Quantitative analysis of the kinetics of denaturation and renaturation of barstar in the folding transition zone.

The fluorescence-monitored kinetics of folding and unfolding of barstar by guanidine hydrochloride (GdnHCl) in the folding transition zone, at pH 7, 25 degrees C, have been quantitatively analyzed using a 3-state mechanism: U(S)<-->UF<-->N. U(S) and UF are slow-refolding and fast-refolding unfolded forms of barstar, and N is the native protein. U(S) and UF probably differ in possessing trans and cis conformations, respectively, of the Tyr 47-Pro 48 bond. The 3-state model could be used because the kinetics of folding and unfolding of barstar show 2 phases, a fast phase and a slow phase, and because the relative amplitudes of the 2 phases depend only on the final refolding conditions and not on the initial conditions. Analysis of the observed kinetics according to the 3-state model yields the values of the 4 microscopic rate constants that describe the transitions between the 3 states at different concentrations of GdnHCl. The value of the equilibrium unfolded ratio U(S):UF (K21) and the values of the rate constants of the U(S)-->UF and UF-->U(S) reactions, k12 and k21, respectively, are shown to be independent of the concentration of GdnHCl. K21 has a value of 2.1 +/- 0.1, and k12 and k21 have values of 5.3 x 10(-3) s-1 and 11.2 x 10(-3) s-1, respectively. Double-jump experiments that monitor reactions that are silent to fluorescence monitoring were used to confirm the values of K21, k12, and k21 obtained from the 3-state analysis and thereby the validity of the 3-state model.(ABSTRACT TRUNCATED AT 250 WORDS)     

Deletion of Glutamate Delta-1 Receptor in Mouse Leads to Enhanced Working Memory and Deficit in Fear Conditioning

Glutamate delta-1 (GluD1) receptors are expressed throughout the forebrain during development with high levels in the hippocampus during adulthood. We have recently shown that deletion of GluD1 receptor results in aberrant emotional and social behaviors such as hyperaggression and depression-like behaviors and social interaction deficits. Additionally, abnormal expression of synaptic proteins was observed in amygdala and prefrontal cortex of GluD1 knockout mice (GluD1 KO). However the role of GluD1 in learning and memory paradigms remains unknown. In the present study we evaluated GluD1 KO in learning and memory tests. In the eight-arm radial maze GluD1 KO mice committed fewer working memory errors compared to wildtype mice but had normal reference memory. Enhanced working memory in GluD1 KO was also evident by greater percent alternation in the spontaneous Y-maze test. No difference was observed in object recognition memory in the GluD1 KO mice. In the Morris water maze test GluD1 KO mice showed no difference in acquisition but had longer latency to find the platform in the reversal learning task. GluD1 KO mice showed a deficit in contextual and cue fear conditioning but had normal latent inhibition. The deficit in contextual fear conditioning was reversed by D-Cycloserine (DCS) treatment. GluD1 KO mice were also found to be more sensitive to foot-shock compared to wildtype. We further studied molecular changes in the hippocampus, where we found lower levels of GluA1, GluA2 and GluK2 subunits while a contrasting higher level of GluN2B in GluD1 KO. Additionally, we found higher postsynaptic density protein 95 (PSD95) and lower glutamate decarboxylase 67 (GAD67) expression in GluD1 KO. We propose that GluD1 is crucial for normal functioning of synapses and absence of GluD1 leads to specific abnormalities in learning and memory. These findings provide novel insights into the role of GluD1 receptors in the central nervous system.     

Early events during folding of wild-type staphylococcal nuclease and a single-tryptophan variant studied by ultrarapid mixing

A continuous-flow mixing device with a dead time of 100 micros coupled with intrinsic tryptophan and 1-anilinonaphthalene-8-sulfonate (ANS) fluorescence was used to monitor structure formation during early stages of the folding of staphylococcal nuclease (SNase). A variant with a unique tryptophan fluorophore in the N-terminal beta-barrel domain (Trp76 SNase) was obtained by replacing the single Trp140 in wild-type SNase with His in combination with Trp substitution of Phe76. A common background of P47G, P117G and H124L mutations was chosen in order to stabilize the protein and prevent accumulation of cis proline isomers under native conditions. In contrast to WT(*) SNase, which shows no changes in tryptophan fluorescence prior to the rate-limiting folding step ( approximately 100 ms), the F76W/W140H variant shows additional changes (enhancement) during an early folding phase with a time constant of 75 micros. Both proteins exhibit a major increase in ANS fluorescence and identical rates for this early folding event. These findings are consistent with the rapid accumulation of an ensemble of states containing a loosely packed hydrophobic core involving primarily the beta-barrel domain while the specific interactions in the alpha-helical domain involving Trp140 are formed only during the final stages of folding. The fact that both variants exhibit the same number of kinetic phases with very similar rates confirms that the folding mechanism is not perturbed by the F76W/W140H mutations. However, the Trp at position 76 reports on the rapid formation of a hydrophobic cluster in the N-terminal beta-sheet region while the wild-type Trp140 is silent during this early stage of folding. Quantitative modeling of the (un)folding kinetics and thermodynamics of these two proteins versus urea concentration revealed that the F76W/W140H mutation selectively destabilizes the native state relative to WT(*) SNase while the stability of transient intermediates remains unchanged, leading to accumulation of intermediates under equilibrium conditions at moderate denaturant concentrations.     

Structural and kinetic characterization of early folding events in beta-lactoglobulin

We have defined the structural and dynamic properties of an early folding intermediate of beta-lactoglobulin known to contain non-native alpha-helical structure. The folding of beta-lactoglobulin was monitored over the 100 micros--10 s time range using ultrarapid mixing techniques in conjunction with fluorescence detection and hydrogen exchange labeling probed by heteronuclear NMR. An initial increase in Trp fluorescence with a time constant of 140 micros is attributed to formation of a partially helical compact state. Within 2 ms of refolding, well protected amide protons indicative of stable hydrogen bonded structure were found only in a domain comprising beta-strands F, G and H, and the main alpha-helix, which was thus identified as the folding core of beta-lactoglobulin. At the same time, weak protection (up to approximately 10-fold) of amide protons in a segment spanning residues 12--21 is consistent with formation of marginally stable non-native alpha-helices near the N-terminus. Our results indicate that efficient folding, despite some local non-native structural preferences, is insured by the rapid formation of a native-like alpha/beta core domain.     

Ultrarapid mixing experiments reveal that Im7 folds via an on-pathway intermediate

Many proteins populate partially organized structures during folding. Since these intermediates often accumulate within the dead time (2-5 ms) of conventional stopped-flow and quench-flow devices, it has been difficult to determine their role in the formation of the native state. Here we use a microcapillary mixing apparatus, with a time resolution of approximately 150 micros, to directly follow the formation of an intermediate in the folding of a four-helix protein, Im7. Quantitative kinetic modeling of folding and unfolding data acquired over a wide range of urea concentrations demonstrate that this intermediate ensemble lies on a direct path from the unfolded to the native state.     

A Systems Genetics Approach Identifies CXCL14, ITGAX,and LPCAT2 as Novel Aggressive Prostate Cancer Susceptibility Genes

Although prostate cancer typically runs an indolent course, a subset of men develop aggressive, fatal forms of this disease. We hypothesize that germline variation modulates susceptibility to aggressive prostate cancer. The goal of this work is to identify susceptibility genes using the C57BL/6-Tg(TRAMP)8247Ng/J (TRAMP) mouse model of neuroendocrine prostate cancer. Quantitative trait locus (QTL) mapping was performed in transgene-positive (TRAMPxNOD/ShiLtJ) F2 intercross males (n = 228), which facilitated identification of 11 loci associated with aggressive disease development. Microarray data derived from 126 (TRAMPxNOD/ShiLtJ) F2 primary tumors were used to prioritize candidate genes within QTLs, with candidate genes deemed as being high priority when possessing both high levels of expression-trait correlation and a proximal expression QTL. This process enabled the identification of 35 aggressive prostate tumorigenesis candidate genes. The role of these genes in aggressive forms of human prostate cancer was investigated using two concurrent approaches. First, logistic regression analysis in two human prostate gene expression datasets revealed that expression levels of five genes (CXCL14, ITGAX, LPCAT2, RNASEH2A, and ZNF322) were positively correlated with aggressive prostate cancer and two genes (CCL19 and HIST1H1A) were protective for aggressive prostate cancer. Higher than average levels of expression of the five genes that were positively correlated with aggressive disease were consistently associated with patient outcome in both human prostate cancer tumor gene expression datasets. Second, three of these five genes (CXCL14, ITGAX, and LPCAT2) harbored polymorphisms associated with aggressive disease development in a human GWAS cohort consisting of 1,172 prostate cancer patients. This study is the first example of using a systems genetics approach to successfully identify novel susceptibility genes for aggressive prostate cancer. Such approaches will facilitate the identification of novel germline factors driving aggressive disease susceptibility and allow for new insights into these deadly forms of prostate cancer.     

Transformation of ferulic acid to 4-vinyl guaiacol as a major metabolite: a microbial approach

The majority of the flavours and fragrances used worldwide are produced by chemical synthesis at low price. However, consumers prefer natural compounds because of increasing health and nutrition awareness in routine life. Hence, biotransformation is an alternative process to produce natural aroma compounds. Microorganisms have been gradually used more to produce natural aroma compounds with various applications in food, agriculture and pharmaceutical industries. This paper reviews the role of microorganisms in the transformation of ferulic acid to 4-vinyl guaiacol. The microbial processes based on biocatalytic method are discussed in terms of their advantages over chemical synthesis, plant cell cultures and enzyme catalyzed reactions. Thus, the transformation of ferulic acid by microorganisms could have possible use in food, pharmaceutical industry and become an increasingly important platform for the production of natural aroma compounds.     

Production of natural value-added compounds: an insight into the eugenol biotransformation pathway

During the past few years, the production of natural value-added compounds from microbial sources has gained tremendous importance. Due to an increase in consumer demand for natural products, various food and pharmaceutical industries are continuously in search of novel metabolites obtained from microbial biotransformation. The exploitation of microbial biosynthetic pathways is both feasible and cost effective in the production of natural compounds. The environmentally compatible nature of these products is one major reason for their increasing demand. Novel approaches for natural product biogeneration will take advantage of the current studies on biotechnology, biochemical pathways and microbiology. The interest of the scientific community has shifted toward the use of microbial bioconversion for the production of valuable compounds from natural substrates. The present review focuses on eugenol biotransformation by microorganisms resulting in the formation of various value-added products such as ferulic acid, coniferyl alcohol, vanillin and vanillic acid.     

Governing stem cell therapy in India: regulatory vacuum or jurisdictional ambiguity?

Stem cell treatments are being offered in Indian clinics although preclinical evidence of their efficacy and safety is lacking. This is attributed to a governance vacuum created by the lack of legally binding research guidelines. By contrast, this paper highlights jurisdictional ambiguities arising from trying to regulate stem cell therapy under the auspices of research guidelines when treatments are offered in a private market disconnected from clinical trials. While statutory laws have been strengthened in 2014, prospects for their implementation remain weak, given embedded challenges of putting healthcare laws and professional codes into practice. Finally, attending to the capacities of consumer law and civil society activism to remedy the problem of unregulated treatments, the paper finds that the very definition of a governance vacuum needs to be reframed to clarify whose rights to health care are threatened by the proliferation of commercial treatments and individualized negligence-based remedies for grievances.