A glycosidic germacradienolide from Eupatorium altissimum

The isolation and structure determination of a glycosidic germacradienolide from Eupatorium altissimum are reported. Eupatorin and 5-hydroxy-3′,4′,6,7-tetramethoxyflavone were also found.     

Memory switches in chemical reaction space

Just as complex electronic circuits are built from simple Boolean gates, diverse biological functions, including signal transduction, differentiation, and stress response, frequently use biochemical switches as a functional module. A relatively small number of such switches have been described in the literature, and these exhibit considerable diversity in chemical topology. We asked if biochemical switches are indeed rare and if there are common chemical motifs and family relationships among such switches. We performed a systematic exploration of chemical reaction space by generating all possible stoichiometrically valid chemical configurations up to 3 molecules and 6 reactions and up to 4 molecules and 3 reactions. We used Monte Carlo sampling of parameter space for each such configuration to generate specific models and checked each model for switching properties. We found nearly 4,500 reaction topologies, or about 10% of our tested configurations, that demonstrate switching behavior. Commonly accepted topological features such as feedback were poor predictors of bistability, and we identified new reaction motifs that were likely to be found in switches. Furthermore, the discovered switches were related in that most of the larger configurations were derived from smaller ones by addition of one or more reactions. To explore even larger configurations, we developed two tools: the “bistabilizer,” which converts almost-bistable systems into bistable ones, and frequent motif mining, which helps rank untested configurations. Both of these tools increased the coverage of our library of bistable systems. Thus, our systematic exploration of chemical reaction space has produced a valuable resource for investigating the key signaling motif of bistability.     

Assessing quality of hybridized RNA in Affymetrix GeneChip experiments using mixed-effects models

The technology for hybridizing archived tissue specimens and the use of laser-capture microdissection for selecting cell populations for RNA extraction have increased over the past few years. Both these methods contribute to RNA degradation. Therefore, quality assessments of RNA hybridized to microarrays are becoming increasingly more important. Existing methods for estimating the quality of RNA hybridized to a GeneChip, from resulting microarray data, suffer from subjectivity and lack of estimates of variability. In this article, a method for assessing RNA quality for a hybridized array which overcomes these drawbacks is proposed. The effectiveness of the proposed method is demonstrated by the application of the method to two microarray data sets for which external verification of RNA quality is known.     

Protein stabilization by introduction of cross-strand disulfides

Disulfides cross-link residues in a protein that are separated in primary sequence and stabilize the protein through entropic destabilization of the unfolded state. While the removal of naturally occurring disulfides leads to protein destabilization, introduction of engineered disulfides does not always lead to significant stabilization of a protein. We have analyzed naturally occurring disulfides that span adjacent antiparallel strands of beta sheets (cross-strand disulfides). Cross-strand disulfides have recently been implicated as redox-based conformational switches in proteins such as gp120 and CD4. The propensity of these disulfides to act as conformational switches was postulated on the basis of the hypothesis that this class of disulfide is conformationally strained. In the present analysis, there was no evidence to suggest that cross-strand disulfides are more strained compared to other disulfides as assessed by their torsional energy. It was also observed that these disulfides occur solely at non-hydrogen-bonded (NHB) registered pairs of adjacent antiparallel strands and not at hydrogen-bonded (HB) positions as suggested previously. One of the half-cystines involved in cross-strand disulfide formation often occurs at an edge strand. Experimental confirmation of the stabilizing effects of such disulfides was carried out in Escherichia coli thioredoxin. Four pairs of cross-strand cysteines were introduced, two at HB and two at NHB pairs. Disulfides were formed in all four cases. However, as predicted from our analysis, disulfides at NHB positions resulted in an increase in melting temperature of 7-10 degrees C, while at HB positions there was a corresponding decrease of -7 degrees C. The reduced state of all proteins had similar stability.     

N-terminal myristoylation alters the calcium binding pathways in neuronal calcium sensor-1

Neuronal calcium sensor-1 (NCS-1) interacts with many membranes and cytosolic proteins, both in a Ca²⁺-dependent and in a Ca₂₊-independent manner, and its physiological role is governed by its N-terminal myristoylation. To understand the role of myristoylation in altering Ca²⁺ response and other basic biophysical properties, we have characterized the Ca²⁺ filling pathways in both myristoylated (myr) and non-myristoylated (non-myr) forms of NCS-1. We have observed that Ca²⁺ binds simultaneously to all three active EF-hands in non-myr NCS-1, whereas in the case of myr NCS-1, the process is sequential, where the second EF-hand is filled first, followed by the third and fourth EF-hands. In the case of myr NCS-1, the observed sequential Ca²⁺ binding process becomes more prominent in the presence of Mg²⁺. Besides, the analysis of ¹⁵N-relaxation data reveals that non-myr NCS-1 is more dynamic than myr NCS-1. The overall molecular tumbling correlation time increases by approximately 20% upon myristoylation. Comparing the apo forms of non-myr NCS-1 and myr NCS-1, we found the possibility of existence of some substates, which are structurally closer to the holo form of the protein. There are more such substates in the case of non-myr NCS-1 than in the case of the myr NCS-1, suggesting that the former accesses larger volumes of conformational substates compared with the latter. Further, the study reveals that the possibility of Ca²⁺ binding simultaneously to different parts of the protein is more favourable in non-myr NCS-1 than in myr NCS-1.     

High fidelity epigenetic inheritance: Information theoretic model predicts threshold filling of histone modifications post replication

During cell devision, maintaining the epigenetic information encoded in histone modification patterns is crucial for survival and identity of cells. The faithful inheritance of the histone marks from the parental to the daughter strands is a puzzle, given that each strand gets only half of the parental nucleosomes. Mapping DNA replication and reconstruction of modifications to equivalent problems in communication of information, we ask how well enzymes can recover the parental modifications, if they were ideal computing machines. Studying a parameter regime where realistic enzymes can function, our analysis predicts that enzymes may implement a critical threshold filling algorithm which fills unmodified regions of length at most k. This algorithm, motivated from communication theory, is derived from the maximum à posteriori probability (MAP) decoding which identifies the most probable modification sequence based on available observations. Simulations using our method produce modification patterns similar to what has been observed in recent experiments. We also show that our results can be naturally extended to explain inheritance of spatially distinct antagonistic modifications.     

Human β-Defensin 4 with Non-Native Disulfide Bridges Exhibit Antimicrobial Activity

Human defensins play multiple roles in innate immunity including direct antimicrobial killing and immunomodulatory activity. They have three disulfide bridges which contribute to the stability of three anti-parallel β-strands. The exact role of disulfide bridges and canonical β-structure in the antimicrobial action is not yet fully understood. In this study, we have explored the antimicrobial activity of human β-defensin 4 (HBD4) analogs that differ in the number and connectivity of disulfide bridges. The cysteine framework was similar to the disulfide bridges present in μ-conotoxins, an unrelated class of peptide toxins. All the analogs possessed enhanced antimicrobial potency as compared to native HBD4. Among the analogs, the single disulfide bridged peptide showed maximum potency. However, there were no marked differences in the secondary structure of the analogs. Subtle variations were observed in the localization and membrane interaction of the analogs with bacteria and Candida albicans, suggesting a role for disulfide bridges in modulating their antimicrobial action. All analogs accumulated in the cytosol where they can bind to anionic molecules such as nucleic acids which would affect several cellular processes leading to cell death. Our study strongly suggests that native disulfide bridges or the canonical β-strands in defensins have not evolved for maximal activity but they play important roles in determining their antimicrobial potency.     

Haplotype analysis of the polymorphic 17 YSTR markers in Kerala nontribal populations

The origin of the Kerala non tribal population has been a matter of contention for centuries. While some claim that Negritos were the first inhabitants, some historians suggest a Dravidian origin for all Keralites. The aim of our study has been to provide sufficient scientific evidence based on Y chromosome short tandem repeat (Y STR) analysis for tracing the paternal lineage and also to create a database of the Y STR haplotype of the male population for future forensic analysis. Whole blood samples (n = 168) were collected from unrelated healthy men of the Kerala non-tribal population over a period of 2 years from October 2009. Genomic DNA was extracted by salting out method. All samples were genotyped for the 17 Y STR loci by the AmpFLSTR Y-filer PCR Amplification Kit. The haplotype and allele frequencies were determined by direct counting and analyzed using Arlequin 3.1 software, and molecular variance was calculated with the Y chromosome haplotype reference database online analysis tool, . Haplotype diversity was calculated using HaPYDive (). The majority of haplotypes were unique (149/168). The variant allele 17.1 was observed in DYS 385 loci in three samples. Fifteen samples (8.93%) showed the presence of alleles that are not within the established marker range denoted as outside marker range (OMR). The allele frequency of Kerala non tribal population ranged from 0.00003 to 0.5809. The most polymorphic single locus marker was DYS 458. The haplotype diversity value for Kerala non tribal population was 0.9978. The pairwise difference value ranged from 0.0531 to 0.0854 on comparison of the haplotypes of the Kerala non tribals with other Indian populations. The multi dimensional scaling plot depicted the proximity of Kerala non tribal population with Vasterbotten population (Swedish) and Paiwan, Patyal population of Taiwan, Thailand, and Zhuang population of China. The results of the study indicate towards a European paternal lineage in the non tribal Kerala population.     

Microbial biofilm inoculants benefit growth and yield of chrysanthemum varieties under protected cultivation through enhanced nutrient availability

Protected cultivation of ornamental flowers, as a commercial venture, becomes less profitable with excessive use of fertilizers. The present study examined the influence of microbial biofilm inoculants (Anabaena–Azotobacter, Anabaena–Trichoderma and Trichoderma–Azotobacter) on the availability of soil nutrients and structure of rhizosphere microbial communities in three varieties of chrysanthemum (var. White Star, Thai Chen Queen and Zembla). Varietal-specific responses in growth, enzyme activities, flower yield of plants and availability of soil nutrients were recorded. Dehydrogenase activity was highest in var. White Star treated with the Anabaena–Trichoderma biofilm inoculants. The Anabaena–Azotobacter inoculant enhanced the availability of nitrogen, phosphorus and micronutrients in the soil, besides 40–50% increase in soil organic carbon, as compared to carrier alone or no inoculation. PCR-DGGE profiling of the cyanobacterial communities and qPCR quantification of 16S rRNA abundance of bacteria, archaea and cyanobacteria in the rhizosphere soils, revealed the stronger influences of these inoculants, especially in var. Zembla. Principal Component Analysis (PCA) helped to illustrate that the enhanced microbe-mediated availability of soil macro-and micronutrients, except iron content (Fe), was the most influential factor facilitating improved plant growth and yield parameters. The Anabaena–Azotobacter, and Anabaena–Trichoderma biofilm inoculants, proved superior in all three chrysanthemum varieties.