MOLECULAR PHYLOGENY OF GRACILARIA SPECIES INFERRED FROM MOLECULAR MARKERS BELONGING TO THREE DIFFERENT GENOMES1

The nucleotide sequence data of molecular markers 18S rRNA, RUBISCO spacer, and cox2‐3 intergenic spacer were integrated to infer the phylogeny of Gracilaria species, collected from the western coast of India, reducing the possibility of misidentification and providing greater phylogenetic resolution. A phylogenetic tree was constructed using cox2‐3 and RUBISCO spacer sequences, exhibiting the same clustering but differing slightly from that of the rRNA‐based phylogenetic tree. The phylogeny inferred from the combined data set confers an analogous pattern of clustering, compared with those of trees constructed from individual data sets. The combined data set resulted in a phylogeny with better resolution, which supported the clade with higher consistency index, retention index, and bootstrap values. It was observed that Gracilaria foliifera (Forssk.) Børgesen is closer to G. corticata (J. Agardh) J. Agardh varieties, while G. salicornia (C. Agardh) E. Y. Dawson and G. fergusonii J. Agardh both originated from the same clade. The position of G. textorii (Suringar) De Toni faltered and toppled between G. salicornia and G. dura (C. Agardh) J. Agardh; however, G. gracilis (Stackh.) M. Steentoft, L. M. Irvine et W. F. Farnham was evidently distant from the rest of the species.     

Genetic control of leaf-blade morphogenesis by the <Emphasis Type="Italic">INSECATUS</Emphasis> gene in <Emphasis Type="Italic">Pisum sativum</Emphasis>

To understand the role of INSECATUS (INS) gene in pea, the leaf blades of wild-type, ins mutant and seven other genotypes, constructed by recombining ins with uni-tac, af, tl and mfp gene mutations, were quantitatively compared. The ins was inherited as a recessive mutant allele and expressed its phenotype in proximal leaflets of full size leaf blades. In ins leaflets, the midvein development was arrested in distal domain and a cleft was formed in lamina above this point. There was change in the identity of ins leaflets such that the intercalary interrupted midvein bore a leaf blade. Such adventitious blades in ins, ins tl and ins tl mfp were like the distal segment of respective main leaf blade. The ins phenotype was not seen in ins af and ins af uni-tac genotypes. There was epistasis of uni-tac over ins. The ins, tl and mfp mutations interacted synergistically to produce highly pronounced ins phenotype in the ins tl mfp triple mutant. The role(s) of INS in leaf-blade organogenesis are: positive regulation of vascular patterning in leaflets, repression of UNI activity in leaflet primordia for ectopic growth and in leaf-blade primordium for indeterminate growth of rachis, delimitation of proximal leaflet domain and together with TL and MFP homeostasis for meristematic activity in leaflet primordia. The variant apically bifid shape of the affected ins leaflets demonstrated that the leaflet shape is dependent on the venation pattern.     

Computational Design of a PAK1 Binding Protein

We describe a computational protocol, called DDMI, for redesigning scaffold proteins to bind to a specified region on a target protein. The DDMI protocol is implemented within the Rosetta molecular modeling program and uses rigid-body docking, sequence design, and gradient-based minimization of backbone and side-chain torsion angles to design low-energy interfaces between the scaffold and target protein. Iterative rounds of sequence design and conformational optimization were needed to produce models that have calculated binding energies that are similar to binding energies calculated for native complexes. We also show that additional conformation sampling with molecular dynamics can be iterated with sequence design to further lower the computed energy of the designed complexes. To experimentally test the DDMI protocol, we redesigned the human hyperplastic discs protein to bind to the kinase domain of p21-activated kinase 1 (PAK1). Six designs were experimentally characterized. Two of the designs aggregated and were not characterized further. Of the remaining four designs, three bound to the PAK1 with affinities tighter than 350 μM. The tightest binding design, named Spider Roll, bound with an affinity of 100 μM. NMR-based structure prediction of Spider Roll based on backbone and ¹³C^β chemical shifts using the program CS-ROSETTA indicated that the architecture of human hyperplastic discs protein is preserved. Mutagenesis studies confirmed that Spider Roll binds the target patch on PAK1. Additionally, Spider Roll binds to full-length PAK1 in its activated state but does not bind PAK1 when it forms an auto-inhibited conformation that blocks the Spider Roll target site. Subsequent NMR characterization of the binding of Spider Roll to PAK1 revealed a comparably small binding ‘on-rate’ constant (? 10⁵ M^− 1 s^− 1). The ability to rationally design the site of novel protein-protein interactions is an important step towards creating new proteins that are useful as therapeutics or molecular probes.     

Spontaneous plant regeneration in transformed roots and calli from Tylophora indica: changes in morphological phenotype and tylophorine accumulation associated with transformation by Agrobacterium rhizogenes

We examined the effects of genetic transformation by Agrobacterium rhizogenes on the production of tylophorine, a phenanthroindolizidine alkaloid, in the Indian medicinal plant, Tylophora indica. Transformed roots induced by the bacterium grew in axenic culture and produced shoots or embryogenic calli in the absence of hormone treatments. However, hormonal treatment was required to regenerate shoots in root explants of wild type control plants. Transformed plants showed morphological features typically seen in transgenic plants produced by A. rhizogenes, which include, short internodes, small and wrinkled leaves, more branches and numerous plagiotropic roots. Plants regenerated from transformed roots showed increased biomass accumulation (350–510% in the roots and 200–320% in the whole plants) and augmented tylophorine content (20–60%) in the shoots, resulting in a 160–280% increase in tylophorine production in different clones grown in vitro.     

Investigations on feasibility of in situ development of amphotericin B liposomes for industrial applications

Amphotericin B (AmB) liposome formulations are very successful in the treatment of fungal infections and leishmaniasis. But higher cost limits its widespread use among people in developing countries. Therefore, we have developed a modified ethanol-injection method for the preparation of AmB liposomes. Two liposomal formulations were developed with dimyristoyl phosphatidylcholine [F-1a] and soya phosphatidylcholine [F-2a], along with egg phosphatidyl glycerol and cholesterol. AmB was dissolved in acidified dimethyl acetamide and mixed with ethanolic lipid solution and rapidly injected in 5% dextrose to prepare liposomes. Liposomes were characterized on the basis of size (~100?nm), zeta (-43.3?±?2.8 mV) and percent entrapment efficiency (>95%). The in vitro release study showed an insignificant difference (P?≥?0.05) for 24-hour release between marketed AmB liposomes (AmBisome) and F-1a and F-2a. Proliposome concentrate, used for the preparation of in situ liposomes, was physically stable for more than 3 months at experimental conditions. Similarly, AmB showed no sign of degradation in reconstituted liposomes stored at 2-8°C for more than 3 months. IC(50) value of Ambisome (0.18 μg/mL) was comparatively similar to F-1a (0.17 μg/mL) and F-2a (0.16 μg/mL) against intramacrophagic amastigotes. Under experimental conditions, a novel modified method for AmB liposomes is a great success and generates interest for development as a platform technology for many therapeutic drug products.     

Influence of main channel structure on H(2)O(2) access to the heme cavity of catalase KatE of Escherichia coli

The main channel for H₂O₂ access to the heme cavity in large subunit catalases is twice as long as in small subunit catalases and is divided into two distinct parts. Like small subunit catalases, the 15 Å of the channel adjacent to the heme has a predominantly hydrophobic surface with only weak water occupancy, but the next 15 Å extending to the protein surface is hydrophilic and contains a complex water matrix in multiple passages. At the approximate junction of these two sections are a conserved serine and glutamate that are hydrogen bonded and associated with H₂O₂ in inactive variants. Mutation of these residues changed the dimensions of the channel, both enlarging and constricting it, and also changed the solvent occupancy in the hydrophobic, inner section of the main channel. Despite these structural changes and the prominent location of the residues in the channel, the variants exhibited less than a 2-fold change in the k_cat and apparent K_M kinetic constants. These results reflect the importance of the complex multi-passage structure of the main channel. Surprisingly, mutation of either the serine or glutamate to an aliphatic side chain interfered with heme oxidation to heme d.     

Quantized Water Access to the HIV-1 Protease Active Site as a Proposed Mechanism for Cooperative Mutations in Drug Affinity

The development of resistance to different drugs remains a major problem for a wide range of infections. In particular, combinations of specific mutations, which individually demonstrate no effect, exhibit significant cooperativity. Here we show that changes to the energy of ligand binding in different resistant HIV-1 proteases are correlated with the creation of water binding sites in the active site. This correlation is conserved across two drugs (ritonavir and lopinavir). We propose that individual mutations induce changes in flap packing that are insufficient to allow water binding but in combination allow access, leading to the observed cooperative resistance.     

Impact of low and high fluence rates of UV-B radiation on growth and oxidative stress in Phormidium foveolarum and Nostoc muscorum under copper toxicity: differential display of antioxidants system

In the present study, impact of low (UV-B_L) and high (UV-B_H) fluence rates of UV-B on growth, oxidative stress and antioxidant system was studied in two cyanobacteria i.e. Phormidium foveolarum and Nostoc muscorum under Cu (2 and 5???M) toxicity after 24 and 72?h of experiments. UV-B_H and Cu treatment decreased growth of both the cyanobacteria and Cu induced decrease in growth was accompanied by a significant increase in Cu accumulation. Levels of reactive oxygen species (ROS), i.e. superoxide radicals (SOR; $ \text O_{2}^{\cdot\,-} $ ) and hydrogen peroxide (H₂O₂) were significantly increased by Cu and UV-B_H exposure which in turn accelerated lipid peroxidation (malondialdehyde: MDA) and protein oxidation (reactive carbonyl groups: RCG). Activities of enzymatic antioxidants, such as superoxide dismutase (SOD), peroxidase (POD) and glutathione-S-transferase (GST) were increased by both doses of Cu as well as UV-B. Conversely, Cu and UV-B_H drastically decreased catalase (CAT) activity. After the commencement of 24?h of treatment with Cu alone and together with UV-B_H, non-protein thiols (NP-SH) contents were decreased while after 72?h, a reverse trend was noticed. Unlike NP-SH, cysteine content decreased appreciably during the treatments. In contrast to this, low dose (UV-B_L) of UV-B did not influence growth, SOR, H₂O₂, MDA and RCG contents. An improvement in CAT activity and NP-SH content was observed under Cu and UV-B_L treatment; hence, UV-B_L treatment resulted into certain degree of protection against Cu toxicity in both the organisms. Thus, the results showed that UV-B_H and UV-B_L exerted differential effects on both the organisms under Cu toxicity, and compared to N. muscorum, P. foveolarum was less affected by Cu and UV-B_H.