Combining ganglion cell topology and data of patients with glaucoma to determine a structure-function map

PURPOSE: To introduce techniques for deriving a map that relates visual field locations to optic nerve head (ONH) sectors and to use the techniques to derive a map relating Medmont perimetric data to data from the Heidelberg Retinal Tomograph. METHODS: Spearman correlation coefficients were calculated relating each visual field location (Medmont M700) to rim area and volume measures for 10 degrees ONH sectors (HRT III software) for 57 participants: 34 with glaucoma, 18 with suspected glaucoma, and 5 with ocular hypertension. Correlations were constrained to be anatomically plausible with a computational model of the axon growth of retinal ganglion cells (Algorithm GROW). GROW generated a map relating field locations to sectors of the ONH. The sector with the maximum statistically significant (P < 0.05) correlation coefficient within 40 degrees of the angle predicted by GROW for each location was computed. Before correlation, both functional and structural data were normalized by either normative data or the fellow eye in each participant. RESULTS: The model of axon growth produced a 24-2 map that is qualitatively similar to existing maps derived from empiric data. When GROW was used in conjunction with normative data, 31% of field locations exhibited a statistically significant relationship. This significance increased to 67% (z-test, z = 4.84; P < 0.001) when both field and rim area data were normalized with the fellow eye. CONCLUSIONS: A computational model of axon growth and normalizing data by the fellow eye can assist in constructing an anatomically plausible map connecting visual field data and sectoral ONH data.

Structure-Function Studies of GDNF Family Ligand-RET signalling

Glial cell line-derived neurotrophic factor (GDNF) and its family members neurturin (NRTN), artemin (ARTN) and persephin (PSPN) are growth factors, which are involved in the development, differentiation and maintenance of many neuron types. In addition, they function outside of the nervous system, e.g. in the development of kidney, testis and liver. GDNF family ligand (GFL) signalling happens through a tetrameric receptor complex, which includes two glycosylphosphatidylinositol (GPI)-anchored GDNF family receptor (GFRα) molecules and two RET (rearranged during transfection) receptor tyrosine kinases. Each of the ligands binds preferentially one of the four GFRα receptors: GDNF binds to GFRα1, NRTN to GFRα2, ARTN to GFRα3 and PSPN to GFRα4. The signal is then delivered by RET, which cannot bind the GFLs on its own, but can bind the GFL-GFRα complex. Under normal cellular conditions, RET is only phosphorylated on the cell surface after ligand binding. At least the GDNF-GFRα1 complex is believed to recruit RET to lipid rafts, where downstream signalling occurs. In general, GFRαs consist of three cysteine-rich domains, but all GFRα4s except for chicken GFRα4 lack domain 1 (D1). We characterised the biochemical and cell biological properties of mouse PSPN receptor GFRα4 and showed that it has a significantly weaker capacity than GFRα1 to recruit RET to the lipid rafts. In spite of that, it can phosphorylate RET in the presence of PSPN and contribute to neuronal differentiation and survival. Therefore, the recruitment of RET to the lipid rafts does not seem to be crucial for the biological activity of all GFRα receptors. Secondly, we demonstrated that GFRα1 D1 stabilises the GDNF-GFRα1 complex and thus affects the phosphorylation of RET and contributes to the biological activity. This may be important in physiological conditions, where the concentration of the ligand or the soluble GFRα1 receptor is low. Our results also suggest a role for D1 in heparin binding and, consequently, in the biodistribution of released GFRα1 or in the formation of the GFL-GFRα-RET complex. We also presented the crystallographic structure of GDNF in the complex with GFRα1 domains 2 and 3. The structure differs from the previously published ARTN-GFRα3 structure in three significant ways. The biochemical data verify the structure and reveal residues participating in the interactions between GFRα1 and GDNF, and preliminarily also between GFRα1 and RET and heparin. Finally, we showed that, the precursor of the oncogenic MEN 2B (multiple endocrine neoplasia type 2) form of RET gets phosphorylated already during its synthesis in the endoplasmic reticulum (ER). We also demonstrated that it associates with Src homology 2 domain-containing protein (SHC) and growth factor receptor-bound protein (GRB2) in the ER, and has the capacity to activate several downstream signalling molecules.

Use of protein A gene fusions for the analysis of structure-function relationship of the transactivator protein C of bacteriophage Mu

A sensitive dimerization assay for DNA binding proteins has been developed using gene fusion technology. For this purpose, we have engineered a gene fusion using protein A gene of Staphylococcus aureus and C gene, the late gene transactivator of bacteriophage Mu. The C gene was fused to the 3' end of the gene for protein A to generate an A- C fusion. The overexpressed fusion protein was purified in a single step using immunoglobulin affinity chromatography. Purified fusion protein exhibits DNA binding activity as demonstrated by electrophoretic mobility shift assays. When the fusion protein A-C was mixed with C and analyzed for DNA binding, in addition to C and A-C specific complexes, a single intermediate complex comprising of a heterodimer of C and A-C fusion proteins was observed. Further, the protein A moiety in the fusion protein A-C does not contribute to DNA binding as demonstrated by proteolytic cleavage and circular dichroism (CD) analysis. The assay has also been applied to analyze the DNA binding domain of C protein by generating fusions between protein A and N- and C-terminal deletion mutants of C. The results indicate a role for the region towards the carboxy terminal of the protein in DNA binding. The general applicability of this method is discussed.

Structure, function and intracellular dynamics of alphavirus replication complexes

Intracellular membrane alterations are hallmarks of positive-sense RNA (+RNA) virus replication. Strong evidence indicates that within these exotic compartments, viral replicase proteins engage in RNA genome replication and transcription. To date, fundamental questions such as the origin of altered membranes, mechanisms of membrane deformation and topological distribution and function of viral components, are still waiting for comprehensive answers. This study addressed some of the above mentioned questions for the membrane alterations induced during Semliki Forest virus (SFV) infection of mammalian cells. With the aid of electron and fluorescence microscopy coupled with radioactive labelling and immuno-cytochemistry techniques, our group and others showed that few hours after infection the four non structural proteins (nsP1-4) and newly synthesized RNAs of SFV colocalized in close proximity of small membrane invaginations, designated as spherules . These 50-70 nm structures were mainly detected in the perinuclear area, at the limiting membrane of modified endosomes and lysosomes, named CPV-I (cytopathic vacuoles type I). More rarely, spherules were also found at the plasma membrane (PM). In the first part of this study I present the first three-dimensional reconstruction of the CPV-I and the spherules, obtained by electron tomography after chemical or cryo-fixation. Different approaches for imaging these macromolecular assemblies to obtain better structure preservation and higher resolution are presented as unpublished data. This study provides insights into spherule organization and distribution of viral components. The results of this and other experiments presented in this thesis will challenge currently accepted models for virus replication complex formation and function. In a revisitation of our previous models, the second part of this work provides the first complete description of the biogenesis of the CPV-I. The results demonstrate that these virus-induced vacuoles, where hundreds of spherules accumulate at late stages during infection, represent the final phase of a journey initiated at the PM, which apparently serves as a platform for spherule formation. From the PM spherules were internalized by an endocytic event that required the activity of the class I PI3K, caveolin-1, cellular cholesterol and functional actin-myosin network. The resulting neutral endocytic carrier vesicle delivered the spherules to the membrane of pre-existing acidic endosomes via multiple fusion events. Microtubule based transport supported the vectorial transfer of these intermediates to the pericentriolar area where further fusions generated the CPV-I. A signal for spherule internalization was identified in one of the replicase proteins, nsP3. Infections of cells with viruses harbouring a deletion in a highly phosphorylated region of nsP3 did not result in the formation of CPV-Is. Instead, thousands of spherules remained at the PM throughout the infection cycle. Finally, the role of the replicase protein nsP2 during viral RNA replication and transcription was investigated. Three enzymatic activities, protease, NTPase and RNA-triphosphatase were studied with the aid of temperature sensitive mutants in vitro and, when possible, in vivo. The results highlighted the interplay of the different nsP2 functions during different steps of RNA replication and sub-genomic promoter regulation, and suggest that the protein could have different activities when participating in the replication complex or as a free enzyme.

Mycobacterial Stress Regulation: The Dps ``Twin Sister'' Defense Mechanism and Structure-Function Relationship

In this work, we have tried to emphasize the connection between mycobacterial growth and regulation of gene expression. Utilization of multiple carbon sources and diauxic growth helps bacteria to regulate gene expression at an optimum level so that the inhospitable conditions encountered during nutrient depletion can be circumvented. These aspects will be discussed with respect to mycobacterial growth in subsequent sections. Identification and characterization of genes induced under such conditions is helpful to understand the physiology of the bacterium. Although it is necessary to compare the total expression profile of proteins as they transit from vegetative growth to stationary phase, at times a lot of insights can be deciphered from the expression pattern of one or two proteins. We have compared the protein expression and sigma factor selectivity of two such proteins in M. smegmatis to understand the differential regulation of genes playing diverse function in the same species. Some newer insights on the structure and function of one of the Dps proteins are also explained.

Structure-function relationships of icosahedral plant

X-ray diffraction studies on single crystals of a few viruses have led to the elucidation of their three dimensional structure at near atomic resolution. Both the tertiary structure of the coat protein subunit and the quaternary organization of the icosahedral capsid in these viruses are remarkably similar. These studies have led to a critical re-examination of the structural principles in the architecture of isometric viruses and suggestions of alternative mechanisms of assembly. Apart from their role in the assembly of the virus particle, the coat proteins of certian viruses have been shown to inhibit the replication of the cognate RNA leading to cross-protection. The coat protein amino acid sequence and the genomic sequence of several spherical plant RNA viruses have been determined in the last decade. Experimental data on the mechanisms of uncoating, gene expression and replication of several classes of viruses have also become available. The function of the non-structural proteins of some viruses have been determined. This rapid progress has provided a wealth of information on several key steps in the life cycle of RNA viruses. The function of the viral coat protein, capsid architecture, assembly and disassembly and replication of isometric RNA plant viruses are discussed in the light of this accumulated knowledge.

Isoscalar axial-vector renormalization constant and polarized proton structure function

The isoscalar axial-vector renormalization constant is reevaluated using the QCD sum-rule method. It is found to be substantially different from the anomaly-free octet axial-vector u¯γμγ5+d¯γμγ5-2s¯γμγ5 coupling. Combining this determination with the known values of the isovector coupling GA and the F/D ratio for the octet current, we find the integral of the polarized proton structure function to be Gp=Fgp1(x)dx=0.135, in agreement with recent measurement by the European Muon Collaboration.

Structure-function relationships of icosahedral plant viruses

X-ray diffraction studies on single crystals of a few viruses have led to the elucidation of their three dimensional structure at near atomic resolution. Both the tertiary structure of the coat protein subunit and the quaternary morganization of the icosahedral capsid in these viruses are remarkably similar. These studies have led to a critical re-examination of the structural principles in the architecture of isometric viruses and suggestions of alternative mechanisms of assembly. Apart from their role in the assembly of the virus particle, the coat proteins of certian viruses have been shown to inhibit the replication of the cognate RNA leading to cross-protection. The coat protein amino acid sequence and the genomic sequence of several spherical plant RNA viruses have been determined in the last decade. Experimental data on the mechanisms of uncoating, gene expression and replication of several classes of viruses have also become available. The function of the non-structural proteins of some viruses have been determined. This rapid progress has provided a wealth of information on several key steps in the life cycle of RNA viruses. The function of the viral coat protein, capsid architecture, assembly and disassembly and replication of isometric RNA plant viruses are discussed in the light of this accumulated knowledge.

Structure, function, and mechanism of HhaI DNA methyltransferases

A vast amount of literature has accumulated on the characterization of DNA methyltransferases. The HhaI DNA methyltransferase, a C5-cytosine methyltransferase, has been the subject of investigation for the last 2 decades. Biochemical and kinetic characterization have led to an understanding of the catalytic and kinetic mechanism of the methyltransfer reaction. The HhaI methyltransferase has also been subjected to extensive structural analysis, with the availability of 12 structures with or without a cofactor and a variety of DNA substrates. The mechanism of base flipping, first described for the HhaI methyltransferase, is conserved among all DNA methyltransferases and is also found to occur in numerous DNA repair enzymes. Studies with other methyltransferase reveal a significant structural and functional similarity among different types of methyltransferases. This review aims to summarize the available information on the HhaI DNA methyltransferase.

Structure-function relationship in serine hydroxymethyltransferase

Serine hydroxymethyltransferase (SHMT), a pyridoxal-5V-phosphate (PLP)-dependent enzyme catalyzes thetetrahydrofolate (H4-folate)- dependent retro-aldol cleavage of serine to form 5,10-methylene H4-folate and glycine. The structure–function relationship of SHMT wasstudied in our laboratory initially by mutation of residues that are conserved in all SHMTs and later by structure-based mutagenesis of residues located in the active site. The analysis of mutants showed that K71, Y72, R80, D89, W110, S202, C203, H304, H306 and H356 residues are involved in maintenance of the oligomeric structure. The mutation of D227, a residue involved in charge relay system, led to the formation of inactive dimers, indicating that this residue has a role in maintaining the tetrameric structure and catalysis. E74, a residue appropriately positioned in the structure of the enzyme to carry out proton abstraction, was shown by characterization of E74Q and E74K mutants to be involved in conversion of the enzyme from an ‘open’ to ‘closed’ conformation rather than proton abstraction from the hydroxylgroup of serine. K256, the residue involved in the formation of Schiffs base with PLP, also plays a crucial role in the maintenance of the tetrameric structure. Mutation of R262 residue established the importance of distal interactions in facilitating catalysis and Y82 is not involved in the formaldehyde transfer via the postulated hemiacetal intermediate but plays a role in stabilizing the quinonoid intermediate.The mutational analysis of scSHMT along with the structure of recombinant Bacillus stearothermophilus SHMT and its substrate(s)complexes was used to provide evidence for a direct transfer mechanism rather than retro-aldol cleavage for the reaction catalyzed by SHMT.

Structure-function hierarchies and von Karman-Howarth relations for turbulence in magnetohydrodynamical equations

We generalize the method of A. M. Polyakov, Phys. Rev. E 52, 6183 (1995)] for obtaining structure-function relations in turbulence in the stochastically forced Burgers equation, to develop structure-function hierarchies for turbulence in three models for magnetohydrodynamics (MHD). These are the Burgers analogs of MHD in one dimension Eur. Phys. J.B 9, 725 (1999)], and in three dimensions (3DMHD and 3D Hall MHD). Our study provides a convenient and unified scheme for the development of structure-function hierarchies for turbulence in a variety of coupled hydrodynamical equations. For turbulence in the three sets of MHD equations mentioned above, we obtain exact relations for third-order structure functions and their derivatives; these expressions are the analogs of the von Karman-Howarth relations for fluid turbulence. We compare our work with earlier studies of such relations in 3DMHD and 3D Hall MHD.