Antibacterial endiandric acid derivatives from Beilschmiedia anacardioides

Three endiandric acid derivatives, beilschmiedic acids A, B and C were isolated from the stem bark of Beilschmiedia anacardioides together with the known β-sitosterol. Their structures were established by means of modern spectroscopic techniques. The relative configuration of compound 1 was determined by single crystal X-ray analysis. The antibacterial activities of compounds A,B,C were evaluated in vitro against five strains of microbes. Compound C showed strong activity against Bacillus subtilis, Micrococcus luteus and Streptococcus faecalis (MICs below 23 μM). This Compound was more active than the reference antibiotic ampicillin against B. subtilis and M. luteus.     

Response of photosynthesis and chlorophyll fluorescence quenching to leaf dichotocarpism in Ligustrum vicaryi, an ornamental herb

Net photosynthetic rate of yellow upper leaves (UL) of Ligustrum vicaryi was slightly, but not significantly higher than that of green lower leaves (LL). Diurnally, maximum photochemical efficiency of photosystem 2, PS2 (F_v/F_m) of LL did not significantly decline but the UL showed fairly great daily variations. Yield of PS2 of UL showed an enantiomorphous variation to the photosynthetically active radiation and was significantly lower than in the LL. Unlike F_v/F_m, the efficiency of energy conversion in PS2 and both non-photosynthetic and photosynthetic quenching did not differ in UL and LL. Significant differences between UL and LL were found in contents of chlorophyll (Chl) a, b, and carotenoids (Car) and ratios of Chl a/b, Chl b/Chl (a+b), and Car/Chl (a+b). Leaf colour dichotocarpism in L. vicaryi was mainly caused by different photon utilization; sunflecks affected the LL.     

Alterations of <Emphasis Type="Italic">ROBO1/DUTT1</Emphasis> and <Emphasis Type="Italic">ROBO2</Emphasis> loci in early dysplastic lesions of head and neck: clinical and prognostic implications

Deletion of chromosomal 3p12.3 was suggested to be associated with dysplastic lesions of head and neck. This region harbors two candidate tumor suppressors ROBO1/DUTT1, ROBO2 and two non-coding RNAs (ncRNAs) located at intron 2 of ROBO1/DUTT1. Aim of this study is to understand the role of these genes in development of head and neck squamous cell carcinoma. A collection of 72 dysplastic lesions and 116 HNSCC samples and two oral cancer cell lines were analyzed for ROBO1/DUTT1 and ROBO2 deletion and promoter methylation. ROBO1/DUTT1, ROBO2 and two ncRNAs mRNA expression were analyzed by Q-PCR. Immunohistochemical analysis of ROBO1/DUTT1 and ROBO2 was performed. Alterations of these genes were correlated with different clinicopathological parameters. High frequency of molecular alterations (deletion/methylation) was seen in ROBO1/DUTT1 than ROBO2. In mild dysplastic lesions both of these genes showed high molecular alterations and remained more or less constant in subsequent stages. Q-PCR analysis showed reduced expression of these genes and the two ncRNAs. In vitro demethylation experiment by 5-aza-dC showed upregulation of ROBO1/DUTT1 and ROBO2 while the expression of the ncRNAs remained unchanged. Immunohistochemical analysis of ROBO1/DUTT1 and ROBO2 showed concordance with their mRNA expression and molecular alterations. Poor patients’ outcome was predicted in the cases with alteration of ROBO1/DUTT1 along with tobacco addiction and nodal involvement. Our data suggests (a) ROBO1/DUTT1 and the ncRNAs are transcribed from different promoters, and (b) inactivation of ROBO1/DUTT1 could be used as molecular signature for early detection and prognosis of the head and neck cancer. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A comprehensive study on the 5-hydroxytryptamine3A receptor binding of agonists serotonin and m-chlorophenylbiguanidine

Serotonin type 3 receptors (5-HT₃R) are members of the ligand gated ion channel receptor family. In this study, the interactions of the agonists serotonin (5-HT) and m-chlorophenylbiguanidine (mCPBG) at the binding site of the 5-HT_3AR were investigated at an atomic level. Site-directed mutagenesis studies in Loop B and E along with our earlier published results from mutations within Loops A, C, and D provide comprehensive data on the interaction of 5-HT and mCPBG with 5-HT_3ARs. Using this data we have constructed a refined homology model of the 5-HT_3AR that considers all of the available experimental data. 5-HT and mCPBG were docked into the newly constructed homology model and the amino acid residues critical in binding of these agonists were compared and analyzed. Our docking results reveal many similar binding interactions for 5-HT and mCPBG. Namely, residues THR181, TRP183, PHE226, ILE228, TYR234 and GLU129 were all found to play key roles in binding of both 5-HT and mCPBG. However, the results also revealed two important differences that exist between the interactions of the two agonists. In our model, a hydrogen bond is formed between the indole hydrogen of 5-HT and the residue TYR153. This interaction is not present in the case of mCPBG. Conversely, a hydrogen bond exists between SER182 and a protonated nitrogen of mCPBG, which does not exist in 5-HT. Our modeling results were found to be in accordance with experimental data.     

Techniques for integrating ‐omics data

The challenge for -omics research is to tackle the problem of fragmentation of knowledge by integrating several sources of heterogeneous information into a coherent entity. It is widely recognized that successful data integration is one of the keys to improve productivity for stored data. Through proper data integration tools and algorithms, researchers may correlate relationships that enable them to make better and faster decisions. The need for data integration is essential for present ‐omics community, because ‐omics data is currently spread world wide in wide variety of formats. These formats can be integrated and migrated across platforms through different techniques and one of the important techniques often used is XML. XML is used to provide a document markup language that is easier to learn, retrieve, store and transmit. It is semantically richer than HTML. Here, we describe bio warehousing, database federation, controlled vocabularies and highlighting the XML application to store, migrate and validate -omics data.     

Structural and Biochemical Evidence That a TEM-1 ��-Lactamase N170G Active Site Mutant Acts via Substrate-assisted Catalysis

TEM-1 β-lactamase is the most common plasmid-encoded β-lactamase in Gram-negative bacteria and is a model class A enzyme. The active site of class A β-lactamases share several conserved residues including Ser⁷⁰, Glu¹⁶⁶, and Asn¹⁷⁰ that coordinate a hydrolytic water involved in deacylation. Unlike Ser⁷⁰ and Glu¹⁶⁶, the functional significance of residue Asn¹⁷⁰ is not well understood even though it forms hydrogen bonds with both Glu¹⁶⁶ and the hydrolytic water. The goal of this study was to examine the importance of Asn¹⁷⁰ for catalysis and substrate specificity of β-lactam antibiotic hydrolysis. The codon for position 170 was randomized to create a library containing all 20 possible amino acids. The random library was introduced into Escherichia coli, and functional clones were selected on agar plates containing ampicillin. DNA sequencing of the functional clones revealed that only asparagine (wild type) and glycine at this position are consistent with wild-type function. The determination of kinetic parameters for several substrates revealed that the N170G mutant is very efficient at hydrolyzing substrates that contain a primary amine in the antibiotic R-group that would be close to the Asn¹⁷⁰ side chain in the acyl-intermediate. In addition, the x-ray structure of the N170G enzyme indicated that the position of an active site water important for deacylation is altered compared with the wild-type enzyme. Taken together, the results suggest the N170G TEM-1 enzyme hydrolyzes ampicillin efficiently because of substrate-assisted catalysis where the primary amine of the ampicillin R-group positions the hydrolytic water and allows for efficient deacylation.     

SDF2L1, a Component of the Endoplasmic Reticulum Chaperone Complex, Differentially Interacts with ��-, ��-, and ��-Defensin Propeptides

Mammalian defensins are cationic antimicrobial peptides that play a central role in host innate immunity and as regulators of acquired immunity. In animals, three structural defensin subfamilies, designated as α, β, and θ, have been characterized, each possessing a distinctive tridisulfide motif. Mature α- and β-defensins are produced by simple proteolytic processing of their prepropeptide precursors. In contrast, the macrocyclic θ-defensins are formed by the head-to-tail splicing of nonapeptides excised from a pair of prepropeptide precursors. Thus, elucidation of the θ-defensin biosynthetic pathway provides an opportunity to identify novel factors involved in this unique process. We incorporated the θ-defensin precursor, proRTD1a, into a bait construct for a yeast two-hybrid screen that identified rhesus macaque stromal cell-derived factor 2-like protein 1 (SDF2L1), as an interactor. SDF2L1 is a component of the endoplasmic reticulum (ER) chaperone complex, which we found to also interact with α- and β-defensins. However, analysis of the SDF2L1 domain requirements for binding of representative α-, β-, and θ-defensins revealed that α- and β-defensins bind SDF2L1 similarly, but differently from the interactions that mediate binding of SDF2L1 to pro-θ-defensins. Thus, SDF2L1 is a factor involved in processing and/or sorting of all three defensin subfamilies.Mammalian defensins are tridisulfide-containing antimicrobial peptides that contribute to innate immunity in all species studied to date. Defensins are comprised of three structural subfamilies: the α-, β-, and θ-defensins (1). α- and β-Defensins are peptides of about 29–45-amino acid residues with similar three-dimensional structures. Despite their similar tertiary conformations, the disulfide motifs of α- and β-defensins differ. Expression of human α-defensins is tissue-specific. Four myeloid α-defensins (HNP1–4) are expressed predominantly by neutrophils and monocytes wherein they are packaged in granules, while two enteric α-defensins (HD-5 and HD-6) are expressed at high levels in Paneth cells of the small intestine. Myeloid α-defensins constitute about 5% of the protein mass of human neutrophils. HNPs are discharged into the phagosome during phagocytic ingestion of microbial particles. HD-5 and HD-6 are produced and stored as propeptides in Paneth cell granules and are processed extracellularly by intestinal trypsin (2). β-Defensins are produced primarily by various epithelia (e.g. skin, urogenital tract, airway) and are secreted by the producing cells in their mature forms. In contrast to pro-α-defensins, which contain a conserved prosegment of ∼40 amino acids, the prosegments in β-defensins vary in length and sequence. θ-Defensins are found only in Old World monkeys and orangutans and are the only known circular peptides in animals. These 18-residue macrocyclic peptides are formed by ligation of two nonamer sequences excised from two precursor polypeptides, which are truncated versions of ancestral α-defensins. Like myeloid α-defensins, θ-defensins are stored primarily in neutrophil and monocyte granules (3).Numerous laboratories have demonstrated that the antimicrobial properties of defensins derive from their ability to bind and disrupt target cell membranes (4), and studies have shown defensins to be active against Gram-positive and -negative bacteria (5), viruses (6–9), fungi (10, 11), and parasites such as Giardia lamblia (12). Defensins also play a regulatory role in acquired immunity as they are known to chemoattract T lymphocytes, monocytes, and immature dendritic cells (13, 14), act as adjuvants, stimulate B cell responses, and up-regulate proliferation and cytokine production by spleen cells and T helper cells (15, 16).Defensins are produced as pre-propeptides and undergo post-translational processing to form the mature peptides. While much has been learned about regulation of defensin expression, little is known about the factors involved in their biosynthesis. Valore and Ganz (17) investigated the processing of defensins in cultured cells and demonstrated that maturation of HNPs occurs through two proteolytic steps that lead to formation of mature α-defensins, but the proteases involved have yet to be identified. Moreover, there are virtually no published data regarding endoplasmic reticulum (ER)² factors that are responsible for the folding, processing, and sorting steps necessary for defensin maturation and secretion or trafficking to the proper subcellular compartment. It is likely that several chaperones, proteases, and protein-disulfide isomerase (PDI) family proteins are involved. Consistent with this possibility, Gruber et al. (18) recently demonstrated the role of a PDI in biosynthesis of cyclotides, small ∼30-residue macrocyclic peptides produced by plants.The primary structures of α- and θ-defensin precursors are closely related. We therefore undertook studies to identify proteins that interact with representative propeptides of each defensin subfamily with the goal of determining common and unique processes that regulate biosynthesis of α- and θ-defensins. We used two-hybrid analysis to first identify interactors of the θ-defensin precursor, proRTD1a. As described, we identified SDF2L1, a component of the ER-chaperone complex as an interactor, and showed that it also specifically interacts with α- and β-defensins. This suggests that SDF2L1 is involved in the maturation/trafficking of defensins at a step common to all three subfamilies of mammalian defensins.     

Evidence of Late Palaeocene-Early Eocene equatorial rain forest refugia in southern Western Ghats,India

Equatorial rain forests that maintain a balance between speciation and extinction are hot-spots for studies of biodiversity. Western Ghats in southern India have gained attention due to high tropical biodiversity and endemism in their southern most area. We attempted to track the affinities of the pollen flora of the endemic plants of Western Ghat area within the fossil palynoflora of late Palaeocene-early Eocene (∼55–50 Ma) sedimentary deposits of western and northeastern Indian region. The study shows striking similarity of extant pollen with twenty eight most common fossil pollen taxa of the early Palaeogene. Widespread occurrences of coal and lignite deposits during early Palaeogene provide evidence of existence of well diversified rain forest community and swampy vegetation in the coastal low lying areas all along the western and northeastern margins of the Indian subcontinent. Prevalence of excessive humid climate during this period has been seen as a result of equatorial positioning of Indian subcontinent, superimposed by a long term global warming phase (PETM and EECO) during the early Palaeogene. The study presents clear evidence that highly diversified equatorial rain forest vegetation once widespread in the Indian subcontinent during early Palaeogene times, are now restricted in a small area as a refugia in the southernmost part of the Western Ghat area. High precipitation and shorter periods of dry months seem to have provided suitable environment to sustain lineages of ancient tropical vegetation in this area of Western Ghats in spite of dramatic climatic changes subsequent to the post India-Asia collision and during the Quaternary and Recent times.     

Regulation of Mitochondrial Iron Import through Differential Turnover of Mitoferrin 1 and Mitoferrin 2

Mitoferrin 1 and mitoferrin 2 are homologous members of the mitochondrial solute carrier family. Mitoferrin 1 is required for mitochondrial iron delivery in developing erythrocytes. Here we show that mitoferrin 1 and mitoferrin 2 contribute to mitochondrial iron delivery in a variety of cells. Reductions in mitoferrin 1 and/or mitoferrin 2 levels by RNA interference result in decreased mitochondrial iron accumulation, heme synthesis, and iron-sulfur cluster synthesis. The ectopic expression of mitoferrin 1 in nonerythroid cells silenced for mitoferrin 2 or the expression of mitoferrin 2 in cells silenced for mitoferrin 1 restored heme synthesis to “baseline” levels. The ectopic expression of mitoferrin 2, however, did not support hemoglobinization in erythroid cells deficient in mitoferrin 1. Mitoferrin 2 could not restore heme synthesis in developing erythroid cells because of an inability of the protein to accumulate in mitochondria. The half-life of mitoferrin 1 was increased in developing erythroid cells, while the half-life of mitoferrin 2 did not change. These results suggest that mitochondrial iron accumulation is tightly regulated and that controlling mitoferrin levels within the mitochondrial membrane provides a mechanism to regulate mitochondrial iron levels.Iron is a required element for all eukaryotes, but iron can be toxic at high concentrations. Consequently, the cellular acquisition of iron is highly regulated, as is the concentration of free iron in biological fluids. The regulation of iron concentration is extended to cellular organelles that either store or utilize iron. Mitochondria utilize iron for the synthesis of heme and iron-sulfur (Fe-S) clusters. These prosthetic groups are used within the mitochondria and are exported for use by cytosolic and nuclear proteins. The mechanisms that regulate mitochondrial iron levels are not known, although it is clear that mitochondrial iron levels must be regulated. For example, the loss of function mutations in genes that encode enzymes required for Fe-S cluster synthesis or the Atm1 transporter that exports Fe-S clusters, results in excessive mitochondrial iron accumulation in yeast and humans (for a review, see reference 11).The mechanisms that regulate mitochondrial iron pools are not well defined. Mitochondrial iron pools might be regulated at the level of import. Mitoferrin 1 (Mfrn1) has been shown to be required for mitochondrial iron import in developing erythroid cells. A mutation in zebrafish Mfrn1 (frascati) or the deletion of mouse Mfrn1 leads to defects in hemoglobinization due to a deficit in mitochondrial iron uptake (17). The phenotype of frascati zebrafish is restricted to developing red blood cells; other cell types showed no evidence of a mitochondrial iron phenotype. Mfrn1 has a paralogue, Mfrn2, and both genes have homologues MRS3 and MRS4 in Saccharomyces cerevisiae. Yeast with deletions of MRS3 and MRS4 grows poorly under low iron conditions due to impaired mitochondrial iron acquisition (5, 10, 13, 23). In yeast, the expression of Mfrn1 or Mfrn2 in Δmrs3 Δmrs4 cells can correct the poor growth under low iron conditions. The expression of either mouse or zebrafish Mfrn1 as a transgene in frascati zebrafish corrected the hemoglobin deficiency in cells, but the expression of Mfrn2 did not (17). These observations raise three questions. (i) What is the role of Mfrn2 in mitochondrial iron metabolism? (ii) Is iron transport into mitochondria regulated? (iii) If Mfrn2 transports iron into the mitochondria of vertebrate cells, why doesn''t Mfrn2 rescue the mitochondrial defect in Mfrn1-deficient zebrafish?Here, we show that Mfrn1 and Mfrn2 can transport iron into the mammalian mitochondria of nonerythroid cells. The ectopic expression of either Mfrn1 or Mfrn2 can restore mitochondrial iron transport in cells silenced for Mfrn2 and -1, respectively, but ectopic expression has little effect on increasing mitochondrial iron levels above the baseline values. Mitochondrial iron levels do not increase over the baseline because the levels of Mfrns are regulated posttranslationally. Mfrn1 accumulates in the mitochondria of developing red blood cells as a result of an increased protein half-life. In contrast, Mfrn2 does not accumulate in developing red blood cells or other cells, as the half-life of Mfrn2 protein remains constant.