA; 2.2–22.1 μM) alone or in combination with α-naphthalene acetic acid (NAA; 2.6 μM) with 80% frequency within a period of 2–3 wk. Subculture of embryos on medium containing BA (16.0 μM) supplemented with indole-3-acetic acid (IAA: 2.8–5.7 μM) and/or kinetin (4.6 μM) gave rise to clusters of secondary somatic embryos along with maturation of primary embryos. In subsequent subculture on hormone-free half-strength WPM, the embryo clusters germinated with an increase in the number of secondary somatic embryos. About 70% of somatic embryos germinated into complete plantlets, which were successfully established under greenhouse conditions.     

Catechin is a phytotoxin and a pro-oxidant secreted from the roots of Centaurea stoebe

When applied to the roots of Arabidopsis thaliana, the phytotoxin (±)-catechin triggers a wave of reactive oxygen species (ROS), leading to a cascade of genome-wide changes in gene expression and, ultimately, death of the root system. Biochemical links describing the root secreted phytotoxin, (±)-catechin, represent one of most well studied systems to describe biochemically based negative plant-plant interactions, but of late have also sparked controversies on phytotoxicity and pro-oxidant behavior of (±)-catechin. The studies originating from two labs^1–3 maintained that (±)-catechin is not at all phytotoxic but has strong antioxidant activity. The step-wise experiments performed and the highly correlative results reported in the present study clearly indicate that (±)-catechin indeed is phytotoxic against A. thaliana and Festuca idahoensis. Our results show that catechin dissolved in both organic and aqueous phase inflicts phytotoxic activity against both A. thaliana and F. idahoensis. We show that the deviation in results highlighted by the two labs^1–3 could be due to different media conditions and a group effect in catechin treated seedlings. We also determined the presence of catechin in the growth medium of C. stoebe to support the previous studies. One of the largest functional categories observed for catechin-responsive genes corresponded to gene families known to participate in cell death and oxidative stress. Our results showed that (±)-catechin treatment to A. thaliana plants resulted in activation of signature cell death genes such as accelerated cell death (acd2) and constitutively activated cell death 1 (cad1). Further, we confirmed our earlier observation of (±)-catechin induced ROS mediated phytotoxicity in A. thaliana. We also provide evidence that (±)-catechin induced ROS could be aggravated in the presence of divalent transition metals. These observations have significant impact on our understanding regarding catechin phytotoxicity and pro-oxidant activity. Our data also illustrates that precise conditions are needed to evaluate the effect of catechin phytotoxicity.Key words: Arabidopsis thaliana, cell death genes, (±)-catechin, phytotoxin, reactive oxygen species (ROS)     

Influence of hydrophobic and hydrophilic spacer-containing enzyme conjugates on functional parameters of steroid immunoassay

Introduction of spacers in coating steroid antigen or enzyme conjugates or immunogen is known to exert an influence on the sensitivity of steroid enzyme immunoassays. We have introduced hydrophobic and hydrophilic spacers between enzyme and steroid moieties and studied their effects on functional parameters of enzyme immunoassays, using cortisol as a model steroid. Cortisol-3-O-carboxymethyloxime-bovine serum albumin (F-3-O-CMO-BSA) was used as immunogen to raise the antiserum in New Zealand white rabbits. Three enzyme conjugates were prepared using cortisol-21-hemisuccinate (F-21-HS) as carboxylic derivative of cortisol and horseradish peroxidase (HRP) as an enzyme label. These were F-21-HS-HRP (without spacer), F-21-HS-adipic acid dihydrazide-HRP (adipic acid dihydrazide as hydrophobic spacer), and F-21-HS-urea-HRP (urea as hydrophilic spacer). The influence of hydrophobic and hydrophilic spacers on the functional parameters of assays such as lower detection limit, ED50, and specificity was studied with reference to enzyme conjugate without spacer. The results of the present investigation revealed that the presence of a hydrophilic spacer in the enzyme conjugate decreases the lower detection limit, decreases the ED50, and marginally improves the specificity of assays. These improvements in functional parameters of assays may be due to the decreased magnitude of the overall hydrophobic interactions existing between the spacer in enzyme conjugate and the antigen binding site of the antibody.     

Salt dependent resistance against chemical denaturation of alkaline protease from a newly isolated haloalkaliphilic Bacillus sp

Only few enzymes from haloalkaliphiles are biochemically characterized for their kinetic behaviour and stability. In view of this realization, an alkaline protease from Bacillus sp. AH-6, displaying salt-dependent resistance against chemical denaturation by Urea and Guanidium hydrochloride was investigated for denaturation and in vitro protein folding. The crude enzyme was highly resistant against urea (8 M) denaturation up to 72 h; however, on purification, it turned sensitive and got denatured within 2 h. Interestingly, the purified enzyme regained the resistance in the presence of NaCl. Effective refolding of the purified enzyme was achieved with glycerol; however, other approaches such as lower protein concentrations, rapid dilution and slow removal of the denaturant did not further add to refolding. The results are important from the viewpoint that only few enzymes from haloalkaliphilic bacteria are characterized. Since the resistance against chemical denaturation is a rare phenomenon, the findings would enrich the knowledge on protein stability and denaturation. Besides, such biocatalysts would definitely have novel applications under harsh chemical environments.     

Investigation of the biotransformation of pentachlorophenol and pulp paper mill effluent decolorisation by the bacterial strains in a mixed culture

Mixed culture of two bacterial strains Bacillus sp. and Serratia marcescens showed potential pentachlorophenol (PCP) degradation and decolorisation of pulp paper mill effluent. The physico-chemical quality of pulp paper mill effluent has been analyzed after 168 h incubation period degraded by mixed culture. The study revealed that it has decreased high load of BOD, COD, TS, TDS, TSS, sulphate, phosphate, total nitrogen, total phenols, metals and different salts (i.e. chloride, sodium, nitrate, potassium) at 168 h incubation period. PCP degradation in pulp paper mill effluent was confirmed by HPLC analysis. Mixed culture was found to degrade PCP up to (94%) present in pulp paper mill effluent with 1% glucose and 0.5% peptone (w/v) at 30 ± 1 °C, pH 8.0 ± 0.2 at 120 rpm in 168 h incubation period. The simultaneous release of chloride ion up to 1200 mg/l at 168 h emphasized the bacterial dechlorination in the medium. The pulp paper mill effluent degradation was also supported by decline in pH, AOX (absorbable organic halides), color, D.O., BOD, COD and PCP. The analysis of pulp paper mill effluent degradation products by GC–MS analysis revealed the formation of low molecular weight compound like 2-chlorophenol (RT = 3.8 min) and tetrachlorohydroquinone (RT = 11.86 min) from PCP extracted degraded sample. Further, mixed culture may be used for bioremediation of PCP containing pulp paper mill waste in the environment.     

Non-nucleoside inhibitors of the hepatitis C virus NS5B RNA-dependant RNA polymerase: 2-aryl-3-heteroaryl-1,3-thiazolidin-4-one derivatives

Hepatitis C virus (HCV) NS5B RNA polymerase is crucial for replicating the HCV RNA genome and is an attractive target for developing anti-HCV drugs. A novel series of 2,3-diaryl-1,3-thiazolidin-4-one derivatives were evaluated for their ability to inhibit HCV NS5B. Of this series, compounds 4c, 5b, 5c and 6 emerged as more potent, displaying over 95% inhibition of NS5B RNA polymerase activity in vitro. The two most active compounds 4c and 5c exhibited an IC(50) of 31.9 microM and 32.2 microM, respectively, against HCV NS5B.