Genetic Structure of Indian Valerian (Valeriana jatamansi) Populations in Western Himalaya Revealed by AFLP

Valeriana jatamansi Jones is a natural tetraploid species indigenous to the Indian Himalaya. To assess its genetic diversity and population structure, we analyzed six natural populations from the western Himalayan region using amplified fragment length polymorphism. An analysis of molecular variance found that 93% of the genetic variation of V. jatamansi was within populations and 7% among populations. The correlation between genetic and geographic distances (r = 0.14) was not significant. Though the populations are well separated, the lack of distinct genetic variation between populations may be due to either recent rapid fragmentation from a wide and continuous area resulting in genetically similar populations or wide dispersal of seed by wind, since the follicles are feathery. Polyploidy may be the reason for the lack of genetic impoverishment due to fragmentation.     

Hyperglycemia Enhances the Proliferation of Non-Tumorigenic and Malignant Mammary Epithelial Cells through Increased leptin/IGF1R Signaling and Activation of AKT/mTOR

Obesity and diabetes are associated with increased breast cancer risk and worse disease progression once cancer is diagnosed; however, the exact etiology behind these observations remains to be fully elucidated. Due to the global obesity/diabetes pandemic, it is imperative to understand how these diseases promote and enhance breast cancer and other common cancers. In this study we demonstrate that hyperglycemia promotes breast cancer by altering leptin/IGF1R and AKT/mTOR signaling. To our knowledge, we show for the first time that in breast epithelial cells, hyperglycemia alone directly impacts leptin signaling. Hyperglycemia increased proliferation of both non-tumorigenic and malignant mammary epithelial cells. These observations coincided with increased leptin receptor and IGF1R receptor, as well as, increased levels of GRB2, pJAK2, pSTAT3, pIRS1/2, pAKT, and p-mTOR. Moreover, pJAK2 was almost completely colocalized with leptin receptor under high glucose conditions. These results demonstrate how hyperglycemia can potentially increase the risk of breast cancer in premalignant lesions and enhance cancer progression in malignant cells.     

Functional characterization of a new holin-like antibacterial protein coding gene <Emphasis Type="Italic">tmp1</Emphasis> from goat skin surface metagenome

We have identified a holin-like gene from a goat skin surface metagenome. The ORF designated tmp1 coding for 34 amino acids shared sequence similarity with putative holin-like toxin genes. To analyze the antibacterial activity of tmp1 encoded protein, this ORF was cloned and expressed in Escherichia coli BL21(DE3). The expressed gene product Tmp1 exhibited antibacterial activity against Gram-positive bacteria but not to Gram-negative bacteria. A single transmembrane domain (TMD) was identified within Tmp1 and deletion analysis of the N-terminal region and TMD indicated TMD to be responsible for antibacterial activity. The TMD-dependent antibacterial activity was validated using a synthetic peptide with the amino acid sequence of TMD. Besides antibacterial activity, Tmp1 also complemented the function of holin in a lysis-defective bacteriophage lambda. To broaden the spectrum of antibacterial activity, a mutant library of tmp1 was generated by random mutagenesis. Four mutants with amino acid substitutions at the N-terminus of Tmp1 exhibited increased antibacterial activity against Gram-positive and Gram-negative bacteria and were not hemolytic. An improved activity of these mutant proteins is attributed to their increased hydrophobicity.     

Chromosome identification and karyotype analysis of Podophyllum hexandrum Roxb. ex Kunth using FISH

Podophyllum hexandrum is an important high altitude medicinal plant from Himalaya. Somatic chromosomes of this species were studied to delineate and physical mapping of repetitive rDNA sites to provide landmarks in chromosome identification. The karyotype formula of this species was found to be 6m + 2sm + 2st + 2t with secondary constriction in the chromosome 1 and 7. The FISH analysis of rDNA sites showed 4 sites for 18S rDNA and 2 sites for 5S rDNA. The chromosome number 1, 2, 5 and 6 can be identified based on 18S rDNA sites in their short arm and chromosome 1 and 2 can be identified by 5S rDNA site in the centromere region. The estimated genome size of this plant is 16.07 pg (1C).     

A conserved cysteine residue of Pichia pastoris Pex20p is essential for its recycling from the peroxisome to the cytosol

We identified a cysteine residue, conserved near the N terminus of Pex5p- and Pex20p-like proteins, that is essential for the cytosolic relocation of peroxisomal Pex20p. Surprisingly, this residue is not completely essential for the function of the protein; its point mutation into a serine in Pex20p(C8S) causes the accumulation of the protein at the peroxisome membrane, but this is quickly followed by its subsequent degradation by an ubiquitin-dependent quality control pathway called RADAR (receptor accumulation and degradation in the absence of recycling). This degradative pathway allows partial growth of the Pex20p(C8S) mutant on peroxisome-requiring medium. Mutation of cysteine 8 (C8S) and lysine 19 (K19R), the target residue of the RADAR pathway within Pex20p, leads to a stable but non-functional protein because it fails to recycle to the cytosol. This suggests a role for Cys-8 in Pex20p recycling and that constitutive degradation of peroxisomal receptors can be a partially functional alternative to receptor recycling. In addition, expression of this mutant protein in wild-type cells confers a dominant-negative, oleate-specific growth defect, which is a useful tool for a better understanding of peroxisomal receptor recycling.     

Peroxisomal Pex3 Activates Selective Autophagy of Peroxisomes via Interaction with the Pexophagy Receptor Atg30

Pexophagy is a process that selectively degrades peroxisomes by autophagy. The Pichia pastoris pexophagy receptor Atg30 is recruited to peroxisomes under peroxisome proliferation conditions. During pexophagy, Atg30 undergoes phosphorylation, a prerequisite for its interactions with the autophagy scaffold protein Atg11 and the ubiquitin-like protein Atg8. Atg30 is subsequently shuttled to the vacuole along with the targeted peroxisome for degradation. Here, we defined the binding site for Atg30 on the peroxisomal membrane protein Pex3 and uncovered a role for Pex3 in the activation of Atg30 via phosphorylation and in the recruitment of Atg11 to the receptor protein complex. Pex3 is classically a docking protein for other proteins that affect peroxisome biogenesis, division, and segregation. We conclude that Pex3 has a role beyond simple docking of Atg30 and that its interaction with Atg30 regulates pexophagy in the yeast P. pastoris.     

Distinct Actions of Rab3 and Rab27 GTPases on Late Stages of Exocytosis of Insulin

Rab GTPases associated with insulin‐containing secretory granules (SGs) are key in targeting, docking and assembly of molecular complexes governing pancreatic β‐cell exocytosis. Four Rab3 isoforms along with Rab27A are associated with insulin granules, yet elucidation of the distinct roles of these Rab families on exocytosis remains unclear. To define specific actions of these Rab families we employ Rab3GAP and/or EPI64A GTPase‐activating protein overexpression in β‐cells from wild‐type or Ashen mice to selectively transit the entire Rab3 family or Rab27A to a GDP‐bound state. Ashen mice carry a spontaneous mutation that eliminates Rab27A expression. Using membrane capacitance measurements we find that GTP/GDP nucleotide cycling of Rab27A is essential for generation of the functionally defined immediately releasable pool (IRP) and central to regulating the size of the readily releasable pool (RRP). By comparison, nucleotide cycling of Rab3 GTPases, but not of Rab27A, is essential for a kinetically rapid filling of the RRP with SGs. Aside from these distinct functions, Rab3 and Rab27A GTPases demonstrate considerable functional overlap in building the readily releasable granule pool. Hence, while Rab3 and Rab27A cooperate to generate release‐ready SGs in β‐cells, they also direct unique kinetic and functional properties of the exocytotic pathway.      

Equilibrium binding and kinetic characterization of putative tetracycline repressor family transcription regulator Fad35R from Mycobacterium tuberculosis

Fatty acids play critical role in the survival and virulence of Mycobacterium?tuberculosis (Mtb). Activation of fatty acids by acyl-CoA synthetases (Fad) into fatty acyl-CoA is the first and one of the crucial steps in fatty acid metabolism. Mtb possesses 36 fatty acyl-CoA synthetases, unlike Escherichia?coli, which has single enzyme. However, the mechanisms by which the expression of these multiple Fad genes is regulated remain uncharacterized. We characterized the DNA- and ligand-binding properties of a putative tetracycline repressor family regulator, named Fad35R, located upstream of the Fad35 gene and ScoA-citE operon. We identified a palindromic regulatory motif upstream of Fad35 and characterized the binding of Fad35R to this motif. Equilibrium binding studies show that Fad35R binds to this motif with high affinity (K(d) ～?0.033?μm) and the specificity of binding was confirmed by an electromobility gel shift assay. Kinetic studies indicate that faster association (k(a,avg) ～?5.4?×?10(4) m(-1) ·s(-1) ) and slower dissociation rates (k(d,avg) ～?5.84?×?10(-4) s(-1) ) confer higher affinity. The affinity for the promoter is maximum at 300?mm NaCl but decreases rapidly beyond this range. Ligand-binding studies indicate that Fad35R binds specifically to tetracycline and also binds to fatty acid derivatives. The promoter-binding affinity is decreased significantly in the presence of palmityl-CoA, suggesting that Fad35R can sense the levels of activated fatty acids and alter its DNA-binding activity. Our results suggest that Fad35R may be the functional homologue of FadR and controls the expression of genes in a metabolite-dependent manner. Structured digital abstract ? Fad35R?binds to?palindromic sequence?shown by surface plasmon resonance ? Fad35R?binds to?tetracycline?and?activated fatty acids?as shown by fluorescence spectroscopy.     

β-sheet topology prediction with high precision and recall for β and mixed α/β proteins

The prediction of the correct β-sheet topology for pure β and mixed α/β proteins is a critical intermediate step toward the three dimensional protein structure prediction. The predicted beta sheet topology provides distance constraints between sequentially separated residues, which reduces the three dimensional search space for a protein structure prediction algorithm. Here, we present a novel mixed integer linear optimization based framework for the prediction of β-sheet topology in β and mixed α/β proteins. The objective is to maximize the total strand-to-strand contact potential of the protein. A large number of physical constraints are applied to provide biologically meaningful topology results. The formulation permits the creation of a rank-ordered list of preferred β-sheet arrangements. Finally, the generated topologies are re-ranked using a fully atomistic approach involving torsion angle dynamics and clustering. For a large, non-redundant data set of 2102 β and mixed α/β proteins with at least 3 strands taken from the PDB, the proposed approach provides the top 5 solutions with average precision and recall greater than 78%. Consistent results are obtained in the β-sheet topology prediction for blind targets provided during the CASP8 and CASP9 experiments, as well as for actual and predicted secondary structures. The β-sheet topology prediction algorithm, BeST, is available to the scientific community at http://selene.princeton.edu/BeST/.     

Self-destruction in the line of duty.

Three cellular processes, microautophagy, macroautophagy, and the cytoplasm-to-vacuole (Cvt) pathway, are involved in the cargo delivery from the cytosol to the vacuole or lysosome. Recent findings have identified Cvt19 at the receptor for specific cargo binding in the Cvt pathway.