Analysis of tail-anchored protein translocation pathway in plants

Tail-anchored (TA) proteins are a special class of membrane proteins that carry out vital functions in all living cells. Targeting mechanisms of TA proteins are investigated as the best example for post-translational protein targeting in yeast. Of the several mechanisms, Guided Entry of Tail-anchored protein (GET) pathway plays a major role in TA protein targeting. Many in silico and in vivo analyses are geared to identify TA proteins and their targeting mechanisms in different systems including Arabidopsis thaliana. Yet, crop plants that grow in specific and/or different conditions are not investigated for the presence of TA proteins and GET pathway. This study majorly investigates GET pathway in two crop plants, Oryza sativa subsp. Indica and Solanum tuberosum, through detailed in silico analysis. 508 and 912 TA proteins are identified in Oryza sativa subsp. Indica and Solanum tuberosum respectively and their localization with respect to endoplasmic reticulum (ER), mitochondria, and chloroplast has been delineated. Similarly, the associated GET proteins are identified (Get1, Get3 and Get4) and their structural inferences are elucidated using homology modelling. Get3 models are based on yeast Get3. The cytoplasmic Get3 from O. sativa is identified to be very similar to yeast Get3 with conserved P-loop and TA binding groove. Three cytoplasmic Get3s are identified for S. tuberosum. Taken together, this is the first study to identify TA proteins and GET components in Oryza sativa subsp. Indica and Solanum tuberosum, forming the basis for any further experimental characterization of TA targeting and GET pathway mechanisms in crop plants.     

Development of a fluorescent transgenic zebrafish biosensor for sensing aquatic heavy metal pollution

We report a transgenic zebrafish (Danio rerio) designed to respond to heavy metals using a metal-responsive promoter linked to a fluorescent reporter gene (DsRed2). The metallothionein MT-Ia1 promoter containing metal-responsive elements was derived from the Asian green mussel, Perna viridis. The promoter is known to be induced by a broad spectrum of heavy metals. The promoter-reporter cassette cloned into the Tol2 transposon vector was microinjected into zebrafish embryos that were then reared to maturity. A transgene integration rate of 28 % was observed. The confirmed transgenics were mated with wild-type counterparts, and pools of F₁ embryos were exposed to sub-lethal doses of Cd²⁺, Cu²⁺, Hg²⁺, Pb²⁺ and Zn²⁺. The red fluorescence response of zebrafish embryos was observed 8 h post- exposure to these sub-lethal doses of heavy metals using a fluorescence microscope. Reporter expression estimated by real-time PCR revealed eightfold, sixfold and twofold increase on exposure to highest concentrations of Hg²⁺, Cd²⁺ and Cu²⁺, while Pb²⁺ and Zn²⁺ had no effect. This biosensor could be a first-level screening method for confirming aquatic heavy metal bio-toxicity to eukaryotes.     

The muscular dysgenesis mutation in mice leads to arrest of the genetic program for muscle differentiation

Muscular dysgenesis (mdg) is a mutation in mice which causes the failure of excitation-contraction coupling in skeletal muscle. Although the sarcolemma, the sarcoplasmic reticulum, and the contractile apparatus all maintain nearly normal function, sarcolemmal depolarization fails to cause calcium release from the sarcoplasmic reticulum. Recently, the primary genetic defect in this mutation was shown to be located in the structural gene for the dihydropyridine receptor. We have examined the developmental expression from Fetal Day 15 onward, in normal and mutant muscle, of several unidentified genes as well as genes which are known markers of muscle differentiation. We find that the majority of mRNA sequences are found at similar concentrations in normal and dysgenic muscles at birth. Many differentiation-related genes also are expressed at normal levels early during myogenesis in mutant mice. However, as late fetal development progresses in dysgenic muscle, the mRNA concentrations for these genes fail to undergo the rapid rise which is characteristic of normal muscle. Several additional, unidentified genes, which normally would be down-regulated during development, remain expressed at a high level in dysgenic muscle. Thus, the primary absence of a functional dihydropyridine receptor appears to prevent the changes in gene expression which are necessary for maturation of skeletal muscle.     

Cyclic AMP does not inhibit A23187-induced prostaglandin biosynthesis in cultured rabbit renal microvascular endothelial cells.

We have previously shown that cultured rabbit renal preglomerular microvascular endothelial cells have the ability to synthesize a number of common prostaglandins. In the present study we have examined whether endogenous cyclic AMP is involved in the regulation of PGI2 and PGE2 biosynthesis in these cultured cells. Isoproterenol and forskolin produced an increase in cyclic AMP accumulation in these cells but had no effect on PGI2 or PGE2 biosynthesis either in the presence or absence of A23187. Similar results were noted in the presence of 3-isobutyl-1-methylxanthine, a cyclic AMP-phosphodiesterase inhibitor. These studies suggested that endogenous cyclic AMP does not regulate the biosynthesis of PGI2 or PGE2 in cultured renal preglomerular microvascular endothelial cells either under basal or A23187-stimulated condition. They further suggested that the effect of 3-isobutyl-1-methylxanthine on prostaglandin biosynthesis in these cultured cells was not secondary to its effects on phosphodiesterase.     

Cytochalasin D-mediated hyperinduction of the substrate-associated 52-kilodalton protein p52 in rat kidney fibroblasts

Regulation of certain differentiated and housekeeping functions in cultured mammalian cells is significantly influenced by cell shape. The shape-modulating agent cytochalasin D (CD) was used, therefore, to elucidate potential cytoarchitectural influences affecting synthesis of a major 52 kDa secreted/substrate-associated protein (p52) of normal rat kidney (NRK) fibroblasts. Biosynthetic labeling experiments indicated that treatment of NRK cells with CD increased, by 10-18-fold, the medium content of an Mr 52,000 protein. Two-dimensional gel electrophoresis and peptide fragment mapping confirmed that the 52 kDa protein produced in abundance as a consequence of CD treatment was identical to p52 constitutively expressed by NRK cells. A lower mw protein (p50; Mr 50,000) was also resolved which, based on pl microheterogeneity, protease fragmentation profile, and sensitivity to tunicamycin, could be identified as a less-glycosylated form of p52. p50 and p52 were both detected in the matrix and medium compartments of NRK and NRK/CD cells. The matrix p52 content of CD-induced and uninduced cells, however, was significantly greater (by 200-500-fold) than the corresponding medium levels. This differential compartmentalization, the time course of p52 accumulation in the matrix of NRK/CD cells compared to its appearance in the medium, and the kinetics of p52 pulse-chase from the matrix collectively indicated that the matrix is the initial site of p52 deposition. Low levels of CD (1 microM) produced extensive disruptions of cellular microfilaments but did not result in an overall cell shape change nor a hyperinduction of p52. Morphologic rounding (seen in 10-100 microM CD) coincided with augmented p52 production. Transition from a flat to a round phenotype in NRK cells, or at least the generation of sufficient microfilament fragmentation to compromise cell-substrate adhesivity, appears to be an essential aspect of CD-mediated p52 hyperinduction.     

Side-to-side differences in anterior cruciate ligament volume in healthy control subjects

Examination of anterior cruciate ligament (ACL) anatomy is of great interest both in studying injury mechanisms and surgical reconstruction. However, after a typical acute ACL rupture it is not possible to measure the dimensions of the ACL itself due to concomitant or subsequent degeneration of the remaining ligamentous tissue. The contralateral ACL may be an appropriate surrogate for measuring anatomical dimensions, but it remains unknown whether side-to-side differences preclude using the contralateral as a valid surrogate for the ruptured ACL. This study examined whether the ACL volume is significantly different between the left and right knees of uninjured subjects. ACL volumes were calculated for the left and right sides of 28 individuals using a previously validated MRI-based method. The mean ACL volume was not significantly different (p=0.2331) between the two sides in this population. Side-to-side ACL volume was also well correlated (correlation=0.91, p<0.0001). The results of this study show that the volume of the contralateral ACL is a valid surrogate measure for a missing ACL on the injured side. This non-invasive, in vivo technique for measuring ACL volume may prove useful in future large-scale comprehensive studies of potential risk factors for ACL rupture, in quantifying potential loading effects on ACL size as a prophylactic measure against ACL rupture, and in the use of ACL volume as a screening tool for assessing risk of injury.     

Studies on the covalent binding of an intermediate(s) in prostaglandin biosynthesis to tissue macromolecules.

We investigated the covalent binding of intermediates in prostaglandin biosynthesis to tissue macromolecules. Following incubation of [1-14C]arachidonic acid with the microsomal fraction from guinea pig lung, ram or bovine seminal vesicle, human platelets, rabbit kidney, or rat stomach fundus, the amount of covalent binding of arachidonic acid metabolites expressed as percentage of total arachidonic acid metabolized varied from tissue to tissue ranging from 3% in human platelets to 18.2% in ram seminal vesicles. In general, the thromboxane synthesizing tissues had less covalently bound metabolites than the other tissues. The amount of covalently bound metabolites was increased in the guinea pig lung microsomes when the thromboxane synthetase inhibitor, N-0164, was added to the incubation mixture. The covalent binding of arachidonic acid metabolite(s) was greatly reduced by the addition of glutathione to the incubation mixture. In addition to the covalently bound metabolites, water-soluble metabolites derived from arachidonic acid metabolism were also observed. The amount of water-soluble metabolites was small in each tissue except for the rat stomach fundus. In the rat stomach fundus the water-soluble metabolites accounted for over 50% of the total metabolites. Conditions which would tend to increase or decrease the levels of free prostaglandin endoperoxides during the incubation of arachidonic acid with the microsomes gave increased or decreased levels of covalent binding. Our data suggest that the prostaglandin endoperoxides are responsible for the covalent binding observed during prostaglandin biosynthesis. This covalent binding to tissue macromolecules may be of physiological and pathological significance.     

ATP-competitive inhibitors of the mitotic kinesin KSP that function via an allosteric mechanism

The mitotic kinesin KSP (kinesin spindle protein, or Eg5) has an essential role in centrosome separation and formation of the bipolar mitotic spindle. Its exclusive involvement in the mitotic spindle of proliferating cells presents an opportunity for developing new anticancer agents with reduced side effects relative to antimitotics that target tubulin. Ispinesib is an allosteric small-molecule KSP inhibitor in phase 2 clinical trials. Mutations that attenuate ispinesib binding to KSP have been identified, which highlights the need for inhibitors that target different binding sites. We describe a new class of selective KSP inhibitors that are active against ispinesib-resistant forms of KSP. These ATP-competitive KSP inhibitors do not bind in the nucleotide binding pocket. Cumulative data from generation of resistant cells, site-directed mutagenesis and photo-affinity labeling suggest that they compete with ATP binding via a novel allosteric mechanism.     

Genomic and proteomic studies on the effects of the insect growth regulator diflubenzuron in the model beetle species Tribolium castaneum

Several benzoylphenyl urea-derived insecticides such as diflubenzuron (DFB, Dimilin) are in wide use to control various insect pests. Although this class of compounds is known to disrupt molting and to affect chitin content, their precise mode of action is still not understood. To gain a broader insight into the mechanism underlying the insecticidal effects of benzoylphenyl urea compounds, we conducted a comprehensive study with the model beetle species and stored product pest Tribolium castaneum (red flour beetle) utilizing genomic and proteomic approaches. DFB was added to a wheat flour-based diet at various concentrations and fed to larvae and adults. We observed abortive molting, hatching defects and reduced chitin amounts in the larval cuticle, the peritrophic matrix and eggs. Electron microscopic examination of the larval cuticle revealed major structural changes and a loss of lamellate structure of the procuticle. We used a genomic tiling array for determining relative expression levels of about 11,000 genes predicted by the GLEAN algorithm. About 6% of all predicted genes were more than 2-fold up- or down-regulated in response to DFB treatment. Genes encoding enzymes involved in chitin metabolism were unexpectedly unaffected, but many genes encoding cuticle proteins were affected. In addition, several genes presumably involved in detoxification pathways were up-regulated. Comparative 2D gel electrophoresis of proteins extracted from the midgut revealed 388 protein spots, of which 7% were significantly affected in their levels by DFB treatment as determined by laser densitometry. Mass spectrometric identification revealed that UDP-N-acetylglucosamine pyrophosphorylase and glutathione synthetase were up-regulated. In summary, the red flour beetle turned out to be a good model organism for investigating the global effects of bioactive materials such as insect growth regulators and other insecticides. The results of this study recapitulate all of the different DFB-induced symptoms in a single model insect, which have been previously found in several different insect species, and further illustrate that DFB treatment causes a wide range of effects at the molecular level.