Crystal structure of a Vip3B family insecticidal protein reveals a new fold and a unique tetrameric assembly

Vegetatively expressed insecticidal proteins (VIPs) produced by Bacillus thuringiensis fall into several classes of which the third, VIP3, is known for their activity against several key Lepidopteran pests of commercial broad acre crops and because their mode of action does not overlap with that of crystalline insecticidal proteins. The details of the VIP3 structure and mode of action have remained obscure for the quarter century that has passed since their discovery. In the present article, we report the first crystal structure of a full‐length VIP3 protein. Crystallization of this target required multiple rounds of construct optimization and screening—over 200 individual sequences were expressed and tested. This protein adopts a novel global fold that combines domains with hitherto unreported topology and containing elements seemingly borrowed from carbohydrate‐binding domains, lectins, or from other insecticidal proteins.     

Optimization of treatment of patients with chronic cardiac insufficiency of the ischemic genesis and hobnail liver

Clinical efficacy of combined therapy including the use of rifaximin and L-ornithin-l-aspartate, as well as the dynamics of the biochemical indices, the manifestation levels of portal-systemic-encephalopathy and intestinal microbiocynosis were investigated in patients with chronic cardiac insufficiency of ischemic genesis and hobnail liver. The combined therapy resulted in improvement of the patients clinical state, lower levels of the portal-systemic encephalopathy manifectation by decreasing hyperammonium, normalization of the large intestine microflora, and blood serum biochemical parameters.     

Study of bacteriophage T4-encoded Dam DNA (adenine-N6)-methyltransferase binding with substrates by rapid laser UV cross-linking

DNA methyltransferases of the Dam family (including bacteriophage T4-encoded Dam DNA (adenine-N(6))-methyltransferase (T4Dam)) catalyze methyl group transfer from S-adenosyl-L-methionine (AdoMet), producing S-adenosyl-L-homocysteine (AdoHcy) and methylated adenine residues in palindromic GATC sequences. In this study, we describe the application of direct (i.e. no exogenous cross-linking reagents) laser UV cross-linking as a universal non-perturbing approach for studying the characteristics of T4Dam binding with substrates in the equilibrium and transient modes of interaction. UV irradiation of the enzyme.substrate complexes using an Nd(3+):yttrium aluminum garnet laser at 266 nm resulted in up to 3 and >15% yields of direct T4Dam cross-linking to DNA and AdoMet, respectively. Consequently, we were able to measure equilibrium constants and dissociation rates for enzyme.substrate complexes. In particular, we demonstrate that both reaction substrates, specific DNA and AdoMet (or product AdoHcy), stabilized the ternary complex. The improved substrate affinity for the enzyme in the ternary complex significantly reduced dissociation rates (up to 2 orders of magnitude). Several of the parameters obtained (such as dissociation rate constants for the binary T4Dam.AdoMet complex and for enzyme complexes with a nonfluorescent hemimethylated DNA duplex) were previously inaccessible by other means. However, where possible, the results of laser UV cross-linking were compared with those of fluorescence analysis. Our study suggests that rapid laser UV cross-linking efficiently complements standard DNA methyltransferase-related tools and is a method of choice to probe enzyme-substrate interactions in cases in which data cannot be acquired by other means.     

Modifications of plastic properties and metaplasticity of glutamatergic synapses in the rat cortex and hippocampus under conditions of reserpine-induced behavioral depression

Intraperitoneal injection of 1 mg/kg reserpine into rats caused the development of behavioral depression that was especially clearly pronounced 24 h after injection. Under such conditions, induction of long-term potentiation of synaptic transmission was suppressed, the development of long-term depression in glutamatergic synapses of pyramidal neurons of the hippocampal CA1 area and layers II/III of the parietal cortex was facilitated, and metaplasticity threshold (θ_M) was shifted to the right. Such modifications of plasticity and metaplasticity of glutamatergic synapses were determined by changes in the functional state of postsynaptic NMDA receptors, which was confirmed by a decrease in the duration of NMDA component of field EPSPs generated in the studied neurons and by an increase in the sensitivity of this component to the action of a nonselective blocker of NMDA receptors, ketamine. Simultaneously, the sensitivity to zinc and haloperidol, which are selective with respect to NMDA receptors with the subunit composition NR1/NR2B, decreased. It is hypothesized that, under conditions of depression, either replacement of a part of NR2B subunits in the structure of NMDA receptors by NR2A subunits or biochemical inactivation of NMDA receptors containing NR2B subunit, as well as a decrease in the clearance of transmitter in glutamatergic synapses, occur; these events determine the impairment of plastic properties of the latter contacts. Neirofiziologiya/Neurophysiology, Vol. 39, No. 3, pp. 214–221, May–June, 2007.     

Antiproliferative and apoptosis inducing properties of pyrano[3,2-c]pyridones accessible by a one-step multicomponent synthesis

4-Arylpyrano-[3,2-c]-pyridones have been prepared by a one-step cyclocondensation of 4-hydroxy-1,6-dimethylpyridin-2(1H)-one with various substituted benzaldehydes and malononitrile. These heterocycles exhibit micromolar and submicromolar antiproliferative activity in HeLa and induce apoptosis in Jurkat cell lines. Structure-activity studies performed on a small library of these compounds show a pronounced cytotoxicity enhancing effect of the bromo substituent at the meta position of the C4 aromatic moiety.     

Unique cell type-specific junctional complexes in vascular endothelium of human and rat liver sinusoids

Liver sinusoidal endothelium is strategically positioned to control access of fluids, macromolecules and cells to the liver parenchyma and to serve clearance functions upstream of the hepatocytes. While clearance of macromolecular debris from the peripheral blood is performed by liver sinusoidal endothelial cells (LSECs) using a delicate endocytic receptor system featuring stabilin-1 and -2, the mannose receptor and CD32b, vascular permeability and cell trafficking are controlled by transcellular pores, i.e. the fenestrae, and by intercellular junctional complexes. In contrast to blood vascular and lymphatic endothelial cells in other organs, the junctional complexes of LSECs have not yet been consistently characterized in molecular terms. In a comprehensive analysis, we here show that LSECs express the typical proteins found in endothelial adherens junctions (AJ), i.e. VE-cadherin as well as α-, β-, p120-catenin and plakoglobin. Tight junction (TJ) transmembrane proteins typical of endothelial cells, i.e. claudin-5 and occludin, were not expressed by rat LSECs while heterogenous immunreactivity for claudin-5 was detected in human LSECs. In contrast, junctional molecules preferentially associating with TJ such as JAM-A, B and C and zonula occludens proteins ZO-1 and ZO-2 were readily detected in LSECs. Remarkably, among the JAMs JAM-C was considerably over-expressed in LSECs as compared to lung microvascular endothelial cells. In conclusion, we show here that LSECs form a special kind of mixed-type intercellular junctions characterized by co-occurrence of endothelial AJ proteins, and of ZO-1 and -2, and JAMs. The distinct molecular architecture of the intercellular junctional complexes of LSECs corroborates previous ultrastructural findings and provides the molecular basis for further analyses of the endothelial barrier function of liver sinusoids under pathologic conditions ranging from hepatic inflammation to formation of liver metastasis.     

Effect of base analog substitutions in the specific GATC site on binding and methylation of oligonucleotide duplexes by the bacteriophage T4 Dam DNA-[N6-adenine] methyltransferase.

The interaction of the phage T4 Dam DNA-[N6-adenine] methyltransferase with 24mer synthetic oligonucleotide duplexes having different purine base substitutions in the palindromic recognition sequence, GATC, was investigated by means of gel shift and methyl transfer assays. The substitutions were introduced in either the upper or lower strand: guanine by 7-deazaguanine (G-->D) or 2-aminopurine (G-->N) and target adenine by purine (A-->P) or 2-aminopurine (A-->N). The effects of each base modification on binding/methylation were approximately equivalent for both strands. G-->D and G-->N substitutions resulted in a sharp decrease in binary complex formation. This suggests that T4 Dam makes hydrogen bonds with either the N7- or O6-keto groups (or both) in forming the complex. In contrast, A-->P and A-->N substitutions were much more tolerant for complex formation. This confirms our earlier observations that the presence of intact 5'-G:C base pairs at both ends of the methylation site is critical, but that base substitutions within the central A:T base pairs show less inhibition of complex formation. Addition of T4 Dam to a complete substrate mixture resulted in a burst of [3H]methylated product. In all cases the substrate dependencies of bursts and methylation rates were proportional to each other. For the perfect 24mer k cat = 0.014/s and K m = 7.7 nM was obtained. In contrast to binary complex formation the two guanine substitutions exerted relatively minor effects on catalytic turnover (the k cat was reduced at most 2. 5-fold), while the two adenine substitutions showed stronger effects (5- to 15-fold reduction in k cat). The effects of base analog substitutions on K m(DNA) were more variable: A-->P (decreased); A-->N and G-->D (unchanged); G-->N (increased).     

A compact form of DNA in solution. III. Influence of the ion composition of the solution on the compactization process of double-stranded DNA in the presence of peg]

The data showing the features of the DNA compactization process in PEG-containing solutions of chlorides of different alkaline metals (LiCl, KCl, RbCl and CsCl) and an ammonium salt (CH3-(CH2)17-N-(CH3)3Br) are presented. The data indicate that the formation of a compact form of the double-stranded DNA in PEG-containing water-salt solutions depends not only on the PEG concentration and ionic strength but on tha cation nature as well. The compactization occurs most easily in the presence of Na+-ions. This indicates a specific character of interaction between Na+-ions and DNA phosphate groups which may be due to an optimum structural fit between the hydrated Na+-ions and orientation of the phosphate groups in the DNA molecule. The nature of forces involved in the processes of the intramolecular compactization and intermolecular aggregation of double-stranded DNA molecules in water-salt solution is discussed. The difference between the effect of Na+ and that of K+-ions on the compactization process at the ionic strengths close to physiological values makes it possible to suggest that the changes of the tertiary structure of double-stranded DNA which accompany its function in vivo may take place under conditions of a decreased water activity at the expense of relatively slight changes in ion composition of the water surrounding DNA.     

Macroscopic fluctuations of the kinetic activity of alcohol dehydrogenase

Macroscopic fluctuations of liver alcohol dehydrogenase enzymic activity in complex reaction consisting in ethanol oxidation and butyraldehyde reduction were studied. It was found that maximal fluctuation amplitude was observed when the rates of both reactions were equal. Such dependence indicates connection between macroscopic fluctuations in alcohol dehydrogenase reaction and oscillations of relative affinity of the enzyme to oxidized and reduced coenzyme forms.