A missense point mutation in nerve growth factor (NGFR100W) results in selective peripheral sensory neuropathy.

The R100W mutation in nerve growth factor is associated with hereditary sensory autonomic neuropathy V in a Swedish family. These patients develop severe loss of perception to deep pain but with apparently normal cognitive functions. To better understand the disease mechanism, we examined a knockin mouse model of HSAN V. The homozygous mice showed significant structural deficits in intra-epidermal nerve fibers (IENFs) at birth. These mice had a total loss of pain perception at ∼2 months of age and often failed to survive to adulthood. Heterozygous mutant mice developed a progressive degeneration of small sensory fibers both behaviorally and functionally: they showed a progressive loss of IENFs starting at the age of 9 months accompanied with progressive loss of perception to painful stimuli such as noxious temperature. Quantitative analysis of lumbar 4/5 dorsal root ganglia revealed a significant reduction in small size neurons, while analysis of sciatic nerve fibers revealed the heterozygous mutant mice had no reduction in myelinated nerve fibers. Significantly, the amount of NGF secreted from mouse embryonic fibroblasts were reduced from both heterozygous and homozygous mice compared to their wild-type littermates. Interestingly, the heterozygous mice showed no apparent structural alteration in the brain: neither the anterior cingulate cortex nor the medial septum including NGF-dependent basal forebrain cholinergic neurons. Accordingly, these animals did not develop appreciable deficits in tests for brain function. Our study has thus demonstrated that the NGFR100W mutation likely affects the structure and function of peripheral sensory neurons.

Antigenic properties of porcine teschovirus 1 (PTV-1) Talfan strain and molecular strategy for serotyping of PTVs.

For reliable diagnosis of porcine teschovirus (PTV) infection we created an RT-PCR-based molecular strategy for serotyping that encompassed the dominant neutralizing antigenic site of PTV, followed by phylogenetic analyses of amplicons. We identified neutralizing antigenic sites of PTV-1 Talfan strain through epitope mapping of neutralizing monoclonal antibodies (MAbs), using synthetic peptides spanning the capsid proteins. All 11 MAbs obtained recognized peptides in the EF loop ("puff") of VP2 protein. Two MAbs concurrently reacted to peptides, one in the GH loop of VP1 and one in the VP1 C terminus. Three-dimensional modeling of Talfan capsid protein predicted exposure of all these sites on the virion surface in a close line centered around puff. We then designed a single pair of degenerate primers to VP2 and amplified the region of approximately 320 bp encompassing puff in 8 PTV prototype strains and 6 field isolates. Phylogenetic analyses of the puff sequences of 11 prototype strains and 34 field isolates obtained from databanks showed that all homotypic strains (both field and prototype) were always monophyletic, except for one 'untypable' Japanese strain. This RT-PCR-based strategy appears to be a reliable surrogate for serotyping and could facilitate the diagnosis and epidemiological study of PTV infection.

Good response of membranous lupus nephritis to tacrolimus.

A 22-year-old woman hospitalized for polyarthralgia was diagnosed with systemic lupus erythematosus (SLE). She was treated with prednisolone, and her clinical manifestations improved. However, she was re-admitted for renal biopsy because of persistent hypocomplementemia and development of proteinuria. The biopsy revealed segmental spike formation of basement membrane and subepithelial immune complex deposition, and membranous lupus nephritis (class V) was diagnosed. When tacrolimus was added to prednisolone, the serum complement titer quickly improved and proteinuria disappeared after about 11 months. Nevertheless, when tacrolimus was replaced examination showed cyclosporine due to gastrointestinal symptoms, she complained about arthralgia. Examination showed drop in the serum complement titer and recurrence of proteinuria. Renal biopsy at the time of recurrence showed increased subepithelial immune complex deposition in the capillary loops as compared to the first biopsy, a high degree of thickening of the basement membrane, and segmental circumferential interposition in some of the glomeruli. Membranous lupus nephritis (classes V + III) was diagnosed. By changing to tacrolimus and higher doses of steroids, the serum complement titer improved and proteinuria disappeared. This case indicates that tacrolimus can be an effective therapeutic agent for membranous lupus nephritis.

A case of monoclonal immunoglobulin light- and heavy-chain deposition disease exhibiting atypical deposition with fibrillary structures, successfully treated with chemotherapy.

We report a case of light and heavy chain deposition disease (LHCDD), a rather rare monoclonal immunoglobulin deposition disease (MIDD) with successful therapeutic effect. A 58-year-old woman suffered from proteinuria and renal insufficiency (serum creatinine 1.0 mg/dl, creatinine clearance 49.2 ml/min) in February 2003. In serum and urine samples, monoclonal IgG-kappa was detected. A bone marrow aspiration showed a slightly hypocellular marrow and plasma cell population was increased to 7.0%. Renal histological findings revealed lobulated glomeruli with nodular lesions on light microscopy, characteristic findings of MIDD. Intense deposition of IgG heavy chains in the linear pattern in the glomerular and tubular basement membranes was observed. Immunohistochemistry revealed both kappa and lambda light chain depositions in glomeruli. Electron-microscopic examination revealed fine granular electron-dense deposits accompanied by microfibrils. Based on these findings, this patient was diagnosed as LHCDD. She received three courses of melphalan and prednisone chemotherapy, resulting in disappearance of proteinuria, prevention of renal functional deterioration and the decrease of monoclonal immunoglobulin. This case clearly demonstrates that the earlier and accurate diagnosis and initiation of chemotherapy at the early stage with serum creatinine level below 4.0 mg/dl are necessary to improve renal and patient outcome.

Interrelations between the efficiency of translation start sites and other sequence features of yeast mRNAs.

The translation start site (TSS) plays an important role in the control of the translational efficiency and cytoplasmic stability of eukaryotic mRNAs. The efficiency of TSS recognition is known to be influenced by sequence context, and mRNAs with "weak" TSSs are relatively abundant. We analyzed a sample of 4113 yeast genes in a search for features that might serve to compensate for the inefficient recognition of "weak" TSSs by initiating ribosomes. The first feature found to correlate with variations in TSS strength is differences in the stability of secondary structure upstream and downstream of the start AUG codon. The second feature concerns the characteristics of AUG triplets found at the beginning of the coding sequence, i.e., downstream of the predicted TSS. In particular, the proximal downstream AUG lies in frame with the CDS significantly more often if the TSS itself is located in a "weak" context. The accuracy of TSS annotation, the possibility of polypeptide heterogeneity due to the use of alternative downstream AUGs, and the influence of related features of mRNA sequences are discussed.

[Conformational effects of DNA stretching]. / Konformatsionnye éffekty pri rastiazhenii DNK.

DNA is an extensible molecule, and an extended conformation of DNA is involved in some biological processes. We have examined the effect of elongation stress on the conformational properties of DNA base pairs by conformational analysis. The calculations show that stretching does significantly affect the conformational properties and flexibilities of base pairs. In particular, we have found that the propeller twist in base pairs reverses its sign upon stretching. The energy profile analysis indicates that electrostatic interactions make a major contribution to the stabilization of the positive-propeller-twist configuration in stretched DNA. This stretching also results in a monotonic decrease in the helical twist angle, tending to unwind the double helix. Fluctuations in most variables initially increase upon stretching, because of unstacking of base pairs, but then the fluctuations decrease as DNA is stretched further, owing to the formation of specific interactions between base pairs induced by the positive propeller twist. Thus, the stretching of DNA has particularly significant effects upon DNA flexibility. These changes in both the conformation and flexibility of base pairs probably have a role in functional interactions with proteins.

[The context organization of functional regions in yeast genes with high-level expression]. / Kontekstnaia organizatsiia funktsional'nykh raionov genov s vysokim urovnem ékspressii u drozhzhei.

With the example of yeast genes, context organization was compared for functional gene regions (promoter, 5'-UTR, 3'-UTR) and tested for association with the level of gene expression. Several parameters (nucleotide composition, dinucletoide content bias) proved to correlate with expression level, each functional region having its specific features. Context optimization of a functional region was assumed to be essential for highly efficient interaction with the expression system of the cell. Specific context features were considered as dispersed signals important for high-level gene expression.

Thermodynamic database for protein-nucleic acid interactions (ProNIT).

MOTIVATION: Protein-nucleic acid interactions are fundamental to the regulation of gene expression. In order to elucidate the molecular mechanism of protein-nucleic acid recognition and analyze the gene regulation network, not only structural data but also quantitative binding data are necessary. Although there are structural databases for proteins and nucleic acids, there exists no database for their experimental binding data. Thus, we have developed a Thermodynamic Database for Protein-Nucleic Acid Interactions (ProNIT). RESULTS: We have collected experimentally observed binding data from the literature. ProNIT contains several important thermodynamic data for protein-nucleic acid binding, such as dissociation constant (K(d)), association constant (K(a)), Gibbs free energy change (DeltaG), enthalpy change (DeltaH), heat capacity change (DeltaC(p)), experimental conditions, structural information of proteins, nucleic acids and the complex, and literature information. These data are integrated into a relational database system together with structural and functional information to provide flexible searching facilities by using combinations of various terms and parameters. A www interface allows users to search for data based on various conditions, with different display and sorting options, and to visualize molecular structures and their interactions. AVAILABILITY: ProNIT is freely accessible at the URL http://www.rtc.riken.go.jp/jouhou/pronit/pronit.html.

Filling a cavity dramatically increases pressure stability of the c-Myb R2 subdomain.

Cavities or packing defects in proteins may generally be related with the dynamics and function of a protein. In the c-Myb R2 subdomain, its single cavity has been shown to be crucial for its DNA recognition. Cavities are also considered important in determining the pressure stability of a protein. In the present work, high-pressure proton nuclear magnetic resonance ((1)H NMR) spectroscopy at 750 MHz is used to study the effect of a cavity-filling mutation (V103L) on the stability of the c-Myb R2 subdomain in the pressure range between 1 and 3,700 bar at 5 degrees C. A dramatic increase in the pressure stability of the c-Myb R2 subdomain is attained, from which we estimate the cavity size to be 35.3 A(3), in good agreement with literature values. We also evaluated the increase in thermodynamic stability DeltaG(0)(1bar) from 5.35 kJ/mol to 7.34 kJ/mol by the mutation, giving a clear example of the effect of a cavity on the global stability of a globular protein.

Two consecutive zinc fingers in Sp1 and in MAZ are essential for interactions with cis-elements.

The zinc finger proteins Sp1 and Myc-associated zinc finger protein (MAZ) are transcription factors that control the expression of various genes. Regulation of transcription by these factors is based on interactions between GC-rich DNA-binding sites (GGGCGG for Sp1 and GGGAGGG for MAZ) and the carboxyl-terminal zinc finger motifs of the two proteins. Sp1 and MAZ have three and six zinc fingers, respectively, and the details of their interactions with cis-elements remain to be clarified. We demonstrate here that Sp1 and MAZ interact with the same GC-rich DNA-binding sites, apparently sharing DNA-binding sites with each other. We found that the DNA binding activities of Sp1 and MAZ depended mainly on consecutive zinc fingers, namely the second and third zinc fingers in Sp1 and the third and fourth zinc fingers in MAZ. Furthermore, the interactions of the zinc finger proteins with the same cis-elements appear to play a critical role in the regulation of gene expression. It seems plausible that two consecutive zinc finger motifs in a zinc finger protein might be essential for interaction of the protein with DNA.

ORI-GENE: gene classification based on the evolutionary tree.

MOTIVATION: Genome projects have produced large amounts of data on the sequences of new genes whose functions are as yet unknown. The functions of new genes are usually inferred by comparing their sequences with those of known genes, but evaluation of the sequence homology of individual genes does not make the most of the available sequence information. Therefore, new methods and tools for extracting more biological information from homology searches would be advantageous. RESULTS: We have developed a computational tool, ORI-GENE, to analyze the results of sequence homology searches from the perspective of the evolution of selected sets of new genes. ORI-GENE has a graphical interface and accomplishes two important tasks: first, based on the output of homology searches, it identifies species with similar genes and displays their pattern of distribution on the phylogenetic tree. This function enables one to infer the way in which a given gene may have propagated among species over time. Second, from the distribution patterns, it predicts the point at which a given gene may have been first acquired (i.e. its 'origin'), then classifies the gene on that basis. Because it makes use of available evolutionary information to show the way in which genes cluster among species, ORI-GENE should be an effective tool for the screening and classification of new genes revealed by genome analysis. AVAILABILITY: ORI-GENE is retrievable via the Internet at: http://www.rtc.riken.go.jp/jouhou/ORI-GENE.

ACTIVITY: a database on DNA/RNA sites activity adapted to apply sequence-activity relationships from one system to another.

ACTIVITY is a database on DNA/RNA site sequences with known activity magnitudes, measurement systems, sequence-activity relationships under fixed experimental conditions and procedures to adapt these relationships from one measurement system to another. This database deposits information on DNA/RNA affinities to proteins and cell nuclear extracts, cutting efficiencies, gene transcription activity, mRNA translation efficiencies, mutability and other biological activities of natural sites occurring within promoters, mRNA leaders, and other regulatory regions in pro- and eukaryotic genomes, their mutant forms and synthetic analogues. Since activity magnitudes are heavily system-dependent, the current version of ACTIVITY is supplemented by three novel sub-databases: (i) SYSTEM, measurement systems; (ii) KNOWLEDGE, sequence-activity relationships under fixed experimental conditions; and (iii) CROSS_TEST, procedures adapting a relationship from one measurement system to another. These databases are useful in molecular biology, pharmacogenetics, metabolic engineering, drug design and biotechnology. The databases can be queried using SRS and are available through the Web, http://wwwmgs. bionet.nsc.ru/systems/Activity/.

rSNP_Guide, a database system for analysis of transcription factor binding to target sequences: application to SNPs and site-directed mutations.

rSNP_Guide is a novel curated database system for analysis of transcription factor (TF) binding to target sequences in regulatory gene regions altered by mutations. It accumulates experimental data on naturally occurring site variants in regulatory gene regions and site-directed mutations. This database system also contains the web tools for SNP analysis, i.e., active applet applying weight matrices to predict the regulatory site candidates altered by a mutation. The current version of the rSNP_Guide is supplemented by six sub-databases: (i) rSNP_DB, on DNA-protein interaction caused by mutation; (ii) SYSTEM, on experimental systems; (iii) rSNP_BIB, on citations to original publications; (iv) SAMPLES, on experimentally identified sequences of known regulatory sites; (v) MATRIX, on weight matrices of known TF sites; (vi) rSNP_Report, on characteristic examples of successful rSNP_Tools implementation. These databases are useful for the analysis of natural SNPs and site-directed mutations. The databases are available through the Web, http://wwwmgs.bionet.nsc.ru/mgs/systems/rsnp/.

Thermodynamic databases for proteins and protein-nucleic acid interactions.

Thermodynamic data regarding proteins and their interactions are important for understanding the mechanisms of protein folding, protein stability, and molecular recognition. Although there are several structural databases available for proteins and their complexes with other molecules, databases for experimental thermodynamic data on protein stability and interactions are rather scarce. Thus, we have developed two electronically accessible thermodynamic databases. ProTherm, Thermodynamic Database for Proteins and Mutants, contains numerical data of several thermodynamic parameters of protein stability, experimental methods and conditions, along with structural, functional, and literature information. ProNIT, Thermodynamic Database for Protein-Nucleic Acid Interactions, contains thermodynamic data for protein-nucleic acid binding, experimental conditions, structural information of proteins, nucleic acids and the complex, and literature information. These data have been incorporated into 3DinSight, an integrated database for structure, function, and properties of biomolecules. A WWW interface allows users to search for data based on various conditions, with different display and sorting options, and to visualize molecular structures and their interactions. These thermodynamic databases, together with structural databases, help researchers gain insight into the relationship among structure, function, and thermodynamics of proteins and their interactions, and will become useful resources for studying proteins in the postgenomic era.

Evaluation of free energy landscape for base-amino acid interactions using ab initio force field and extensive sampling.

Structural data of protein-DNA complex show redundancy and flexibility in base-amino acid interactions. To understand the origin of the specificity in protein-DNA recognition, we calculated the interaction free energy, enthalpy, entropy, and minimum energy maps for AT-Asn, GC-Asn, AT-Ser, and GC-Ser by means of a set of ab initio force field with extensive conformational sampling. We found that the most preferable interactions in these pairs are stabilized by hydrogen bonding, and are mainly enthalpy driven. However, minima in the free energy maps are not necessarily the same as those in the minimum energy map or enthalpy maps, due to the entropic effect. The effect of entropy is particularly important in the case of GC-Asn. Experimentally observed structures of base-amino acid interactions are within preferable regions in the calculated free energy maps, where there are many different interaction configurations with similar energy. The full geometry optimization procedure using ab initio molecular orbital method was applied to get the optimal interaction geometries for AT-Asn, GC-Asn, AT-Ser, and GC-Ser. We found that there are various base-amino acid combinations with similar interaction energies. These results suggest that the redundancy and conformational flexibility in the base-amino acid interactions play an important role in the protein-DNA recognition.

[mRNA-FAST (mRNA-Function, Activity, STructure) computer system]. / Komp'iuternaia sistema mRNA-FAST (mRNA-Function, Activity, Structure).

Computer system mRNA-FAST (mRNA--Function, Activity, STructure; http://wwwmgs.bionet.nsc.ru/mgs/dbases/trsig/) is described. The system has been developed to analyze nucleotide sequences of mRNA and to measure their essential properties. The system compiles the data base on translation signals including nucleotide sequences of the regulatory regions with structural and experimental information on their specific activities. It also contains programs to search for local homology between mRNA and translation signals, to search for potential signals basing on analysis of the oligonucleotide dictionaries, and to model secondary RNA structure. Possible applications of the system mRNA-FAST are discussed.

[Analysis of structural motifs of proteins using sets of codes, describing local structures]. / Analiz strukturnykh motivov belkov s pomoshch'iu naborov kodov, opisyvaiushchikh lokal'nye struktury.

An amino acid sequence pattern conserved among a family of proteins is called motif. It is usually related to the specific function of the family. On the other hand, functions of proteins are achieved by their 3D structures. Specific local structures, called structural motifs, are considered related to their functions. However, searching for common structural motifs in different proteins is much more difficult than for common sequence motifs. We are attempting in this study to convert the information about the structural motifs into a set of one-dimensional digital strings, i.e., a set of codes, to compare them more easily by computer and to investigate their relationship to functions more quantitatively. By applying the Delaunay tessellation to a 3D structure of a protein, we can assign each local structure to a unique code that is defined so as to reflect its structural feature. Since a structural motif is defined as a set of the local structures in this paper, the structural motif is represented by a set of the codes. In order to examine the ability of the set of the codes to distinguish differences among the sets of local structures with a given PROSITE pattern that contain both true and false positives, we clustered them by introducing a similarity measure among the set of the codes. The obtained clustering shows a good agreement with other results by direct structural comparison methods such as a superposition method. The structural motifs in homologous proteins are also properly clustered according to their sources. These results suggest that the structural motifs can be well characterized by these sets of the codes, and that the method can be utilized in comparing structural motifs and relating them with function.

Importance of surrounding residues for protein stability of partially buried mutations.

For understanding the factors influencing protein stability, we have analyzed the relationship between changes in protein stability caused by partially buried mutations and changes in 48 physico-chemical, energetic and conformational properties of amino acid residues. Multiple regression equations were derived to predict the stability of protein mutants and the efficiency of the method has been verified with both back-check and jack-knife tests. We observed a good agreement between experimental and computed stabilities. Further, we have analyzed the effect of sequence window length from 1 to 12 residues on each side of the mutated residue to include the sequence information for predicting protein stability and we found that the preferred window length for obtaining the highest correlation is different for each secondary structure; the preferred window length for helical, strand and coil mutations are, respectively, 0, 9 and 4 residues on both sides of the mutant residues. However, all the secondary structures have significant correlation for a window length of one residue on each side of the mutant position, implying the role of short-range interactions. Extraction of surrounding residue information for various distances (3 to 20A) around the mutant position showed the highest correlation at 8A, 6A and 7A, respectively, for mutations in helical, strand and coil segments. Overall, the information about the surrounding residues within the sphere of 7 to 8A, may explain better the stability in all subsets of partially buried mutations implying that this distance is sufficient to accommodate the residues influenced by major intramolecular interactions for the stability of protein structures.

Solvent density and long-range dipole field around a DNA-binding protein studied by molecular dynamics.

The distribution and orientation of solvent around a DNA-binding protein, 434 Cro, were investigated by molecular dynamics simulations with a periodic-boundary condition. The protein was treated in two states: charged and neutral. The computed high-density sites of the solvent around the protein correlated well with the experimentally determined crystal-water sites, in both the charged and neutral states. A local density map, introduced to investigate the solvent density around the highly mobile regions of the protein, showed a hydration shell around hydrophobic sidechains and hydrogen-bondable sites around hydrophilic sidechains, and also showed that the solvent density is sensitive to the slight concaves of the sidechain surface. The long-range solvent-dipole field was observed around the protein, where the pattern of the dipole ordering was considerably different between the charged and neutral states. A local solvent-dipole field was introduced, and the pattern of the dipole ordering was different between the hydrophobic and hydrophilic sidechains. The dipole field from the charged state provided a higher correlation to the electrostatic field obtained from the Poisson-Boltzmann's equation than that from the neutral state, although the correlation become weak quickly for the both states with increasing the protein-solvent distance.

[The cumulative risk index in stroke prevention]. / Kumulatív kockázati index a stroke-prevencióban.

The screening and special management of high risk patient, is possible strategy to reduce the high morbidity and mortality of stroke. In the II. district of Budapest we examined high vascular risk patients cooperating with general practitioners. We let the severity of risk with arithmetic of the cumulative risk index. Significant relation was found between of high cumulative risk index and 50% or higher carotis stenosis, as well as the cumulative risk index and low Se HDL level. Increased blood pressure level has been found in among the treated hypertonic patient. Reduction of high blood pressure was documented in the 6 month control examination. Finally there was very low level of smoking in the high vascular risk patient group.