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.

Genetic studies of the PRP17 gene of Saccharomyces cerevisiae: a domain essential for function maps to a nonconserved region of the protein

The PRP17 gene product is required for the second step of pre-mRNA splicing reactions. The C-terminal half of this protein bears four repeat units with homology to the beta transducin repeat. Missense mutations in three temperature-sensitive prp17 mutants map to a region in the N-terminal half of the protein. We have generated, in vitro, 11 missense alleles at the beta transducin repeat units and find that only one affects function in vivo. A phenotypically silent missense allele at the fourth repeat unit enhances the slow-growing phenotype conferred by an allele at the third repeat, suggesting an interaction between these domains. Although many missense mutations in highly conserved amino acids lack phenotypic effects, deletion analysis suggests an essential role for these units. Only mutations in the N-terminal nonconserved domain of PRP17 are synthetically lethal in combination with mutations in PRP16 and PRP18, two other gene products required for the second splicing reaction. A mutually allele-specific interaction between Prp17 and snr7, with mutations in U5 snRNA, was observed. We therefore suggest that the functional region of Prp17p that interacts with Prp18p, Prp16p, and U5 snRNA is the N terminal region of the protein.

Structure and Function of Hemoglobin Confined Inside Silica Nanotubes

Investigations on the structure and function of hemoglobin (Hb) confined inside sol-gel template synthesized silica nanotubes (SNTs) have been discussed here. Immobilization of hemoglobin inside SNTs resulted in the enhancement of direct electron transfer during an electrochemical reaction. Extent of influence of nanoconfinement on protein activity is further probed via ligand binding and thermal stability studies. Electrochemical investigations show reversible binding of n-donor liquid ligands, such as pyridine and its derivatives, and predictive variation in their redox potentials suggests an absence of any adverse effect on the structure and function of Hb confined inside nanometer-sized channels of SNTs. Immobilization also resulted in enhanced thermal stability of Hb. The melting or denaturation temperature of Hb immobilized inside SNTs increase by approximately 4 degrees C as compared with that of free Hb in solution.

Changes in testicular function following specific deprivation of LH in the adult male rabbit

Sexually mature male rabbits actively immunized against highly purified ovine LH (oLH) were used as a model system to study the effects of endogenous LH deprivation (and therefore testosterone) on spermatogenesis as well as pituitary FSH secretion. Immunization against oLH generated antibody titres capable of cross-reacting and neutralizing rabbit LH and this resulted in a significant reduction (P<0.01) in serum testosterone levels by 2-4 weeks of immunization. A significant increase in circulating FSH concentration (from a basal level of similar to 1 ng to 60-100 ng/ml; P<0.01) was observed within 4-6 weeks of immunization, perhaps a consequence of the negative feedback effect of the lack of testosterone. The effect of LH deprivation on spermatogenesis assessed by DNA flow cytometry and histological analyses of testicular biopsy tissue revealed that lack of testosterone primarily results in a rapid reduction and complete absence of round (1C) and elongated (HC) spermatids. The immediate effect of LH/testosterone deprivation thus appears to be at the step of meiotic transformation of primary spermatocytes (4C) to 1C. A significant reduction (>80%; P<0.01) in the 4C population and a relative accumulation (>90%; P<0.01) in spermatogonia (2C) was also observed, suggesting a need for testosterone during the transformation of 2C to 1C. In all but one of the rabbits, both qualitative and quantitative recovery in spermatogenesis occurred during the recovery phase, even at a time when only a marginal increase in serum testosterone (compared with the preimmunization) levels was observed as a result of a rapid decline in the cross-reactive antibody titres. These results clearly show that LH/testosterone deprivation in addition to primarily affecting the meiotic step also regulates the conversion of 2C to 4C during spermatogenesis.

Post-liquefaction undrained monotonic behaviour of sands: experiments and DEM simulations

This paper presents the results of a series of servo-controlled cyclic triaxial tests and numerical simulations using the three- dimensional discrete element method (DEM) on post-liquefaction undrained monotonic strength of granular materials. In a first test series,undrained monotonic tests were carried out after dissipating the excess pore water pressure developed during liquefaction. The influence of different parameters such as amplitude of axial strain,relative density and confining pressure prior to liquefaction on the post-liquefaction undrained response have been investigated.The results obtained highlight an insignificant influence of amplitude of axial strain, confining pressure and a significant influence of relative density on the post-liquefaction undrained monotonic stress-strain response.In the second series, undrained monotonic tests were carried out on similar triaxial samples without dissipating the excess pore water pressure developed during liquefaction. The results highlight that the amplitude of axial strain prior to liquefaction has a significant influence on the post-liquefaction undrained monotonic response.In addition,DEM simulations have been carried out on an assembly of spheres to simulate post-liquefaction behaviour.The simulations were very similar to the experiments with an objective to understand the behaviour of monotonic strength of liquefied samples from the grain scale. The numerical simulations using DEM have captured qualitatively all the features of the post-liquefaction undrained monotonic response in a manner similar to that of the experiments.In addition,a detailed study on the evolution of micromechanical parameters such as the average coordination number and induced anisotropic coefficients has been reported during the post-liquefaction undrained monotonic loading.

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.

Binding and regulatory properties of phosphofructokinase from swine kidney

The influence of fructose 2,6-bisphosphate on the activation of purified swine kidney phosphofructokinase as a function of the concentration of fructose 6P, ATP and citrate was investigated. The purified enzyme was nearly completely inhibited in the presence of 2 mM ATP. The addition of 20 nM fructose 2,6-P2 reversed the inhibition and restored more than 80% of the activity. In the absence of fructose 2,6-P2 the reaction showed a sigmoidal dependence on fructose-6-phosphate. The addition of 10 nM fructose 2,6-bisphosphate decreased the K0.5 for fructose 6-phosphate from 3 mM to 0.4 mM in the presence of 1.5 mM ATP. These results clearly show that fructose 2,6-bisphosphate increases the affinity of the enzyme for fructose 6-phosphate and decreases the inhibitory effect of ATP. The extent of inhibition by citrate was also significantly decreased in the presence of fructose 2,6-phosphate. The influence of various effectors of phosphofructokinase on the binding of ATP and fructose 6-P to the enzyme was examined in gel filtration studies. It was found that kidney phosphofructokinase binds 5.6 moles of fructose 6-P per mole of enzyme, which corresponds to about one site per subunit of tetrameric enzyme. The KD for fructose 6-P was 13 microM and in the presence of 0.5 mM ATP it increased to 27 microM. The addition of 0.3 mM citrate also increased the KD for fructose 6-P to about 40 microM. AMP, 10 microM, decreased the KD to 5 microM and the addition of fructose 2,6-phosphate decreased the KD for fructose 6-P to 0.9 microM. The addition of these compounds did not effect the maximal amount of fructose 6-P bound to the enzyme, which indicated that the binding site for these compounds might be near, but was not identical to the fructose 6-P binding site. The enzyme bound a maximum of about 12.5 moles of ATP per mole, which corresponds to 3 moles per subunit. The KD of the site with the highest affinity for ATP was 4 microM, and it increased to 15 microM in the presence of fructose 2,6-bisphosphate. The addition of 50 microM fructose 1,6-bisphosphate increased the KD for ATP to 5.9 microM. AMP increased the KD to 5.9 microM whereas 0.3 mM citrate decreased the KD for ATP to about 2 microM.(ABSTRACT TRUNCATED AT 400 WORDS).

CD4+ alpha beta T cell and gamma delta T cell responses to Mycobacterium tuberculosis. Similarities and differences in Ag recognition, cytotoxic effector function, and cytokine production.

CD4+ and gamma delta T cells are activated readily by Mycobacterium tuberculosis. To examine their role in the human immune response to M. tuberculosis, CD4+ and gamma delta T cells from healthy tuberculin-positive donor were studied for patterns of Ag recognition, cytotoxicity, and cytokine production in response to M. tuberculosis-infected mononuclear phagocytes. Both T cell subsets responded to intact M. tuberculosis and its cytosolic Ags. However, CD4+ and gamma delta T cells differed in the range of cytosolic Ags recognized: reactivity to a wide m.w. range of Ags for CD4+ T cells, and a restricted pattern for gamma delta T cells, with dominance of Ags of 10 to 15 kDa. Both T cell subsets were equally cytotoxic for M. tuberculosis-infected monocytes. Furthermore, both CD4+ and gamma delta T cells produced large amounts of IFN-gamma: mean pg/ml of IFN-gamma in supernatants was 2458 +/- 213 for CD4+ and 2349 +/- 245 for gamma delta T cells. By filter-spot ELISA (ELISPOT), the frequency of IFN-gamma-secreting gamma delta T cells was one-half of that of CD4+ T cells in response to M. tuberculosis, suggesting that gamma delta T cells on a per cell basis were more efficient producers of IFN-gamma than CD4+ T cells. In contrast, CD4+ T cells produced more IL-2 than gamma delta T cells, which correlated with diminished T cell proliferation of gamma delta T cells compared with CD4+ T cells. These results indicate that CD4+ and gamma delta T cell subsets have similar effector functions (cytotoxicity, IFN-gamma production) in response to M. tuberculosis-infected macrophages, despite differences in the Ags recognized, IL-2 production, and efficiency of IFN-gamma production.

Computationally efficient optimum weighting function for vector quantization of LSF parameters

We propose a new weighting function which is computationally simple and an approximation to the theoretically derived optimum weighting function shown in the literature. The proposed weighting function is perceptually motivated and provides improved vector quantization performance compared to several weighting functions proposed so far, for line spectrum frequency (LSF) parameter quantization of both clean and noisy speech data.

Inferring molecular function: contributions from functional linkages

In the current era of high-throughput sequencing and structure determination, functional annotation has become a bottleneck in biomedical science. Here, we show that automated inference of molecular function using functional linkages among genes increases the accuracy of functional assignments by >= 8% and enriches functional descriptions in >= 34% of top assignments. Furthermore, biochemical literature supports >80% of automated inferences for previously unannotated proteins. These results emphasize the benefit of incorporating functional linkages in protein annotation.

THE BERGMAN KERNEL FUNCTION

In this note, we point out that a large family of n x n matrix valued kernel functions defined on the unit disc D subset of C, which were constructed recently in [9], behave like the familiar Bergman kernel function on ID in several different ways. We show that a number of questions involving the multiplication operator on the corresponding Hilbert space of holomorphic functions on D can be answered using this likeness.

Structural Studies on the Second Mycobacterium smegmatis Dps: Invariant and Variable Features of Structure, Assembly and Function

A second DNA binding protein from stationary-phase cells of Mycobacterium smegmatis (MsDps2) has been identified from the bacterial genome. It was cloned, expressed and characterised and its crystal structure was determined. The core dodecameric structure of MsDps2 is the same as that of the Dps from the organism described earlier (MsDps1). However, MsDps2 possesses a long N-terminal tail instead of the C-terminal tail in MsDps1. This tail appears to be involved in DNA binding. It is also intimately involved in stabilizing the dodecamer. Partly on account of this factor, MsDps2 assembles straightway into the dodecamer, while MsDps1 does so on incubation after going through an intermediate trimeric stage. The ferroxidation centre is similar in the two proteins, while the pores leading to it exhibit some difference. The mode of sequestration of DNA in the crystalline array of molecules, as evidenced by the crystal structures, appears to be different in MsDps1 and MsDps2, highlighting the variability in the mode of Dps–DNA complexation. A sequence search led to the identification of 300 Dps molecules in bacteria with known genome sequences. Fifty bacteria contain two or more types of Dps molecules each, while 195 contain only one type. Some bacteria, notably some pathogenic ones, do not contain Dps. A sequence signature for Dps could also be derived from the analysis.

The mixed black hole partition function for the STU model

We evaluate the mixed partition function for dyonic BPS black holes using the recently proposed degeneracy formula for the STU model. The result factorizes into the OSV mixed partition function times a proportionality factor. The latter is in agreement with the measure factor that was recently conjectured for a class of N = 2 black holes that contains the STU model.