Comparison of Guanidine Hydrochloride (GdnHCl) and Urea Denaturation on Inactivation and Unfolding of Human Placental Cystatin (HPC)

The activity and conformational change of human placental cystatin (HPC), a low molecular weight thiol proteinase inhibitor (12,500) has been investigated in presence of guanidine hydrochloride (GdnHCl) and urea. The denaturation of HPC was followed by activity measurements, fluorescence spectroscopy and Circular Dichroism (CD) studies. Increasing the denaturant concentration significantly enhanced the inactivation and unfolding of HPC. The enzyme was 50% inactivated at 1.5 M GdnHCl or 3 M urea. Up to 1.5 M GdnHCl concentration there was quenching of fluorescence intensity compared to native form however at 2 M concentration intensity increased and emission maxima had 5 nm red shift with complete unfolding in 4–6 M range. The mid point of transition was in the region of 1.5–2 M. In case of urea denaturation, the fluorescence intensity increased gradually with increase in the concentration of denaturant. The protein unfolded completely in 6–8 M concentration of urea with a mid-point of transition at 3 M. CD spectroscopy shows that the ellipticity of HPC has increased compared to that of native up to 1.5 M GdnHCl and then there is gradual decrease in ellipticity from 2 to 5 M concentration. At 6 M GdnHCl the protein had random coil conformation. For urea the ellipticity decreases with increase in concentration showing a sigmoidal shaped transition curve with little change up to 1 M urea. The protein greatly loses its structure at 6 M urea and at 8 M it is a random coil. The urea induced denaturation follows two-state rule in which Native→Denatured state transition occurs in a single step whereas in case of GdnHCl, intermediates or non-native states are observed at lower concentrations of denaturant. These intermediate states are possibly due to stabilizing properties of guanidine cation (Gdn⁺) at lower concentrations, whereas at higher concentrations it acts as a classical denaturant.     

The SERCA3-type of organellar Ca2+pumps

Of all the SERCA pumps, SERCA3 was the latest to be described and the least well known. Its primary structure deviates more than usual from the other members of the SERCA family. It is not known whether its remarkably low affinity for Ca²⁺ (K_0.5 > 1M) observed upon expression in the COS cell system occurs also in its normal cellular context. SERCA3 is particularly expressed at high levels in different types of blood cells and related cells like platelets, lymphocytes, mast cells and arterial endothelial cells. It is also found in cerebellar Purkinje neurons. The physiological significance of this expression pattern remains unknown.     

The rational design of a novel potent analogue of the 5'-AMP-activated protein kinase inhibitor compound C with improved selectivity and cellular activity

We have designed and synthesized analogues of compound C, a non-specific inhibitor of 5'-AMP-activated protein kinase (AMPK), using a computational fragment-based drug design (FBDD) approach. Synthesizing only twenty-seven analogues yielded a compound that was equipotent to compound C in the inhibition of the human AMPK (hAMPK) α2 subunit in the heterotrimeric complex in vitro, exhibited significantly improved selectivity against a subset of relevant kinases, and demonstrated enhanced cellular inhibition of AMPK.     

Polyelectrolyte multilayer films as substrates for photoreceptor cells

Reconstruction of extracellular matrix substrates for delivery of functional photoreceptors is crucial in pathologies such as retinal degeneration and age-related macular degeneration. In this study, we assembled polyelectrolyte films using the layer-by-layer deposition method. The buildup of three different films composed of poly(L-lysine)/chondroitin sulfate (PLL/CSA), poly(L-lysine)/poly(styrenesulfonate) (PLL/PSS), or poly(L-lysine)/hyaluronic acid (PLL/HA) was followed by means of quartz crystal microbalance measurements, optical waveguide light mode spectroscopy, confocal microscopy, and atomic force microscopy. The exponential growth regime and the diffusion of PLL chains from the bulk through the PLL/CSA, PLL/PSS, and PLL/HA films was examined. Evaluation of photoreceptor cell viability was optimal on one layer of PLL (PLL(1)), followed by 10 bilayers of PLL/HA [(PLL/HA)(10)] and 10 bilayers of PLL/CSA [(PLL/CSA)(10)]. The number of bilayers and the type of terminating layer also had a significant influence on the number of photoreceptor cells attached. Functionalized polyelectrolyte multilayer films were obtained by adsorbing basic fibroblastic factor (bFGF) or the insoluble fraction of interphotoreceptor matrix (IPM) on or within polyelectrolyte multilayers. bFGF and IPM adsorption on top of the (PLL/CSA)(10)/PLL polyelectrolyte films increased the number of photoreceptor cells attached and maintained the differentiation of rod and cone cells.     

Molten globule state of human placental cystatin (HPC) at low pH conditions and the effects of trifluoroethanol (TFE) and methanol.

Acid-induced conformational changes were studied in human placental cystatin (HPC) in terms of circular dichroism (CD) spectroscopy, the binding of hydrophobic dye 1-anilinonapthalene-8-sulphonic acid (ANS), and intrinsic fluorescence measurements. Our results show the formation of an acid-induced molten globule state at pH 2.0, with significant secondary and tertiary interactions that resemble the native state, exposed hydrophobic regions and the effects of trifluoroethanol (TFE) and methanol in conversion of the acid-denatured state of HPC to the alcohol-induced state, which is characterized by increased helical content, disrupted tertiary structure, and the absence of hydrophobic clusters. Alcohol-induced formation of alpha-helical structures at pH 2.0 is evident from the increase in the ellipticity values at 222 nm, with native-like secondary structural features at 40% TFE. The increase in helical content was observed up to 80% TFE concentration. The ability of TFE (40%) to refold acid-denatured HPC to native-state conformation is also supported by intrinsic and ANS fluorescence measurements.     

The N-terminal region of the <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis> RecQ helicase,Rqh1p,physically interacts with Topoisomerase III and is required for Rqh1p function

The Schizosaccharomyces pombe rqh1⁺ gene encodes a member of the RecQ DNA helicase family. Members of this protein family are essential for the maintenance of genetic integrity. Thus, mutations in the genes encoding the human RecQ homologues Blm, Wrn and RecQ4 cause Bloom syndrome, Werner syndrome and Rothmund–Thomson syndrome, respectively—diseases which result from genome instability. S. pombe cells that lack a functional rqh1⁺ gene show reduced viability and display defective chromosome segregation, particularly after UV irradiation or S-phase arrest. In this study we used an rqh1⁺ deletion series to show that the N-terminal portion of Rqh1 is essential for Rqh1 function. Moreover, the conserved Helicase and RNaseD C-terminal (HRDC) domain of Rqh1 also plays a role in allowing cells to tolerate exposure to DNA damaging agents and the S-phase inhibitor hydroxyurea (HU). We also demonstrate that Topoisomerase III (Top3) binds to a site within the first 322 N-terminal amino acids of Rqh1 and that this binding correlates with Rqh1 function. Genetic analysis of rqh1^– top3 mutants reveals that, in the presence of functional or partially functional Rqh1 protein, Top3 is required to maintain genome integrity and cell viability.     

Serological profiling with Chlamycheck, a commercial multiplex recombinant antigen Western blot assay of chlamydial infections

A new chlamydial test system, the Chlamycheck assay, which uses 4 purified recombinant antigens of Chlamydia trachomatis and Chlamydophila pneumoniae and one antigen of Chlamydophila psittaci, has been developed and commercialized. We investigated the reactivities of the recombinant antigens with sera from a group of 30 patients with acute Chlamydia trachomatis infection, 88 patients consulting for sexually transmitted infections, and 46 patients with serological evidence of Chlamydophila pneumoniae infection. The results obtained from human and infected mouse sera suggest that Chlamycheck serology against multiple proteins may provide additional useful information that is not available by conventional whole elementary body microimmunofluorescence or single-antigen enzyme-linked immunosorbent assay serology. Specific serological profiles were associated with acute versus past Chlamydia trachomatis infection or with Chlamydia trachomatis primo-infection versus infection in a Chlamydophila pneumoniae history context.