A model of direction selectivity in the starburst amacrine cell network

Displaced starburst amacrine cells (SACs) are retinal interneurons that exhibit GABA _A receptor-mediated and Cl ^? cotransporter-mediated, directionally selective (DS) light responses in the rabbit retina. They depolarize to stimuli that move centrifugally through the receptive field surround and hyperpolarize to stimuli that move centripetally through the surround (Gavrikov et al, PNAS 100(26):16047–16052, 2003, PNAS 103(49):18793–18798, 2006). They also play a key role in the activity of DS ganglion cells (DS GC; Amthor et al, Vis Neurosci 19:495–509 2002; Euler et al, Nature 418:845–852, 2002; Fried et al, Nature 420:411– 414, 2002; Gavrikov et al, PNAS 100(26):16047–16052, 2003, PNAS 103(49):18793–18798, 2006; Lee and Zhou, Neuron 51:787–799 2006; Yoshida et al, Neuron 30:771–780, 2001). In this paper we present a model of strong DS behavior of SACs which relies on the GABA-mediated communication within a tightly interconnected network of these cells and on the glutamate signal that the SACs receive from bipolar cells (a presynaptic cell that receives input from cones). We describe how a moving light stimulus can produce a large, sustained depolarization of the SAC dendritic tips that point in the direction that the stimulus moves (i.e., centrifugal motion), but produce a minimal depolarization of the dendritic tips that point in the opposite direction (i.e., centripetal motion). This DS behavior, which is quantified based on the relative size and duration of the depolarizations evoked by stimulus motion at dendritic tips pointing in opposite directions, is robust to changes of many different parameter values and consistent with experimental data. In addition, the DS behavior is strengthened under the assumptions that the Cl^? cotransporters Na^?+?-K^?+?-Cl^?? and K^?+?-Cl^?? are located in different regions of the SAC dendritic tree (Gavrikov et al, PNAS 103(49):18793–18798, 2006) and that GABA evokes a long-lasting response (Gavrikov et al, PNAS 100(26):16047–16052, 2003, PNAS 103(49):18793–18798, 2006; Lee and Zhou, Neuron 51:787–799, 2006). A possible mechanism is discussed based on the generation of waves of local glutamate and GABA secretion, and their postsynaptic interplay as the waves travel between cell compartments.     

Septin 7 forms a complex with CD2AP and nephrin and regulates glucose transporter trafficking

Podocytes are insulin-sensitive and take up glucose in response to insulin. This requires nephrin, which interacts with vesicle-associated membrane protein 2 (VAMP2) on GLUT4 storage vesicles (GSVs) and facilitates their fusion with the plasma membrane. In this paper, we show that the filament-forming GTPase septin 7 is expressed in podocytes and associates with CD2-associated protein (CD2AP) and nephrin, both essential for glomerular ultrafiltration. In addition, septin 7 coimmunoprecipitates with VAMP2. Subcellular fractionation of cultured podocytes revealed that septin 7 is found in both cytoplasmic and membrane fractions, and immunofluorescence microscopy showed that septin 7 is expressed in a filamentous pattern and is also found on vesicles and the plasma membrane. The filamentous localization of septin 7 depends on CD2AP and intact actin organization. A 2-deoxy-d-glucose uptake assay indicates that depletion of septin 7 by small interfering RNA or alteration of septin assembly by forchlorfenuron facilitates glucose uptake into cells and further, knockdown of septin 7 increased the interaction of VAMP2 with nephrin and syntaxin 4. The data indicate that septin 7 hinders GSV trafficking and further, the interaction of septin 7 with nephrin in glomeruli suggests that septin 7 may participate in the regulation of glucose transport in podocytes.     

α-Synuclein Amyloid Fibrils with Two Entwined,Asymmetrically Associated Protofibrils

Parkinson disease and other progressive neurodegenerative conditions are characterized by the intracerebral presence of Lewy bodies, containing amyloid fibrils of α-synuclein. We used cryo-electron microscopy and scanning transmission electron microscopy (STEM) to study in vitro-assembled fibrils. These fibrils are highly polymorphic. Focusing on twisting fibrils with an inter-crossover spacing of 77 nm, our reconstructions showed them to consist of paired protofibrils. STEM mass per length data gave one subunit per 0.47 nm axial rise per protofibril, consistent with a superpleated β-structure. The STEM images show two thread-like densities running along each of these fibrils, which we interpret as ladders of metal ions. These threads confirmed the two-protofibril architecture of the 77-nm twisting fibrils and allowed us to identify this morphotype in STEM micrographs. Some other, but not all, fibril morphotypes also exhibit dense threads, implying that they also present a putative metal binding site. We propose a molecular model for the protofibril and suggest that polymorphic variant fibrils have different numbers of protofibrils that are associated differently.     

Self-assembling protein arrays using electronic semiconductor microchips and in vitro translation

Protein arrays will greatly accelerate research and development in medical and biological sciences. We have used cell-free protein biosynthesis and a parallel immobilization strategy for producing protein biochips. We demonstrate a model two-protein microarray using luciferase and green fluorescent protein, both expressed in a cell-free system and specifically immobilized on CombiMatrix semiconductor oligonucleotide microarrays. This demonstration provides evidence for the appropriate folding, activity, robust presentation, and efficient flexible detection of proteins on the microscale.     

Optimization of casein-based semisynthetic medium for growing of toxigenic Corinebacterium diphtheriae in a fermenter

Diphtheria toxin is produced by growing Corinebacterium diphtheriae either in a semisynthetic casein-based medium or in the Pope-Lingood meat extract based medium. The World Health Organization advises the use of the semisynthetic one, as it has important advantages. Data on the composition of casein-based media and their ability to support high toxin production are not freely available. Important factors affecting toxin production during C. diphtheriae cultivation are the pH of the culture medium and the concentration of casein hydrolysate and of Fe2+. We established that the optimal pH for toxin production is 7.2. The highest yield of toxin was obtained using a casein hydrolysate concentration of 35.0 g/L and a Fe2+ concentration of 0.05-0.41 microg/mL. Under these conditions, diphtheria toxin with higher purity and yield compared with the batches obtained using the meat-based medium of Pope-Lingood was produced.     

Beta-helix model for the filamentous haemagglutinin adhesin of Bordetella pertussis and related bacterial secretory proteins

Bordetella pertussis establishes infection by attaching to epithelial cells of the respiratory tract. One of its adhesins is filamentous haemagglutinin (FHA), a 500-A-long secreted protein that is rich in beta-structure and contains two regions, R1 and R2, of tandem 19-residue repeats. Two models have been proposed in which the central shaft is (i) a hairpin made up of a pairing of two long antiparallel beta-sheets; or (ii) a beta-helix in which the polypeptide chain is coiled to form three long parallel beta-sheets. We have analysed a truncated variant of FHA by electron microscopy (negative staining, shadowing and scanning transmission electron microscopy of unstained specimens): these observations support the latter model. Further support comes from detailed sequence analysis and molecular modelling studies. We applied a profile search method to the sequences adjacent to and between R1 and R2 and found additional "covert" copies of the same motifs that may be recognized in overt form in the R1 and R2 sequence repeats. Their total number is sufficient to support the tenet of the beta-helix model that the shaft domain--a 350 A rod--should consist of a continuous run of these motifs, apart from loop inserts. The N-terminus, which does not contain such repeats, was found to be weakly homologous to cyclodextrin transferase, a protein of known immunoglobulin-like structure. Drawing on crystal structures of known beta-helical proteins, we developed structural models of the coil motifs putatively formed by the R1 and R2 repeats. Finally, we applied the same profile search method to the sequence database and found several other proteins--all large secreted proteins of bacterial provenance--that have similar repeats and probably also similar structures.     

5-alkylvinyl-1,2,4-triazole nucleosides: Synthesis and biological evaluation

Abstract

Some 5-substituted ribavirin analogues have a high antiviral and anticancer activity, but their mechanisms of action are obviously not the same as their parent compound. The SAR studies performed on 3 (5)-substituted 1,2,4-triazole nucleosides have shown a high dependency between the structure of the 3 (5)-substituent and the level of antiviral/anticancer activity. The most active substances of the row contain coplanar with the 1,2,4-triazole ring aromatic substituent which is connected by a rigid ethynyl bond. However, the compounds with the trans-vinyl linker also had antiviral activity. We decided to study the antitumor activity of ribavirin analogues with alkyl/aryl vinyl substituents in the 5th position of the 1,2,4-triazole ring. Protected nucleoside analogues with various 5-alkylvinyl substituents were obtained by Horner-Wadsworth-Emmons reaction from the common precursor and converted to the nucleosides. Arylvinyl nucleosides were synthesised according the reported procedures. All compounds did not show significant antiproliferative activity on several tumour cell lines. Coplanar aromatic motif in the 5-substituent for the anticancer activity manifestation was confirmed.     

Dihydro-resveratrol—A potent dietary polyphenol

Dihydro-resveratrol (dihydro-R), a prominent polyphenol component of red wine, has a profound proliferative effect on hormone-sensitive tumor cell lines such as breast cancer cell line MCF7. We found a significant increase in MCF7 tumor cells growth rates in the presence of picomolar concentrations of this compound. The proliferative effect of dihydro-R was not observed in cell lines that do not express hormone receptors (MDA-MB-231, BT-474, and К-562).     

Cold-active DnaK of an Antarctic psychrotroph Shewanella sp. Ac10 supporting the growth of dnaK-null mutant of Escherichia coli at cold temperatures

Shewanella sp. Ac10 is a psychrotrophic bacterium isolated from the Antarctica that actively grows at such low temperatures as 0°C. Immunoblot analyses showed that a heat-shock protein DnaK is inducibly formed by the bacterium at 24°C, which is much lower than the temperatures causing heat shock in mesophiles such as Escherichia coli. We found that the Shewanella DnaK (SheDnaK) shows much higher ATPase activity at low temperatures than the DnaK of E. coli (EcoDnaK): a characteristic of a cold-active enzyme. The recombinant SheDnaK gene supported neither the growth of a dnaK-null mutant of E. coli at 43°C nor phage propagation at an even lower temperature, 30°C. However, the recombinant SheDnaK gene enabled the E. coli mutant to grow at 15°C. This is the first report of a DnaK supporting the growth of a dnaK-null mutant at low temperatures.