Analogs of diadenosine tetraphosphate (Ap4A)

This review summarizes our knowledge of analogs and derivatives of diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A), the most extensively studied member of the dinucleoside 5',5"'-P1,Pn-polyphosphate (NpnN) family. After a short discussion of enzymes that may be responsible for the accumulation and degradation of Np4)N's in the cell, this review focuses on chemically and/or enzymatically produced analogs and their practical applications. Particular attention is paid to compounds that have aided the study of enzymes involved in the metabolism of Ap4A (Np4N'). Certain Ap4A analogs were alternative substrates of Ap4A-degrading enzymes and/or acted as enzyme inhibitors, some other helped to establish enzyme mechanisms, increased the sensitivity of certain enzyme assays or produced stable enzyme:ligand complexes for structural analysis.     

Insights into the association of FcgammaRII and TCR with detergent-resistant membrane domains: isolation of the domains in detergent-free density gradients facilitates membrane fragment reconstitution

Plasma membrane rafts are routinely isolated as detergent-resistant membranes (DRMs) floating in detergent-free density gradients. Here we show that both the presence and exclusion of TX-100 during the density gradient fractionation have profound effects on the location of FcgammaRII and TCR in DRM fractions. The presence of TX-100 during fractionation promoted solubilization of non-cross-linked FcgammaRII when the receptor was insufficiently dissolved upon cell lysis. In the detergent-supplemented gradients, TX-100 micelles floated, further enhancing dissociation of FcgammaRII and TCR from DRMs and promoting a shift of the receptors toward higher-density fractions. Hence, fractionation of cell lysates over the detergent-containing gradients enables isolation of DRMs devoid of weakly associated proteins, like nonactivated FcgammaRII and TCR. On the other hand, in a detergent-free gradient, non-cross-linked FcgammaRII, fully soluble in 0.2% TX-100, was recovered in DRM fractions. Moreover, employment of the TX-100-free gradient for refractionation of intermediate-density fractions, derived from detergent-supplemented gradients and containing FcgammaRII and TCR, resulted in flotation of the receptors to buoyant fractions. An analysis of the TX-100 concentration revealed that after fractionation of 0.2% TX-100 cell lysates in the absence of detergent, the level of TX-100 in DRM fractions was reduced to 0.01%, below the critical micelle concentration. Therefore, fractionation of detergent cell lysates over detergent-free gradients can mimic conditions for a membrane reconstitution, evoking association of a distinct subset of membrane proteins, including FcgammaRII and TCR, with DRMs.     

Crystal structure of the human GGA1 GAT domain

GGAs are a family of vesicle-coating regulatory proteins that function in intracellular protein transport. A GGA molecule contains four domains, each mediating interaction with other proteins in carrying out intracellular transport. The GAT domain of GGAs has been identified as the structural entity that binds membrane-bound ARF, a molecular switch regulating vesicle-coat assembly. It also directly interacts with rabaptin5, an essential component of endosome fusion. A 2.8 A resolution crystal structure of the human GGA1 GAT domain is reported here. The GAT domain contains four helices and has an elongated shape with the longest dimension exceeding 80 A. Its longest helix is involved in two structural motifs: an N-terminal helix-loop-helix motif and a C-terminal three-helix bundle. The N-terminal motif harbors the most conservative amino acid sequence in the GGA GAT domains. Within this conserved region, a cluster of residues previously implicated in ARF binding forms a hydrophobic surface patch, which is likely to be the ARF-binding site. In addition, a structure-based mutagenesis-biochemical analysis demonstrates that the C-terminal three-helix bundle of this GAT domain is responsible for the rabaptin5 binding. These structural characteristics are consistent with a model supporting multiple functional roles for the GAT domain.     

Accounting for nonignorable verification bias in assessment of diagnostic tests

A "gold" standard test, providing definitive verification of disease status, may be quite invasive or expensive. Current technological advances provide less invasive, or less expensive, diagnostic tests. Ideally, a diagnostic test is evaluated by comparing it with a definitive gold standard test. However, the decision to perform the gold standard test to establish the presence or absence of disease is often influenced by the results of the diagnostic test, along with other measured, or not measured, risk factors. If only data from patients who received the gold standard test were used to assess the test performance, the commonly used measures of diagnostic test performance--sensitivity and specificity--are likely to be biased. Sensitivity would often be higher, and specificity would be lower, than the true values. This bias is called verification bias. Without adjustment for verification bias, one may possibly introduce into the medical practice a diagnostic test with apparent, but not truly, high sensitivity. In this article, verification bias is treated as a missing covariate problem. We propose a flexible modeling and computational framework for evaluating the performance of a diagnostic test, with adjustment for nonignorable verification bias. The presented computational method can be utilized with any software that can repetitively use a logistic regression module. The approach is likelihood-based, and allows use of categorical or continuous covariates. An explicit formula for the observed information matrix is presented, so that one can easily compute standard errors of estimated parameters. The methodology is illustrated with a cardiology data example. We perform a sensitivity analysis of the dependency of verification selection process on disease.     

A minimal physically realistic protein-like lattice model: designing an energy landscape that ensures all-or-none folding to a unique native state

A simple protein model restricted to the face-centered cubic lattice has been studied. The model interaction scheme includes attractive interactions between hydrophobic (H) residues, repulsive interactions between hydrophobic and polar (P) residues, and orientation-dependent P-P interactions. Additionally, there is a potential that favors extended beta-type conformations. A sequence has been designed that adopts a native structure, consisting of an antiparallel, six-member Greek-key beta-barrel with protein-like structural degeneracy. It has been shown that the proposed model is a minimal one, i.e., all the above listed types of interactions are necessary for cooperative (all-or-none) type folding to the native state. Simulations were performed via the Replica Exchange Monte Carlo method and the numerical data analyzed via a multihistogram method.     

Unfolding of globular proteins: monte carlo dynamics of a realistic reduced model

Reduced lattice models of proteins and Monte Carlo dynamics were used to simulate the initial stages of the unfolding of several proteins of various structural types, and the results were compared to experiment. The models semiquantitatively reproduce the approximate order of events of unfolding as well as subtle mutation effects and effects resulting from differences in sequences of similar folds. The short-time mobility of particular residues, observed in simulations, correlates with the crystallographic temperature factor. The main factor controlling unfolding is the native state topology, with sequence playing a less important role. The correlation with various experiments, especially for sequence-specific effects, strongly suggests that properly designed reduced models of proteins can be used for qualitative studies (or prediction) of protein unfolding pathways.     

Substitution pattern of 3-deoxy-D-manno-oct-2-ulosonic acid in bacterial lipopolysaccharides investigated by methylation analysis of whole LPS

3-Deoxy-D-manno-oct-2-ulosonic acid (Kdo) is a constituent of the inner core part of bacterial lipopolysaccharides (LPS). This sugar may contribute to biological activities of the LPS, the type of substitution of Kdo is thus of importance and this work is aimed at the evaluation of a method for monitoring the substitution of Kdo in LPS. The procedure consists of three steps, namely permethylation of the lipopolysaccharide, with iodomethane and sodium methylsulfinylmethanide or NaOH in Me(2)SO, or with methyl triflate, then the product is methanolysed with HCl in MeOH and acetylated with acetic anhydride in pyridine. The resulting partially methylated acetates of Kdo methyl glycosides were analyzed by gas-liquid chromatography-electron impact ionization mass spectrometry (GLC-MS). For several derivatives of Kdo, specific GLC retention times and MS fragmentation patterns were determined. Lipopolysaccharides from several bacterial strains were isolated and analyzed with three different methods of methylation. The complete solubilization of the LPS in the acid form allows diminishing possible undermethylation. Sodium methylsulfinylmethanide is the most efficient agent in the permethylation of the whole LPS, of all the tested procedures. Methylation with methyl triflate allows the detection of base labile substituents on Kdo residues.     

Oogenesis in Vimba vimba (L. 1758) from Drawieński National Park (NW Poland)

The objective of the studies was to analyse the process of oogenesis in vimba from a non-migratory population living in the waters of Drawieński National Park in north-west Poland. The character of spawning of this species is an obstacle in determining the right moment to catch spawners or developing artificial spawning biotechniques. Previtellogenesis of vimba begins about six months after hatching and lasts three years. The trophoplasmatic growth of oocytes (October-March/April) begins when carbohydrate vesicles appear near the nuclei oocytes of sexually mature females (aged 4+). Just before spawning, granulated, lipoprotein-like substances are cumulated. The resorption of pre-ovulation corpora lutea (non-ejected oocytes) and post-ovulation corpora lutea (ruptured theca folds and follicles) begins in the ovary of vimba in the middle of June. These were observed in histological cross sections for about two to three months. Describing the process of oogenesis can provide a foundation for developing practical applications in aquaculture aimed at preserving the biodiversity of the park's waters and this critically endangered species of the Polish ichthyofauna.     

Structure of a new 2-deoxy-2-[(R)-3-hydroxybutyramido]-D-glucose-containing O-specific polysaccharide from the lipopolysaccharide of Citrobacter gillenii PCM 1542

The O-specific polysaccharide of Citrobacter gillenii PCM 1542 from serotype O-12a,12 b is composed of one residue each of D-glucose, D-GlcNAc, 2-deoxy-2-[(R)-3-hydroxybutyramido]-D-glucose (D-GlcNAcyl) and two GalNAc residues. On the basis of sugar and methylation analyses of the intact and Smith degraded polysaccharides, along with 1D and 2D 1H and 13C NMR spectroscopy, the following structure of the branched pentasaccharide repeating unit of the O-specific polysaccharide was established:This structure differs significantly from that of the O-specific polysaccharide of C. gillenii PCM 1544 from the same serotype O-12a,12 b, which has been established earlier (Kübler-Kielz.shtsls;b, J. et al. Carbohydr. Res. 2001, 331, 331-336). Serological studies confirmed that the two O-antigens are not related and suggested that strains PCM 1542 and 1544 should be classified into different O-serogroups.     

Acid-catalyzed isomerization of methyl 2-deoxy-D-arabino-hexosides: equilibria, kinetics and mechanism

Four isomers of methyl 2-deoxy-D-arabino-hexosides were isolated by HPLC as chromatographically homogeneous compounds. The rates of pyranoside isomerization (alpha(p) and beta(p)) at 40 degrees C and of furanoside isomerization (alpha(f) and beta(f)) at 26 degrees C were determined. A mechanism has been suggested for transformations taking place during isomerization of methyl 2-deoxy-D-arabino-hexosides in methanolic solution catalyzed with hydrogen chloride.