Vimentin organization modulates the formation of lamellipodia

Vimentin intermediate filaments (VIF) extend throughout the rear and perinuclear regions of migrating fibroblasts, but only nonfilamentous vimentin particles are present in lamellipodial regions. In contrast, VIF networks extend to the entire cell periphery in serum-starved or nonmotile fibroblasts. Upon serum addition or activation of Rac1, VIF are rapidly phosphorylated at Ser-38, a p21-activated kinase phosphorylation site. This phosphorylation of vimentin is coincident with VIF disassembly at and retraction from the cell surface where lamellipodia form. Furthermore, local induction of photoactivatable Rac1 or the microinjection of a vimentin mimetic peptide (2B2) disassemble VIF at sites where lamellipodia subsequently form. When vimentin organization is disrupted by a dominant-negative mutant or by silencing, there is a loss of polarity, as evidenced by the formation of lamellipodia encircling the entire cell, as well as reduced cell motility. These findings demonstrate an antagonistic relationship between VIF and the formation of lamellipodia.     

Identification and validation of QTLs conferring resistance to sorghum downy mildew (Peronosclerospora sorghi) and Rajasthan downy mildew (P. heteropogoni) in maize

We have mapped the quantitative trait loci (QTLs) conferring resistance to sorghum downy mildew (Peronosclerospora sorghi; SDM) and Rajasthan downy mildew (P. heteropogoni; RDM), two species of DM prevalent throughout India. QTL mapping was carried out on a backcross population of 151 individuals derived from a cross between CM139 (susceptible parent) and NAI116 (highly resistant to both SDM and RDM). Heritability estimates were 0.74 for SDM and 0.67 for RDM. Composite interval mapping combined with a linkage map constructed with 80 simple sequence repeat (SSR) markers resulted in the identification of three QTLs (one each on chromosomes 2, 3 and 6) for SDM resistance and two QTLs (one each on chromosomes 3 and 6) for RDM resistance, all of which were contributed by NAI116. The significance of the major QTL on chromosome 6 (bin 6.05) that confers resistance to diverse DMs in tropical Asia, including SDM and RDM in India, was also verified. The results confirmed that some common QTLs contribute to both SDM and RDM resistance, while additional loci might specifically govern resistance to SDM. The QTL information generated in this study provide information that will aid in undertaking an integrated breeding strategy for the transfer of resistance to SDM and RDM in maize lines using marker-assisted selection.     

Large-scale identification of proteins in human salivary proteome by liquid chromatography/mass spectrometry and two-dimensional gel electrophoresis-mass spectrometry

Human saliva contains a large number of proteins and peptides (salivary proteome) that help maintain homeostasis in the oral cavity. Global analysis of human salivary proteome is important for understanding oral health and disease pathogenesis. In this study, large-scale identification of salivary proteins was demonstrated by using shotgun proteomics and two-dimensinal gel electrophoresis-mass spectrometry (2-DE-MS). For the shotgun approach, whole saliva proteins were prefractionated according to molecular weight. The smallest fraction, presumably containing salivary peptides, was directly separated by capillary liquid chromatography (LC). However, the large protein fractions were digested into peptides for subsequent LC separation. Separated peptides were analyzed by on-line electrospray tandem mass spectrometry (MS/MS) using a quadrupole-time of flight mass spectrometer, and the obtained spectra were automatically processed to search human protein sequence database for protein identification. Additionally, 2-DE was used to map out the proteins in whole saliva. Protein spots 105 in number were excised and in-gel digested; and the resulting peptide fragments were measured by matrix-assisted laser desorption/ionization-mass spectrometry and sequenced by LC-MS/MS for protein identification. In total, we cataloged 309 proteins from human whole saliva by using these two proteomic approaches.     

Regulation of Ca2+-dependent desensitization in the vanilloid receptor TRPV1 by calcineurin and cAMP-dependent protein kinase

The vanilloid receptor TRPV1 is a polymodal nonselective cation channel of nociceptive sensory neurons involved in the perception of inflammatory pain. TRPV1 exhibits desensitization in a Ca2+-dependent manner upon repeated activation by capsaicin or protons. The cAMP-dependent protein kinase (PKA) decreases desensitization of TRPV1 by directly phosphorylating the channel presumably at sites Ser116 and Thr370. In the present study we investigated the influence of protein phosphatase 2B (calcineurin) on Ca2+-dependent desensitization of capsaicin- and proton-activated currents. By using site-directed mutagenesis, we generated point mutations at PKA and protein kinase C consensus sites and studied wild type (WT) and mutant channels transiently expressed in HEK293t or HeLa cells under whole cell voltage clamp. We found that intracellular application of the cyclosporin A.cyclophilin A complex (CsA.CyP), a specific inhibitor of calcineurin, significantly decreased desensitization of capsaicin- or proton-activated TRPV1-WT currents. This effect was similar to that obtained by extracellular application of forskolin (FSK), an indirect activator of PKA. Simultaneous applications of CsA.CyP and FSK in varying concentrations suggested that these substances acted independently from each other. In mutation T370A, application of CsA.CyP did not reduce desensitization of capsaicin-activated currents as compared with WT and to mutant channels S116A and T144A. In a double mutation at candidate protein kinase C phosphorylation sites, application of CsA.CyP or FSK decreased desensitization of capsaicin-activated currents similar to WT channels. We conclude that Ca2+-dependent desensitization of TRPV1 might be in part regulated through channel dephosphorylation by calcineurin and channel phosphorylation by PKA possibly involving Thr370 as a key amino acid residue.     

Cloning, expression, and characterization of an oligoxyloglucan reducing end-specific xyloglucanobiohydrolase from Aspergillus nidulans

An oligoxyloglucan reducing end-specific xyloglucanobiohydrolase from the filamentous fungus Aspergillus nidulans was cloned and expressed in Pichia pastoris as a secreted histidine-tagged protein and purified by affinity chromatography. The enzyme acts on xyloglucan oligomers and releases the first two glycosyl residue segments from the reducing end, provided that neither the first glucose nor the xylose attached to the third glucose residue from the reducing end is not further substituted. The enzyme has a specific activity of 7 U/mg at the pH optimum of 3 and at the temperature optimum of 42 degrees C.     

Purpureotin: a novel di-dimeric C-type lectin-like protein from Trimeresurus purpureomaculatus venom is stabilized by noncovalent interactions

Purpureotin, a novel di-dimeric C-type lectin-like protein (CLP) from Trimeresurus purpureomaculatus, was purified and sequenced. While its native molecular mass was determined to be 63kDa, purpureotin showed a single band of 30kDa on nonreducing SDS-PAGE and two polypeptide chains (16.0 and 14.5kDa) under reducing condition. These results were subsequently confirmed by mass spectrometric analyses. Based on these results, we postulate that purpureotin is a dimer of the alpha,beta-heterodimer which is held together by noncovalent interactions. Molecular modeling studies indicate that a dimer of alpha,beta-heterodimers can be formed where the alpha chains are held together by electrostatic charges and beta chains via hydrophobic interactions. Functionally, purpureotin induced platelet aggregation without any cofactor in a dose-dependent manner. However, the platelet aggregation effect was blocked by echicetin. Therefore, purpureotin is assumed to be a GPIb-binding protein which binds to the same or a closely related GPIb site on platelets as echicetin.     

Hypoxia augments TNF-alpha-mediated endothelin-1 release and cell proliferation in human optic nerve head astrocytes

The effect of hypoxia (24 h) on TNF-alpha-mediated release of endothelin-1 (ET-1) from human optic nerve head astrocytes (hONAs) and TNF-alpha- and ET-1-induced hONA proliferation was determined. ET-1 synthesis and release was quantitated using ELISA while TNF-alpha (10 nM)- and ET-1 (100 nM)-mediated hONA proliferation was assessed by CellTiter 96 aqueous one-solution cell proliferation assay, respectively. hONAs appeared to be more rounded with fewer processes following 24 h hypoxia compared to thodr seen in normoxia. Hypoxia enhanced TNF-alpha-mediated ET-1 synthesis and release (by 5-fold) and also significantly increased TNF-alpha- and ET-1-mediated hONA proliferation. PD142893 (1 microM), an ET(A/B) receptor antagonist, blocked ET-1-mediated hONA proliferation both under normoxia and hypoxia, while doing so only under normoxia following TNF-alpha treatment. Also, U0126 (10 microM; an upstream ERK1/2 inhibitor) completely blocked agonist-induced hONA proliferation in normoxia and partially blocked the same in hypoxia. These results demonstrate for the first time that hONAs secrete ET-1 and that TNF-alpha and hypoxia can regulate its levels. Moreover, hypoxia augments the proliferative responses of hONAs to TNF-alpha and ET-1. These agonist-mediated effects following hypoxia could contribute to astroglial activation as seen in glaucomatous optic nerve heads.