Studies on a chitooligosaccharide-specific lectin from Coccinia indica. Thermodynamics and kinetics of umbelliferyl glycoside binding.

Coccinia indica agglutinin (CIA) is a chitooligosaccharide-specific lectin with two binding sites/homodimer of M(r) 32,000. Quenching studies implied tryptophan involvement in binding activity, which was confirmed by chemical modification experiments (A. R. Sanadi and A. Surolia, submitted for publication). Binding of 4-methylumbelliferyl chitooligosaccharides has been carried out to study their binding by CIA. Reversal experiments confirm the validity of the data previously obtained (A. R. Sanadi and A. Surolia, submitted for publication) from intrinsic fluorescence studies. Surprisingly, unlike wheat germ agglutinin, there is no consistent thermodynamic effect of the chromophoric label on binding activities as compared with the native sugars. From the changes in the optical properties of the chromophoric group upon binding to CIA, it has been possible to confirm that the tryptophan located in the binding site is closest to the fourth subsite. Thermodynamic analysis shows that the binding of the labeled tetrasaccharide is very strongly entropically driven, with the terminal, nonreducing sugar residue protruding from the binding pocket. The results of stopped-flow kinetic studies on the binding of the chromophoric trisaccharide by CIA show that the mechanism of binding is a one-step process.

Topography of the combining region of a Thomsen-Friedenreich-antigen-specific lectin jacalin (Artocarpus integrifolia agglutinin). A thermodynamic and circular-dichroism spectroscopic study

Thermodynamic analysis of carbohydrate binding by Artocarpus integrifolia (jackfruit) agglutinin (jacalin) shows that, among monosaccharides, Me alpha GalNAc (methyl-alpha-N-acetylgalactosamine) is the strongest binding ligand. Despite its strong affinity for Me alpha GalNAc and Me alpha Gal, the lectin binds very poorly when Gal and GalNAc are in alpha-linkage with other sugars such as in A- and B-blood-group trisaccharides, Gal alpha 1-3Gal and Gal alpha 1-4Gal. These binding properties are explained by considering the thermodynamic parameters in conjunction with the minimum energy conformations of these sugars. It binds to Gal beta 1-3GalNAc alpha Me with 2800-fold stronger affinity over Gal beta 1-3GalNAc beta Me. It does not bind to asialo-GM1 (monosialoganglioside) oligosaccharide. Moreover, it binds to Gal beta 1-3GalNAc alpha Ser, the authentic T (Thomsen-Friedenreich)-antigen, with about 2.5-fold greater affinity as compared with Gal beta 1-3GalNAc. Asialoglycophorin A was found to be about 169,333 times stronger an inhibitor than Gal beta 1-3GalNAc. The present study thus reveals the exquisite specificity of A. integrifolia lectin for the T-antigen. Appreciable binding of disaccharides Glc beta 1-3GalNAc and GlcNAc beta 1-3Gal and the very poor binding of beta-linked disaccharides, which instead of Gal and GalNAc contain other sugars at the reducing end, underscore the important contribution made by Gal and GalNAc at the reducing end for recognition by the lectin. The ligand-structure-dependent alterations of the c.d. spectrum in the tertiary structural region of the protein allows the placement of various sugar units in the combining region of the lectin. These studies suggest that the primary subsite (subsite A) can accommodate only Gal or GalNAc or alpha-linked Gal or GalNAc, whereas the secondary subsite (subsite B) can associate either with GalNAc beta Me or Gal beta Me. Considering these factors a likely arrangement for various disaccharides in the binding site of the lectin is proposed. Its exquisite specificity for the authentic T-antigen, Gal beta 1-3GalNAc alpha Ser, together with its virtual non-binding to A- and B-blood-group antigens, Gal beta 1-3GalNAc beta Me and asialo-GM1 should make A. integrifolia lectin a valuable probe for monitoring the expression of T-antigen on cell surfaces.

A simple and rapid method for quantification of hapten specific antibodies

A sensitive and simple method for quantification of antibodies against small molecules is described using DNP-lysozyme as the enzyme conjugate. The anti-DNP antiserum was raised against DNP-bovin serum albumin conjugate. Anti-DNP antibody or its monovalent fragment (Fab) reduced the enzyme activity of DNP-lysozyme conjugate in a concentration-dependent manner. The inhibition of enzyme activity is a specific measure of the antibody and Fab content of the sample. The specificity of the reaction was assessed by reduction of antibody-induced inhibition by DNP-lysine. The ability of DNP-lysine to reduce the antibody-induced inhibition of DNP-lysozyme activity also makes possible a sensitive assay for DNP-lysine.

Lattice vibrations and specific heat of zinc blende

The dispersion relations, frequency distribution function and specific heat of zinc blende have been calculated using Houston's method on (1) A short range force (S. R.) model of the type employed in diamond by Smith and (2) A long range model assuming an effective charge Ze on the ions. Since the elastic constant data on ZnS are not in agreement with one another the following values were used in these calculations: {Mathematical expression}. As compared to the results on the S. R. model, the Coulomb force causes 1. A splitting of the optical branches at (000) and a larger dispersion of these branches; 2. A rise in the acoustic frequency branches the effect being predominant in a transverse acoustic branch along [110]; 3. A bridging of the gap of forbidden frequencies in the S. R. model; 4. A reduction of the moments of the frequency distribution function and 5. A flattening of the Θ- T curve. By plotting (Θ/Θ0) vs. T., the experimental data of Martin and Clusius and Harteck are found to be in perfect coincidence with the curve for the short range model. The values of the elastic constants deduced from the ratio Θ0 (Theor)/Θ0 (Expt) agree with those of Prince and Wooster. This is surprising as several lines of evidence indicate that the bond in zinc blende is partly covalent and partly ionic. The conclusion is inescapable that the effective charge in ZnS is a function of the wave vector {Mathematical expression}.

Identification of Specific DNA Binding Residues in the TCP Family of Transcription Factors in Arabidopsis

The TCP transcription factors control multiple developmental traits in diverse plant species. Members of this family share an similar to 60-residue-long TCP domain that binds to DNA. The TCP domain is predicted to form a basic helix-loop-helix ( bHLH) structure but shares little sequence similarity with canonical bHLH domain. This classifies the TCP domain as a novel class of DNA binding domain specific to the plant kingdom. Little is known about how the TCP domain interacts with its target DNA. We report biochemical characterization and DNA binding properties of a TCP member in Arabidopsis thaliana, TCP4. We have shown that the 58-residue domain of TCP4 is essential and sufficient for binding to DNA and possesses DNA binding parameters comparable to canonical bHLH proteins. Using a yeast-based random mutagenesis screen and site-directed mutants, we identified the residues important for DNA binding and dimer formation. Mutants defective in binding and dimerization failed to rescue the phenotype of an Arabidopsis line lacking the endogenous TCP4 activity. By combining structure prediction, functional characterization of the mutants, and molecular modeling, we suggest a possible DNA binding mechanism for this class of transcription factors.

Drosophila indirect flight muscle specific Act88F actin mutants as a model system for studying congenital myopathies of the human ACTA1 skeletal muscle actin gene

Most human ACTA1 skeletal actin gene mutations cause dominant, congenital myopathies often with severely reduced muscle function and neonatal mortality. High sequence conservation of actin means many mutated ACTA1 residues are identical to those in the Drosophila Act88F, an indirect flight muscle specific sarcomeric actin. Four known Act88F mutations occur at the same actin residues mutated in ten ACTA1 nemaline mutations, A138D/P, R256H/L, G268C/D/R/S and R372C/S. These Act88F mutants were examined for similar muscle phenotypes. Mutant homozygotes show phenotypes ranging from a lack of myofibrils to almost normal sarcomeres at eclosion. Aberrant Z-disc-like structures and serial Z-disc arrays, ‘zebra bodies’, are observed in homozygotes and heterozygotes of all four Act88F mutants. These electron-dense structures show homologies to human nemaline bodies/rods, but are much smaller than those typically found in the human myopathy. We conclude that the Drosophila indirect flight muscles provide a good model system for studying ACTA1 mutations.

Studies on non-specific interference due to serum in the avidin-biotin microELISA for monkey chorionic gonadotropin and a method for its elimination

A study has been carried out on the non-specific interference due to serum in the avidin biotin micro-ELISA for monkey chorionic gonadotropin. Results suggest that it is not due to any proteolytic activity in the serum, but immunoglobulin or associated factors interfering at the level of antigen-antibody interaction. This interference was eliminated by heating samples at 60°C for 30 min.

Molecular Caches: A caching structure for dynamic creation of application-specific Heterogeneous cache regions

CMPs enable simultaneous execution of multiple applications on the same platforms that share cache resources. Diversity in the cache access patterns of these simultaneously executing applications can potentially trigger inter-application interference, leading to cache pollution. Whereas a large cache can ameliorate this problem, the issues of larger power consumption with increasing cache size, amplified at sub-100nm technologies, makes this solution prohibitive. In this paper in order to address the issues relating to power-aware performance of caches, we propose a caching structure that addresses the following: 1. Definition of application-specific cache partitions as an aggregation of caching units (molecules). The parameters of each molecule namely size, associativity and line size are chosen so that the power consumed by it and access time are optimal for the given technology. 2. Application-Specific resizing of cache partitions with variable and adaptive associativity per cache line, way size and variable line size. 3. A replacement policy that is transparent to the partition in terms of size, heterogeneity in associativity and line size. Through simulation studies we establish the superiority of molecular cache (caches built as aggregations of molecules) that offers a 29% power advantage over that of an equivalently performing traditional cache.

Comparison of promoter-specific events during transcription initiation in mycobacteria

DNA protein interactions that occur during transcription initiation play an important role in regulating gene expression. To initiate transcription, RNA polymerase (RNAP) binds to promoters in a sequence-specific fashion. This is followed by a series of steps governed by the equilibrium binding and kinetic rate constants, which in turn determine the overall efficiency of the transcription process. We present here the first detailed kinetic analysis of promoter RNAP interactions during transcription initiation in the sigma(A)-dependent promoters P-rrnAPCL1, P-rrnB and P-gyr of Mycobacterium smegmatis. The promoters show comparable equilibrium binding affinity but differ significantly in open complex formation, kinetics of isomerization and promoter clearance. Furthermore, the two rrn promoters exhibit varied kinetic properties during transcription initiation and appear to be subjected to different modes of regulation. In addition to distinct kinetic patterns, each one of the housekeeping promoters studied has its own rate-limiting step in the initiation pathway, indicating the differences in their regulation.

Plasticity in the primary binding site of galactose/N-acetylgalactosamine-specific lectins - Implication of the C-H center dot center dot center dot O hydrogen bond at the specificity-determining C-4 locus of the saccharide in 4-methoxygalactose recognition by jacalin and winged bean (basic) agglutinin I

It is currently believed that an unsubstituted axial hydroxyl at the specificity-determining C-4 locus of galactose is indispensable for recognition by galactose/N-acetylgalactosamine-specific lectins. Titration calorimetry demonstrates that 4-methoxygalactose retains binding allegiance to the Moraceae lectin jacalin and the Leguminosae lectin, winged bean (basic) agglutinin (WBA I). The binding reactions were driven by dominant favorable enthalpic contributions and exhibited significant enthalpy-entropy compensation. Proton NMR titration of C-methoxygalactose with jacalin and WBA I resulted in broadening of the sugar resonances without any change in chemical shift. The alpha-and beta-anomers of 4-methoxygalactose were found to be in slow exchange with free and lectin-bound states. Both the anomers experience magnetically equivalent environments at the respective binding sites. The binding constants derived from the dependence of NMR line widths on 4-methoxygalactose concentration agreed well with those obtained from titration calorimetry. The results unequivocally demonstrate that the loci corresponding to the axially oriented C-4 hydroxyl group of galactose within the primary binding site of these lectins exhibit plasticity. These analyses suggest, for the first time, the existence of C-H ... O-type hydrogen-bond(s) in protein-carbohydrate interactions in general and between the C-4 locus of galactose derivative and the lectins jacalin and WBA I in particular.

Testis-specific histone H1t is truly a testis-specific variant and not a meiotic-specific variant

Immunoblotting studies using highly specific polyclonal anti-histone H1t-IgG, which was extensively characterized by us previously, did not produce a signal with any of the histone H1 subtypes of either 1-day-old or adult rat ovarian nuclei. The absence of histone H1t in ovarian nuclei was also confirmed by indirect immunofluorescence studies. It is concluded, therefore, that histone H1t is truly a testis-specific histone variant and not a meiotic-specific variant.

Induction of allergen-specific IgE and IgG responses by anti-idiotypic antibodies

In order to explore idiotypic, anti-idiotypic, and anti-anti-idiotypic responses to allergens, BALB/c mice were immunized with affinity- purified human idiotypic antibodies directed against a highly purified shrimp allergen. This resulted in the production of anti-idiotypic antibodies which were quantitated by using rabbit idiotypic antibodies raised against the same purified allergen. The mouse anti-idiotypic antibodies recognized shrimp-specific human idiotypic antibodies of the IgE isotype from 18 of 20 individuals, and IgG antibodies from 14 of 20 shrimp-sensitive patients. Immunization of BALB/c mice with affinity- purified, allergen-specific anti-idiotypic antibodies induced anti- allergen IgE and IgG responses in the absence of the allergen. This paper thus presents evidence that anti-idiotypic antibodies raised against allergen-specific idiotypic antibodies may substitute for the original allergen in the induction of allergen-specific idiotypic antibodies. The demonstration of shared idiotopes on IgG and IgE antibodies in the sera of shrimp-sensitive patients supports the use of allergen-specific anti-idiotypic antibodies as surrogate allergens.

Study of magnetic field effect on the specific heat and entropy in DyBa2Cu3O7-x

The change in the specific heat by the application of magnetic field up to 161 for high temperature superconductor system for DyBa2Cu3O7-x by Revaz et al. [23] is examined through the phenomenological Ginzburg-Landau(G-L) theory of anisotropic Type-II superconductors. The observed specific heat anomaly near T-c with magnetic field is explained qualitatively through the expression <Delta C > = (B-a/T-c) t/(1 - t)(alpha Theta(gamma)lambda(2)(m)(0)), which is the anisotropic formulation of the G-L theory in the London limit developed by Kogan and coworkers; relating to the change in specific heat Delta C for the variation of applied magnetic field for different orientations with c-axis. The analysis of this equation explains satisfactorily the specific heat anomaly near T-c and determines the anisotropic ratio gamma as 5.608, which is close to the experimental value 5.3 +/- 0.5given in the paper of Revaz et al. for this system. (C) 2010 Elsevier B.V. All rights reserved.