Structure of very early protein folding intermediates: new insights through a variant of hydrogen exchange labelling

BACKGROUND: Hydrogen exchange labelling has been a key method in characterizing the structure of transient folding intermediates. In studies of several proteins, however, there has been clear spectroscopic evidence for partial folding of some kind at very early times, before any protection from exchange was measurable. These results, presumably a consequence of limited stability of specific backbone interactions, have made it difficult to assess the extent of native-like folding in the very early intermediates. We have used a variant of the labelling method to investigate marginally stable structures formed within the first few milliseconds of refolding of two such proteins, hen lysozyme and ubiquitin. RESULTS: In lysozyme, population of a subset of native-like secondary structures on this timescale is revealed, thus reconciling the exchange behaviour with circular dichroism measurements and confirming the significance of the rapidly formed embryonic structure as a foundation for the subsequent folding pathway. In the case of ubiquitin, by contrast, no significantly protective structure was detectable, suggesting that here secondary structural elements can be populated only marginally ahead of the major cooperative folding event; this was also supported by stopped-flow circular dichroism measurements. CONCLUSIONS: The hydrogen exchange approach can be extended to probe the formation of native-like structure formed in very early folding intermediates, even when the stability of specific interactions is marginal. In the case of lysozyme, this has provided a new window on an early stage of organization of the alpha-helical domain.     

Crystal structures of Escherichia coli and Salmonella typhimurium 3-isopropylmalate dehydrogenase and comparison with their thermophilic counterpart from Thermus thermophilus

The basis of protein stability has been investigated by the structural comparison of themophilic enzymes with their mesophilic counterparts. A number of characteristics have been found that can contribute to the stabilization of thermophilic proteins, but no one is uniquely capable of imparting thermostability. The crystal structure of 3-isopropylmalate dehydrogenase (IPMDH) from the mesophiles Escherichia coli and Salmonella typhimurium have been determined by the method of molecular replacement using the known structure of the homologous Thermus thermophilus enzyme. The structure of the E. coli enzyme was refined at a resolution of 2.1 A to an R-factor of 17.3%, that of the S. typhimurium enzyme at 1.7 A resolution to an R-factor of 19.8%. The three structures were compared to elucidate the basis of the higher thermostability of the T. thermophilus enzyme. A mutant that created a cavity in the hydrophobic core of the thermophilic enzyme was designed to investigate the importance of packing density for thermostability. The structure of this mutant was analyzed. The main stabilizing features in the thermophilic enzyme are an increased number of salt bridges, additional hydrogen bonds, a proportionately larger and more hydrophobic subunit interface, shortened N and C termini and a larger number of proline residues. The mutation in the hydrophobic core of T. thermophilus IPMDH resulted in a cavity of 32 A3, but no significant effect on the activity and thermostability of the mutant was observed.     

Bestimmung von Haselnußprotein (Corylin) mit Hilfe der Elektroimmundiffusion nach Laurell

European Food Research and Technology - Bei Bestimmungen des Proteingehalts von Haselnüssen in verschiedenen Handelsprodukten wie Nuß-Nougatcremes and Nußmus mit der...     

Cost-effectiveness of self-help smokeless tobacco cessation programs.

This study assessed the cost-effectiveness of two low-intensity programs for quitting smokeless tobacco, based on results of a randomized trial with 1,069 volunteer participants. Cost data were collected for two levels of intervention: manual only (a self-help manual) and assisted self-help (the manual plus a videotape and two supportive phone calls from tobacco cessation counselors). Incremental cost-effectiveness ratios were calculated for assisted self-help vs. quitting on one's own, using the manual-only quit rate and data from another study as alternative proxies for no intervention. A threshold analysis was conducted to determine the spontaneous quit rate at which the manual-only intervention becomes more cost-effective than assisted self-help. The cost to provide and receive the assisted self-help intervention averaged US $56 per participant vs. $20 for the manual-only intervention (societal perspective, Year 2000 dollars). Estimates for incremental cost per quit for the assisted self-help intervention ranged from $922 to $1,758, depending on the proxy used for no intervention. The manual-only intervention was more cost-effective than assisted self-help if quitting among motivated chewers who do not receive treatment does not exceed 3.4%. Support from a wife or partner added little cost to a quit attempt for male chewers ($3-$4). Providing a manual, video, and brief phone counseling to smokeless tobacco users who want to quit is a reasonable use of health care resources. The self-help quitting guide also may be a cost-effective treatment, but it remains to be demonstrated whether it is more effective than quitting on one's own.     

New HPLC Method to Determine Ethyl Carbamate in Alcoholic Beverages Using Fluorescence Detection

ABSTRACT: A new methodology to the quantification of ethyl carbamate (EC) has been developed. This method allows the analysis by HPLC of ethyl carbamate in samples of wine, fortified wine, and wine brandy, by a pre-column derivatization with 9-xanthydrol, and fluorescence detection. This does not require previous sample extraction or concentration. The method presents an average recovery of 96% among samples studied, a detection limit of 4.2μg/L, and an average intermediate precision of 6.3%. The comparison of the results obtained for EC analysis on the same wine brandy samples by GC/MS and HPLC are statistically indistinguishable with 97.5% probability. The results of the analysis of 42 samples are presented.     

Review of Counseling and psychotherapy of work dysfunctions.

Reviews the book, Counseling and psychotherapy of work dysfunctions by Rodney L. Lowman (see record 1993-98413-000). Dr. Lowman's book on work dysfunctions serves as a seminal product for further understanding a much-neglected component of the lives of the many men and women who seek psychological assistance in working through issues around their employment. The book conveys in a well-written, sometimes humorous, style that "work is a natural part of living, and the issues it raises are important parts of character." The overall impression is that of a well-thought-out and most readable book. There is no doubt in this reviewer's mind that it will become essential reading for psychologists, social workers, family therapists, and graduate students who are becoming increasingly involved in this field of study. What this reviewer found very helpful was the clarity of presentation of the assessment techniques as well as the amply illustrated intervention modules that may be utilized. To this reviewer, there is no doubt that this is one area for psychologists' contributions in the future--the work area. Lowman's book gives us basic material from which to continue to build our profession in a new and exciting direction. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Modification of the calcium and calmodulin sensitivity of the type I adenylyl cyclase by mutagenesis of its calmodulin binding domain

The type I adenylyl cyclase is directly stimulated by Ca2+ and calmodulin in vitro, and the enzyme is also stimulated by increases in intracellular Ca2+ in vivo. Ca2+ stimulation of the enzyme in vivo may be due to direct interactions of the enzyme with Ca2+ and calmodulin or to an indirect mechanism involving stimulation of the enzyme by Ca(2+)-activated protein kinases. In this study, we have made several point mutations within the calmodulin binding domain to determine if the Ca2+ sensitivity of the enzyme can be modified by mutagenesis. The catalytic activities of the mutant enzymes were comparable to wild type type I adenylyl cyclase. Substitution of Cys-507 with Ser-507 did not have significant effects on the calmodulin or Ca2+ sensitivity of the enzyme. However, replacement of Lys-504 with Asp caused a 4-fold decrease in sensitivity to Ca2+. Ca2+ and calmodulin stimulation were abolished by substitution of Phe-503 with Arg-503. Stimulation of type I adenylyl cyclase activity in vivo by intracellular Ca2+ was also greatly diminished with the Arg-503 mutant indicating that Ca2+ stimulation of the enzyme in vivo is due primarily to direct interactions with calmodulin and Ca2+. These data demonstrate that the Ca2+ sensitivity of this enzyme can be modulated by point mutagenesis within the putative calmodulin binding domain and indicate that the enzyme can be directly regulated by Ca2+ and calmodulin in vivo.