Phenology of plants in relation to ambient environment in a subalpine forest of Uttarakhand,western Himalaya

Observations on phenology of some representative trees, shrubs, under-shrubs and herbs in a subalpine forest of Uttarakhand, western Himalaya were recorded. With the commencement of favorable growth season in April, occurrence of leaf fall was indicatory growth phenomenon in Quercus semecarpifolia, Q. floribunda and Abies spectabilis. However, active vegetative growth in herbaceous species starts onward April and fruit maturation and seed dehiscence are completed from mid of September to October. In general, vegetative growth and reproductive stages in majority of the studied species seems to be dependent on adequate moisture content and also flowering and fruiting in subalpine plants correlate ambient temperature.     

New Insights on Cyclization Specificity of Fungal Type III Polyketide Synthase,PKSIII<Emphasis Type="Italic">Nc</Emphasis> in <Emphasis Type="Italic">Neurospora crassa</Emphasis>

Type III polyketide synthases (PKSs) biosynthesize varied classes of metabolites with diverse bio-functionalities. Inherent promiscuous substrate specificity, multiple elongations of reaction intermediates and several modes of ring-closure, confer the proteins with the ability to generate unique scaffolds from limited substrate pools. Structural studies have identified crucial amino acid residues that dictate type III PKS functioning, though cyclization specific residues need further investigation. PKSIIINc, a functionally and structurally characterized type III PKS from the fungus, Neurospora crassa, is known to biosynthesize alkyl-resorcinol, alkyl-triketide- and alkyl-tetraketide-α-pyrone products. In this study, we attempted to identify residue positions governing cyclization specificity in PKSIIINc through comparative structural analysis. Structural comparisons with other type III PKSs revealed a motif with conserved hydroxyl/thiol groups that could dictate PKSIIINc catalysis. Site-directed mutagenesis of Cys120 and Ser186 to Ser and Cys, respectively, altered product profiles of mutant proteins. While both C120S and S186C proteins retained wild-type PKSIIINc product activity, S186C favoured lactonization and yielded higher amounts of the α-pyrone products. Notably, C120S gained new cyclization capability and biosynthesized acyl-phloroglucinol in addition to wild-type PKSIIINc products. Generation of alkyl-resorcinol and acyl-phloroglucinol by a single protein is a unique observation in fungal type III PKS family. Mutation of Cys120 to bulky Phe side-chain abrogated formation of tetraketide products and adversely affected overall protein stability as revealed by molecular dynamics simulation studies. Our investigations identify residue positions governing cyclization programming in PKSIIINc protein and provide insights on how subtle variations in protein cores dictate product profiles in type III PKS family.     

An improved sample preparation method for analyzing mycobacterial proteins in two-dimensional gels

Two-dimensional gel electrophoresis (2-DE) is currently a widely used analytical method for resolving complex mixtures of proteins. Sample preparation has a marked influence on 2-DE pattern. To reduce impurities and to increase the low-abundance proteins, protein precipitation is often used for the preparation of samples before 2-DE. In this study, we revealed that addition of SDS prior to TCA precipitation of mycobacterial cell extract proteins increases the resolution of the 2-D gel pattern.     

SIRT3 interacts with the daf-16 homolog FOXO3a in the mitochondria, as well as increases FOXO3a dependent gene expression

Cellular longevity is a complex process relevant to age-related diseases including but not limited to chronic illness such as diabetes and metabolic syndromes. Two gene families have been shown to play a role in the genetic regulation of longevity; the Sirtuin and FOXO families. It is also established that nuclear Sirtuins interact with and under specific cellular conditions regulate the activity of FOXO gene family proteins. Thus, we hypothesize that a mitochondrial Sirtuin (SIRT3) might also interact with and regulate the activity of the FOXO proteins. To address this we used HCT116 cells overexpressing either wild-type or a catalytically inactive dominant negative SIRT3. For the first time we establish that FOXO3a is also a mitochondrial protein and forms a physical interaction with SIRT3 in mitochondria. Overexpression of a wild-type SIRT3 gene increase FOXO3a DNA-binding activity as well as FOXO3a dependent gene expression. Biochemical analysis of HCT116 cells over expressing the deacetylation mutant, as compared to wild-type SIRT3 gene, demonstrated an overall oxidized intracellular environment, as monitored by increase in intracellular superoxide and oxidized glutathione levels. As such, we propose that SIRT3 and FOXO3a comprise a potential mitochondrial signaling cascade response pathway.     

Functionalized polycarbonate derived from tartaric acid: enzymatic ring-opening polymerization of a seven-membered cyclic carbonate

Enantiomerically pure functional polycarbonate was synthesized from a novel seven-membered cyclic carbonate monomer derived from naturally occurring L-tartaric acid. The monomer was synthesized in three steps and screened for polymerization with four commercially available lipases from different sources at 80 degrees C, in bulk. The ring-opening polymerization (ROP) was affected by the source of the enzyme; the highest number-average molecular weight, M(n) = 15500 g/mol (PDI = 1.7; [alpha]D(20) = +77.8, T(m) = 58.8 degrees C) optically active polycarbonate was obtained with lipase Novozyme-435. The relationship between monomer conversion, reaction time, molecular weight, and molecular weight distribution were investigated for Novozyme-435 catalyzed ROP. Deprotection of the ketal groups was achieved with minimal polymer chain cleavage (M(n) = 10000 g/mol, PDI = 2.0) and resulted in optically pure polycarbonate ([alpha]D(20) = +56) bearing hydroxy functional groups. Deprotected poly(ITC) shows T(m) of 60.2 degrees C and DeltaH(f) = 69.56 J/g and similar to that of the poly(ITC), a glass transition temperature was not found. The availability of the pendant hydroxyl group is expected to enhance the biodegradability of the polymer and serves in a variety of potential biomedical applications such as polymeric drug delivery systems.     

Mountain Aspect Influences the Genetic Clustering of Psychrotolerant Phosphate Solubilizing <Emphasis Type="Italic">Pseudomonads</Emphasis> in the Uttarakhand Himalayas

Fourteen cold tolerant phosphate solubilizing bacteria isolated from high altitude representative locations of the two major mountain aspects of the Uttarakhand Himalayas (cooler north and warmer south facing slopes) were selected for this study. The tricalcium phosphate (TCP) solubilizing abilities of the isolates were estimated at three different incubation temperatures viz., 4, 15, and 30°C under in vitro conditions. Irrespective of their geographical origin, all the isolates recorded maximum P release values at 30°C. At 4°C, the isolates from the north facing slope were found to release significantly higher levels of P, as compared to the isolates from the south facing slopes. Alternatively at 15°C, the isolates from the south facing slope were found to release significantly higher levels of P. Initial confirmation of their genus level identity as Pseudomonads was arrived by amplification of a 990 bp fragment of the 16S rRNA gene using genus specific primers. Further putative species level identification was arrived by sequencing of the 16S rRNA gene. The diversity among the isolates was determined by rep-PCR using the primers BOX, ERIC, and ERIC2. A composite dendrogram constructed using the rep-PCR profiles revealed that the isolates from the north and south mountain aspects formed separate major clusters. The extent of diversity was greater among the isolates from the south mountain aspect. This study reveals the potential of rep-PCR in determining the genetic diversity among Pseudomonads selected for a single functional trait, but varying in their geographical origin.     

Phosphate solubilization and growth promotion by <Emphasis Type="Italic">Pseudomonas fragi</Emphasis> CS11RH1 (MTCC 8984), a psychrotolerant bacterium isolated from a high altitude Himalayan rhizosphere

Phosphate solubilization and growth promotion by Pseudomonas fragi CS11RH1 (MTCC 8984), a psychrotolerant bacterium isolated from a high altitude garlic rhizosphere from the Indian Himalayas, are reported here. The identity of the isolate was arrived on the basis of its biochemical features and sequencing of the 16S rRNA gene. The isolate grew and solubilized phosphate at temperatures ranging from 4 to 30°C. Besides solubilizing P it produced indole acetic acid (IAA) and hydrogen cyanide (HCN). Seed bacterization with the isolate significantly increased the percent germination, rate of germination, plant biomass and nutrient uptake of wheat seedlings. While Pseudomonas fragi is normally associated with the spoilage of dairy products stored at cold temperatures, this is an early report on the plant growth promoting ability of the bacterium.     

Ligand migration and hexacoordination in type 1 non-symbiotic rice hemoglobin

Type 1 non-symbiotic rice hemoglobin (rHb1) shows bis-histidyl heme hexacoordination and is capable of binding diatomic ligands reversibly. The biological function is as yet unclear, but the high oxygen affinity makes it unlikely to be involved in oxygen transport. In order to gain insight into possible physiological roles, we have studied CO rebinding kinetics after laser flash photolysis of rHb1 in solution and encapsulated in silica gel. CO rebinding to wt rHb1 in solution occurs through a fast geminate phase with no sign of rebinding from internal docking sites. Encapsulation in silica gel enhances migration to internal cavities. Site-directed mutagenesis of FB10, a residue known to have a key role in the regulation of hexacoordination and ligand affinity, resulted in substantial effects on the rebinding kinetics, partly inhibiting ligand exit to the solvent, enhancing geminate rebinding and enabling ligand migration within the internal cavities. The mutation of HE7, one of the histidyl residues involved in the hexacoordination, prevents hexacoordination, as expected, but also exposes ligand migration through a complex system of cavities. This article is part of a Special Issue entitled: Protein Dynamics: Experimental and Computational Approaches.     

Polymer optoelectronic structures for retinal prosthesis

This commentary highlights the effectiveness of optoelectronic properties of polymer semiconductors based on recent results emerging from our laboratory, where these materials are explored as artificial receptors for interfacing with the visual systems. Organic semiconductors based polymer layers in contact with physiological media exhibit interesting photophysical features, which mimic certain natural photoreceptors, including those in the retina. The availability of such optoelectronic materials opens up a gateway to utilize these structures as neuronal interfaces for stimulating retinal ganglion cells. In a recently reported work entitled “A polymer optoelectronic interface provides visual cues to a blind retina,” we utilized a specific configuration of a polymer semiconductor device structure to elicit neuronal activity in a blind retina upon photoexcitation. The elicited neuronal signals were found to have several features that followed the optoelectronic response of the polymer film. More importantly, the polymer-induced retinal response resembled the natural response of the retina to photoexcitation. These observations open up a promising material alternative for artificial retina applications.