Inhibition of Ribonuclease A by polyphenols present in green tea

We report the effect of the natural polyphenolic compounds from green tea on the catalytic activity of Ribonuclease A (RNase A). The compounds behave as noncompetitive inhibitors of the protein with inhibition constants ranging from 80-1300 microM. The dissociation constants range from 50-150 microM for the RNase A-polyphenol complexes as determined by ultraviolet (UV) and circular dichroism (CD) studies. We have also investigated the changes in the secondary structure of RNase A on complex formation by CD and Fourier transformed infrared (FTIR) spectroscopy. The presence of the gallate moiety has been shown to be important for the inhibition of enzymatic activity. Docking studies for these compounds indicate that the preferred site of binding is the region encompassing residues 34-39 with possible hydrogen bonding with Lys 7 and Arg 10. Finally we have also looked at changes in the accessible surface area of the interacting residues on complex formation for an insight into the residues involved in the interaction.     

Narrowing the conformational space sampled by two-domain proteins with paramagnetic probes in both domains

Calmodulin is a two-domain protein which in solution can adopt a variety of conformations upon reorientation of its domains. The maximum occurrence (MO) of a set of calmodulin conformations that are representative of the overall conformational space possibly sampled by the protein, has been calculated from the paramagnetism-based restraints. These restraints were measured after inclusion of a lanthanide binding tag in the C-terminal domain to supplement the data obtained by substitution of three paramagnetic lanthanide ions to the calcium ion in the second calcium binding loop of the N-terminal domain. The analysis shows that the availability of paramagnetic restraints arising from metal ions placed on both domains, reduces the MO of the conformations to different extents, thereby helping to identify those conformations that can be mostly sampled by the protein.     

Genome-wide analysis of genetic alterations in testicular primary seminoma using high resolution single nucleotide polymorphism arrays

Testicular germ cell tumors (TGCT) represent the most common malignancy among young males. To our knowledge no comprehensive Copy Number Variation (CNVs) studies of TGCT using high-resolution Single Nucleotide Polymorphism (SNP) array have been performed. By a genome-wide analysis of CNV and loss of heterozygosity (LOH) in 25 primary seminomas, we confirmed several previously reported genomic alterations and discovered eight novel genomic alterations including amplifications and homozygous deletions. Moreover, a comparison of genomic alterations of early and late stage seminoma identified CNVs that correlate with progression, which included deletions in chromosomes 4q, 5p, 9q, 13q and 20p and amplifications in chromosomes 9q and 13q. We compared previously perform Affymetrix expression analysis in a subset of samples and found robust correlation between expression and genomic alterations. Furthermore, high correlations (40-75%) were observed between CNV by SNP analysis and quantitative PCR. Our findings may lead to better understanding of TGTC's pathogenesis.     

A systematic screen for micro-RNAs regulating the canonical Wnt pathway

MicroRNAs (miRs) and the canonical Wnt pathway are known to be dysregulated in human cancers and play key roles during cancer initiation and progression. To identify miRs that can modulate the activity of the Wnt pathway we performed a cell-based overexpression screen of 470 miRs in human HEK293 cells. We identified 38 candidate miRs that either activate or repress the canonical Wnt pathway. A literature survey of all verified candidate miRs revealed that the Wnt-repressing miRs tend to be anti-oncomiRs and down-regulated in cancers while Wnt-activating miRs tend to be oncomiRs and upregulated during tumorigenesis. Epistasis-based functional validation of three candidate miRs, miR-1, miR-25 and miR-613, confirmed their inhibitory role in repressing the Wnt pathway and suggest that while miR-25 may function at the level of a-catenin (β-cat), miR-1 and miR-613 act upstream of β-cat. Both miR-25 and miR-1 inhibit cell proliferation and viability during selection of human colon cancer cell lines that exhibit dysregulated Wnt signaling. Finally, transduction of miR-1 expressing lentiviruses into primary mammary organoids derived from Conductin-lacZ mice significantly reduced the expression of the Wnt-sensitive β-gal reporter. In summary, these findings suggest the potential use of Wnt-modulating miRs as diagnostic and therapeutic tools in Wnt-dependent diseases, such as cancer.     

Reactivity of trypsin in reverse micelles: neglected role of aggregate size compared to water-pool components

The catalytic efficiency of trypsin was estimated in cationic reverse micelles as a function of the concentration of water-pool components and aggregate size to determine their independent influence on enzyme activity. The variation in the aggregate size/water-pool size was achieved by changing both the W0 (mole ratio of water to surfactant) and the headgroup area of surfactant through introduction of hydroxyethyl groups at the polar head. The local molar concentrations of water present inside the water-pool ([H2O]wp) of different cationic reverse micelles across varying W0 was estimated using a modified phenyl cation-trapping protocol. The [H2O]wp in cationic reverse micelles (surfactant/isooctane/n-hexanol/water) increases with W0 and attains the molarity of normal water beyond W0=40 irrespective of the nature of headgroup. Concurrently, the catalytic activity of trypsin compartmentalized within the water-pool increases with the increase in [H2O]wp upto an optimal W0=40 in organized solutions of any surfactant. The aggregate size (determined by static light scattering) also increases expectedly with W0 and noticeably with the area of the surfactant headgroup at similar W0. Since the enzyme activity rises both with the increase in water-pool size and [H2O]wp, trypsin's efficiency was compared with these two parameters across reverse micelles of varying surfactant headgroup size at similar W0 to determine their probable independent influence in regulating the enzyme activity. Noticeably, the efficiency of trypsin rises two to ninefold in spite of the [H2O]wp being distinctly lower in case of hydroxyethyl group substituted surfactants compared to cetyltrimethylammonium bromide w/o microemulsions at similar W0. Thus, the influence of the aggregate size possibly plays an important role alongwith the [H2O]wp in modulating the enzyme activity.     

Deuterium oxide promotes assembly and bundling of FtsZ protofilaments

The assembly and bundling of FtsZ protofilaments play an important role during bacterial cell division. Deuterium oxide (D2O) is known to have strong stabilization effects on the assembly dynamics of several proteins including tubulin, a homologue of FtsZ. Here, we found that D2O enhanced the light-scattering intensity of the assembly reaction, increased sedimentable polymer mass, and induced bundling of FtsZ protofilaments. D2O also increased the stability of FtsZ polymers under challenged GTP conditions and suppressed dilution-induced disassembly of protofilaments. D2O enhances the assembly parameters of FtsZ and microtubules albeit differently. For example, D2O induced bundling of FtsZ protofilaments, whereas it did not induce bundling of microtubules in vitro. In addition, D2O strongly suppressed the GTP hydrolysis rate of microtubules, but it had no effect on the initial rate of GTP hydrolysis of the FtsZ assembly. D2O (80%) also increased the helical content of FtsZ by 25% compared to the helical content of FtsZ in aqueous buffer. D2O was shown to reduce the binding of 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) to tubulin. In contrast, we found that D2O strongly enhanced the binding of bis-ANS to FtsZ. The results indicated that D2O promotes assembly and bundling of FtsZ protofilaments by increasing hydrophobic interactions between the protofilaments. The results also suggest that the phosphate release rather than the on-site GTP hydrolysis is the rate-limiting step of the GTP turnover reaction.