Import of a precursor protein into chloroplasts is inhibited by the herbicide glyphosate

Import of the precursor to 5-enolpyruvylshikimate-3-phosphate synthase (pEPSPS) into chloroplasts is inhibited by the herbicide glyphosate. Inhibition of import is maximal at glyphosate concentrations of ≥10 μm and occurs only when pEPSPS is present as a ternary complex of enzyme–shikimate-3-phosphate–glyphosate. Glyphosate alone had no effect on the import of pEPSPS since it is not known to interact with the enzyme in the absence of shikimate-3-phosphate. Experiments with wild-type and glyphosate-resistant mutant forms of pEPSPS show that inhibition of import is directly proportional to the binding constants for glyphosate. Inhibition of import is thus a direct consequence of glyphosate binding to the enzyme–shikimate-3-phosphate complex. The potential for non-specific effects of glyphosate on the chloroplast transport mechanism has been discounted by showing that import of another chloroplast-designated protein was unaffected by high concentrations of glyphosate and shikimate-3-phosphate. The mechanism of import inhibition by glyphosate is consistent with a precursor unfolding/refolding model.     

Differential regulation of protein kinase C isoforms of macrophages by pathogenic and non-pathogenic mycobacteria

Given the fact that Mycobacterium tuberculosis (Mtb) may respond to the intracellular milieu of the macrophage with the induction of environmentally regulated genes required for survival and growth of the bacteria we assumed that the protein kinases may also be the factors in Mycobacterium-macrophage interaction. Since, protein kinases play a major role in various critical cellular processes including regulation of immune responses, we describe the fate of expression and phosphorylation of protein kinase C in macrophage cell lines exposed to Mtb H37Rv and raised the question whether the change in the events of expression and phosphorylation are the results of direct interaction of bacilli with macrophages and/or, are also indirectly mediated by specific cytokines that are induced in response to exposure. Our results show that only novel PKCs are phosphorylated during infection of macrophages by pathogenic and non-pathogenic mycobacteria and the alteration is a result of direct host-bacilli association which is independent of cytokines as mediators. Expression of PKC-alpha (conventional PKC isoform) was down regulated by Mtb H37Rv. In contrast the non-pathogenic fast grower Mycobacterium smegmatis (MS) increased the expression and phosphorylation of PKC-alpha. PKC-alpha was also increased in macrophages treated with serum of mice immunized with Mtb H37Rv. The study has shown that pathogenic and non-pathogenic mycobacteria categorically select the type of protein kinases C for activation/deactivation.     

Correcting improper chromosome-spindle attachments during cell division

For accurate segregation of chromosomes during cell division, microtubule fibres must attach sister kinetochores to opposite poles of the mitotic spindle (bi-orientation). Aurora kinases are linked to oncogenesis and have been implicated in the regulation of chromosome-microtubule attachments. Although loss of Aurora kinase activity causes an accumulation of mal-orientated chromosomes in dividing cells, it is not known how the active kinase corrects improper chromosome attachments. The use of reversible small-molecule inhibitors allows activation of protein function in living vertebrate cells with temporal control. Here we show that by removal of small-molecule inhibitors, controlled activation of Aurora kinase during mitosis can correct chromosome attachment errors by selective disassembly of kinetochore-microtubule fibres, rather than by alternative mechanisms involving initial release of microtubules from either kinetochores or spindle poles. Observation of chromosomes and microtubule dynamics with real-time high-resolution microscopy showed that mal-orientated, but not bi-orientated, chromosomes move to the spindle pole as both kinetochore-microtubule fibres shorten, followed by alignment at the metaphase plate. Our results provide direct evidence for a mechanism required for the maintenance of genome integrity during cell division.     

Evaluation of the suitability of free-energy minimization using nearest-neighbor energy parameters for RNA secondary structure prediction

Background

A detailed understanding of an RNA's correct secondary and tertiary structure is crucial to understanding its function and mechanism in the cell. Free energy minimization with energy parameters based on the nearest-neighbor model and comparative analysis are the primary methods for predicting an RNA's secondary structure from its sequence. Version 3.1 of Mfold has been available since 1999. This version contains an expanded sequence dependence of energy parameters and the ability to incorporate coaxial stacking into free energy calculations. We test Mfold 3.1 by performing the largest and most phylogenetically diverse comparison of rRNA and tRNA structures predicted by comparative analysis and Mfold, and we use the results of our tests on 16S and 23S rRNA sequences to assess the improvement between Mfold 2.3 and Mfold 3.1.

Results

The average prediction accuracy for a 16S or 23S rRNA sequence with Mfold 3.1 is 41%, while the prediction accuracies for the majority of 16S and 23S rRNA structures tested are between 20% and 60%, with some having less than 20% prediction accuracy. The average prediction accuracy was 71% for 5S rRNA and 69% for tRNA. The majority of the 5S rRNA and tRNA sequences have prediction accuracies greater than 60%. The prediction accuracy of 16S rRNA base-pairs decreases exponentially as the number of nucleotides intervening between the 5' and 3' halves of the base-pair increases.

Conclusion

Our analysis indicates that the current set of nearest-neighbor energy parameters in conjunction with the Mfold folding algorithm are unable to consistently and reliably predict an RNA's correct secondary structure. For 16S or 23S rRNA structure prediction, Mfold 3.1 offers little improvement over Mfold 2.3. However, the nearest-neighbor energy parameters do work well for shorter RNA sequences such as tRNA or 5S rRNA, or for larger rRNAs when the contact distance between the base-pairs is less than 100 nucleotides.     

Interaction between Nbp35 and Cfd1 Proteins of Cytosolic Fe-S Cluster Assembly Reveals a Stable Complex Formation in Entamoeba histolytica

Iron-Sulfur (Fe-S) proteins are involved in many biological functions such as electron transport, photosynthesis, regulation of gene expression and enzymatic activities. Biosynthesis and transfer of Fe-S clusters depend on Fe-S clusters assembly processes such as ISC, SUF, NIF, and CIA systems. Unlike other eukaryotes which possess ISC and CIA systems, amitochondriate Entamoeba histolytica has retained NIF & CIA systems for Fe-S cluster assembly in the cytosol. In the present study, we have elucidated interaction between two proteins of E. histolytica CIA system, Cytosolic Fe-S cluster deficient 1 (Cfd1) protein and Nucleotide binding protein 35 (Nbp35). In-silico analysis showed that structural regions ranging from amino acid residues (P33-K35, G131-V135 and I147-E151) of Nbp35 and (G5-V6, M34-D39 and G46-A52) of Cfd1 are involved in the formation of protein-protein complex. Furthermore, Molecular dynamic (MD) simulations study suggested that hydrophobic forces surpass over hydrophilic forces between Nbp35 and Cfd1 and Van-der-Waal interaction plays crucial role in the formation of stable complex. Both proteins were separately cloned, expressed as recombinant fusion proteins in E. coli and purified to homogeneity by affinity column chromatography. Physical interaction between Nbp35 and Cfd1 proteins was confirmed in vitro by co-purification of recombinant Nbp35 with thrombin digested Cfd1 and in vivo by pull down assay and immunoprecipitation. The insilico, in vitro as well as in vivo results prove a stable interaction between these two proteins, supporting the possibility of its involvement in Fe-S cluster transfer to target apo-proteins through CIA machinery in E. histolytica. Our study indicates that initial synthesis of a Fe-S precursor in mitochondria is not necessary for the formation of Cfd1-Nbp35 complex. Thus, Cfd1 and Nbp35 with the help of cytosolic NifS and NifU proteins can participate in the maturation of non-mitosomal Fe-S proteins without any apparent assistance of mitosomes.     

The Genetic Architecture of Climatic Adaptation of Tropical Cattle

Adaptation of global food systems to climate change is essential to feed the world. Tropical cattle production, a mainstay of profitability for farmers in the developing world, is dominated by heat, lack of water, poor quality feedstuffs, parasites, and tropical diseases. In these systems European cattle suffer significant stock loss, and the cross breeding of taurine x indicine cattle is unpredictable due to the dilution of adaptation to heat and tropical diseases. We explored the genetic architecture of ten traits of tropical cattle production using genome wide association studies of 4,662 animals varying from 0% to 100% indicine. We show that nine of the ten have genetic architectures that include genes of major effect, and in one case, a single location that accounted for more than 71% of the genetic variation. One genetic region in particular had effects on parasite resistance, yearling weight, body condition score, coat colour and penile sheath score. This region, extending 20 Mb on BTA5, appeared to be under genetic selection possibly through maintenance of haplotypes by breeders. We found that the amount of genetic variation and the genetic correlations between traits did not depend upon the degree of indicine content in the animals. Climate change is expected to expand some conditions of the tropics to more temperate environments, which may impact negatively on global livestock health and production. Our results point to several important genes that have large effects on adaptation that could be introduced into more temperate cattle without detrimental effects on productivity.