Structure-function analysis of the C-terminal domain of LcrV from Yersinia pestis

LcrV, a multifunctional protein, acts as a positive regulator of effector protein secretion for the type III secretion system (T3SS) in Yersinia pestis by interaction with the negative regulator LcrG. In this study, LcrV was analyzed to identify regions required for LcrG interaction. Random-linker insertion mutagenesis, deletion analysis, and site-directed mutagenesis of hydrophobic amino acids between residues 290 and 311 allowed the isolation of an LcrV mutant (LcrV L291R F308R) defective for LcrG interaction. The new residues identified in LcrG interaction lie in helix 12 of LcrV; residues in helix 7 of LcrV are known to be involved in LcrG interaction. Helix 7 and helix 12 of LcrV interact to form an intramolecular coiled coil; these new results suggest that the intramolecular coiled coil in LcrV is required for LcrG interaction and activation of the T3SS.     

Ovicidal activity of Sesamum indicum (L.) oil against Spodoptera littoralis (Boisd.)

In the research for alternative tools and botanical products to control Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae). Sesamum indicum (L.) (Lamiales: Pedaliaceae) oil was assayed as an ovicide. The mortality increased with existence of fatty acids. Chemical analysis of S. indicum oil using GLC analysis showed palmitic acid as the major fatty acid (51.27%), while the major hydrocarbon and sterols were found to be heneicosane (58.63%) and β-sitosterol (2.60%), respectively. Generally, the values of LC₅₀s indicated that one-day-old egg masses are more susceptible than three-day-old eggs. Also, the leaf dip technique is more efficient than the spraying one. Results showed several features of chorionic surface deformation treated with sesame and KZ oils than control using scanning electron microscopy. Meanwhile, the tested oils caused significant reduction in both total soluble protein and transaminase enzymes as compared to control. Additionally, the oils elongated the incubation period and larval duration than control.     

Expression of tomato thymidine kinase 1 by means of the baculovirus expression vector system

ABSTRACT

Tomato thymidine kinase 1 (ToTK1) is a deoxyribonucleoside kinase (dNK) that has been subject to study because of its potential to phosphorylate the nucleoside analogue 3-azido-2,3-dideoxythymidine (azidothymidine, AZT) equally well as its natural substrate thymidine (dThd). The combination of ToTK1 and AZT has been tested in two animal studies for its efficiency and use in suicide gene therapy for malignant glioma. The determination of the 3D structure of ToTK1 might shed light on the structure–function relationships of nucleoside activation by this enzyme and thereby show routes toward further improvement of ToTK1 and other TK1-like dNKs for suicide gene therapy. Here we report the successful expression of both full-length ToTK1 and a C-terminal truncated ToTK1 in Spodoptera frugiperda and Trichoplusia ni insect cells using the baculovirus expression vector system. This constitutes a further step on the road to determine the 3D structure of the first TK1 of plant origin, but also an enzyme with great potential for dNK-mediated suicide gene therapy.     

Christia vespertilionis extract induced antiproliferation and apoptosis in breast cancer (MCF7) cells

Molecular Biology Reports - C. vespertiliomis extracts were evaluated for antiproliferative and apoptosis effect on breast cancer (MCF7) cells. The leaves extracts were analysed for its...     

The structure of the excisionase (Xis) protein from conjugative transposon Tn916 provides insights into the regulation of heterobivalent tyrosine recombinases

Heterobivalent tyrosine recombinases play a prominent role in numerous bacteriophage and transposon recombination systems. Their enzymatic activities are frequently regulated at a structural level by excisionase factors, which alter the ability of the recombinase to assemble into higher-order recombinogenic nucleoprotein structures. The Tn916 conjugative transposon spreads antibiotic resistance in pathogenic bacteria and is mobilized by a heterobivalent recombinase (Tn916Int), whose activity is regulated by an excisionase factor (Tn916Xis). Unlike the well-characterized (lambda)Xis excisionase from bacteriophage lambda, Tn916Xis stimulates excision in vitro and in Escherichia coli only modestly. To gain insights into this functional difference, we have performed in vitro DNA-binding studies of Tn916Xis and Tn916Int, and we have solved the solution structure of Tn916Xis. We show that the heterobivalent Tn916Int protein is capable of bridging the DR2-type and core-type sites on the left arm of the tranpsoson. Consistent with the notion that Tn916Int is regulated only loosely, we find that Tn916Xis binding does not alter the stability of DR2-Tn916Int-core bridges or the ability of Tn916Int to recognize the arms of the transposon in vitro. Despite a high degree of divergence at the primary sequence level, we show that Tn916Xis and (lambda)Xis adopt related prokaryotic winged-helix structures. However, they differ at their C termini, with Tn916Xis replacing the flexible integrase contacting tail found in (lambda)Xis with a positively charged alpha-helix. This difference provides a structural explanation for why Tn916Xis does not interact cooperatively with its cognate integrase in vitro, and reveals how subtle changes in the winged-helix fold can modulate the functional properties of excisionase factors.     

Analysis of Microtubule-Associated-Proteins during IBA-Mediated Adventitious Root Induction Reveals KATANIN Dependent and Independent Alterations of Expression Patterns

Adventitious roots (AR) are post embryonic lateral organs that differentiate from non-root tissues. The understanding of the molecular mechanism which underlies their differentiation is important because of their central role in vegetative plant propagation. Here it was studied how the expression of different microtubule (MT)-associated proteins (MAPs) is affected during AR induction, and whether expression differences are dependent on MT organization itself. To examine AR formation when MTs are disturbed we used two mutants in the MT severing protein KATANIN. It was found that rate and number of AR primordium formed following IBA induction for three days was reduced in bot1-1 and bot1-7 plants. The reduced capacity to form ARs in bot1-1 was associated with altered expression of MAP-encoding genes along AR induction. While the expression of MAP65-4, MAP65-3, AURORA1, AURORA2 and TANGLED, increased in wild-type but not in bot1-1 plants, the expression of MAP65-8 and MDP25 decreased in wild type plants but not in the bot1-1 plant after two days of IBA-treatment. The expression of MOR1 was increased two days after AR induction in wild type and bot1-1 plants. To examine its expression specifically in AR primordium, MOR1 upstream regulatory sequence was isolated and cloned to regulate GFP. Expression of GFP was induced in the primary root tips and lateral roots, in the pericycle of the hypocotyls and in all stages of AR primordium formation. It is concluded that the expression of MAPs is regulated along AR induction and that reduction in KATANIN expression inhibits AR formation and indirectly influences the specific expression of some MAPs.     

Exome sequencing followed by large-scale genotyping fails to identify single rare variants of large effect in idiopathic generalized epilepsy

Idiopathic generalized epilepsy (IGE) is a complex disease with high heritability, but little is known about its genetic architecture. Rare copy-number variants have been found to explain nearly 3% of individuals with IGE; however, it remains unclear whether variants with moderate effect size and frequencies below what are reliably detected with genome-wide association studies contribute significantly to disease risk. In this study, we compare the exome sequences of 118 individuals with IGE and 242 controls of European ancestry by using next-generation sequencing. The exome-sequenced epilepsy cases include study subjects with two forms of IGE, including juvenile myoclonic epilepsy (n = 93) and absence epilepsy (n = 25). However, our discovery strategy did not assume common genetic control between the subtypes of IGE considered. In the sequence data, as expected, no variants were significantly associated with the IGE phenotype or more specific IGE diagnoses. We then selected 3,897 candidate epilepsy-susceptibility variants from the sequence data and genotyped them in a larger set of 878 individuals with IGE and 1,830 controls. Again, no variant achieved statistical significance. However, 1,935 variants were observed exclusively in cases either as heterozygous or homozygous genotypes. It is likely that this set of variants includes real risk factors. The lack of significant association evidence of single variants with disease in this two-stage approach emphasizes the high genetic heterogeneity of epilepsy disorders, suggests that the impact of any individual single-nucleotide variant in this disease is small, and indicates that gene-based approaches might be more successful for future sequencing studies of epilepsy predisposition.