An Assessment of Relationships between Actual and Perceived Caregiving Demand and Negative Impact Measures

Previous research has focused on the time spent and the tasks involved in caregiving and on care recipient characteristics in measuring caregiving demand and predicting negative impacts (Aneshensel, Pearlin, & Schuler, 1993; McKinlay, Crawford, & Tennstedt, 1995; Stull, Bowman, & Smerglia, 1994). However, the nature of the relationship between objectively measured caregiving demand and negative impacts for caregivers remains unclear. Researchers now note that assessing caregivers\u27 perceptions of demand may be key in understanding negative impacts (Coverman, 1989; Litvin, Albert, Brody, & Hoffman, 1995; Loomis & Booth, 1995). This study assessed relationships between objective and subjective reports of caregiving demand and negative impact measures. Information about subgroups of caregivers and caregiving demand and negative impacts was also made available. The design involved a cross-sectional survey methodology that utilized means tests and correlation statistics for analysis. The findings indicate that caregiver\u27s subjective reports of demand are positively associated with negative impacts while their objective reports are inversely associated with negative impacts. The findings also show that some subgroups of caregivers experience greater negative impacts than others. Implications for practice and policy are discussed

Residues at the Active Site of the Esterase 2 fromAlicyclobacillus acidocaldarius Involved in Substrate Specificity and Catalytic Activity at High Temperature

The recently solved three-dimensional structure of the thermophilic esterase 2 from Alicyclobacillus acidocaldarius allowed us to have a snapshot of an enzyme-sulfonate complex, which mimics the second stage of the catalytic reaction, namely the covalent acyl-enzyme intermediate. The aim of this work was to design, by structure-aided analysis and to generate by site-directed and saturation mutagenesis, EST2 variants with changed substrate specificity in the direction of preference for monoacylesters whose acyl-chain length is greater than eight carbon atoms. Positions 211 and 215 of the polypeptide chain were chosen to introduce mutations. Among five variants with single and double amino acid substitutions, three were obtained, M211S, R215L, and M211S/R215L, that changed the catalytic efficiency profile in the desired direction. Kinetic characterization of mutants and wild type showed that this change was achieved by an increase in k(cat) and a decrease in K(m) values with respect to the parental enzyme. The M211S/R215L specificity constant for p-nitrophenyl decanoate substrate was 6-fold higher than the wild type. However, variants M211T, M211S, and M211V showed strikingly increased activity as well as maximal activity with monoacylesters with four carbon atoms in the acyl chain, compared with the wild type. In the case of mutant M211T, the k(cat) for p-nitrophenyl butanoate was 2.4-fold higher. Overall, depending on the variant and on the substrate, we observed improved catalytic activity at 70 degrees C with respect to the wild type, which was a somewhat unexpected result for an enzyme with already high k(cat) values at high temperature. In addition, variants with altered specificity toward the acyl-chain length were obtained. The results were interpreted in the context of the EST2 three-dimensional structure and a proposed catalytic mechanism in which k(cat), e.g. the limiting step of the reaction, was dependent on the acyl chain length of the ester substrate

A Novel Aspartyl Proteinase from Apocrine Epithelia and Breast Tumors

Abstract GCDFP-15 (gross cysticdisease fluid protein,15 kDa) is a secretory marker of apocrine differentiation in breast carcinoma. In human breast cancer cell lines, gene expression is regulated by hormones, including androgens and prolactin. The protein is also known under different names in different body fluids such as gp17 in seminal plasma. GCDFP-15/gp17 is a ligand of CD4 and is a potent inhibitor of T-cell apoptosis induced by sequential CD4/T-cell receptor triggering. We now report that GCDFP-15/gp17 is a protease exhibiting structural properties relating it to the aspartyl proteinase superfamily. Unexpectedly, GCDFP-15/gp17 appears to be related to the retroviral members rather than to the known cellular members of this class. Site-specific mutagenesis of Asp22 (predicted to be catalytically important for the active site) and pepstatin A inhibition confirmed that the protein is an aspartic-type protease. We also show that, among the substrates tested, GCDFP-15/gp17 is specific for fibronectin. The study of GCDFP-15/gp17-mediated proteolysis may provide a handle to understand phenomena as diverse as mammary tumor progression and fertilization

The Aes protein and the monomeric alpha-galactosidase from Escherichia coli form a non-covalent complex. Implications for the regulation of carbohydrate metabolism.

Aes, a 36-kDa acetylesterase fromEscherichia coli, belongs to the hormone-sensitive lipase family, and it is involved in the regulation of MalT, the transcriptional activator of the maltose regulon. The activity of MalT is depressed through a direct protein-protein interaction with Aes. Although the effect is clear-cut, the meaning of this interaction and the conditions that trigger it still remain elusive. To perform a comparative thermodynamic study between the mesophilic Aes protein and two homologous thermostable enzymes, Aes was overexpressed in E. coli and purified. At the last step of the purification procedure the enzyme was eluted from a Mono Q HR 5/5 column as a major form migrating, anomalously, at 56 kDa on a calibrated Superdex 75 column. A minor peak that contains the Aes protein and a polypeptide of 50 kDa was also detected. By a combined analysis of size-exclusion chromatography and surface-enhanced laser desorption ionization-time of flight mass spectrometry, it was possible to demonstrate the presence in this peak of a stable 87-kDa complex, containing the Aes protein itself and the 50-kDa polypeptide in a 1:1 ratio. The homodimeric molecular species of Aes and of the 50-kDa polypeptide were also detected. The esterase activity associated with the 87-kDa complex, when assayed with p-nitrophenyl butanoate as substrate, proved 6-fold higher than the activity of the major Aes form of 56 kDa. Amino-terminal sequencing highlighted that the 50-kDa partner of Aes in the complex was the α-galactosidase from E. coli. TheE. coli cells harboring plasmid pT7-SCII-aesand, therefore, expressing Aes were hampered in their growth on a minimal medium containing raffinose as a sole carbon source. Because α-galactosidase is involved in the metabolism of raffinose, the above findings suggest a potential role of Aes in the regulation of carbohydrate metabolism in E. coli

SSo

Previously, we reported from the Sulfolobus solfataricus open reading frame (ORF) SSO2517 the cloning, overexpression and characterization of an esterase belonging to the hormone-sensitive lipase (HSL) family and apparently having a deletion at the N-terminus, which we named SsoNΔ. Searching the recently reported Sulfolobus acidocaldarius genome by sequence alignment, using SSO2517 as a query, allowed identity of a putative esterase (ORF SAC1105) sharing high sequence similarity (82%) with SSO2517. This esterase displays an N-terminus and total length similar to other known esterases of the HSL family. Analysis of the upstream DNA sequence of SS02517 revealed the possibility of expressing a longer version of the protein with an extended N-terminus; however, no clear translation signal consistent with a longer protein version was detected. This new version of SSO2517 was cloned, over-expressed, purified and characterized. The resulting protein, named SsoNΔlong, was 15-fold more active with the substrate p-nitrophenyl hexanoate than SsoNΔ. Furthermore, SsoNΔlong and SsoNΔ displayed different substrate specificities for triacylglycerols. These results and the phylogenetic relationship between S. solfataricus and S. acidocaldarius suggest a common origin of SSO2517 and SAC1105 from an ancestral gene, followed by divergent evolution. Alternatively, a yet-to-be discovered mechanism of translation that directs the expression of SsoNΔlong under specific metabolic conditions could be hypothesized

A Substrate-induced Switch in the Reaction Mechanism of a Thermophilic Esterase KINETIC EVIDENCES AND STRUCTURAL BASIS

The reaction mechanism of the esterase 2 (EST2) from Alicyclobacillus acidocaldarius was studied at the kinetic and structural level to shed light on the mechanism of activity and substrate specificity increase previously observed in its double mutant M211S/R215L. In particular, the values of kinetic constants (k1, k(-1), k2, and k3) along with activation energies (E1, E(-1), E2, and E3) were measured for wild type and mutant enzyme. The previously suggested substrate-induced switch in the reaction mechanism from kcat=k3 with a short acyl chain substrate (p-nitrophenyl hexanoate) to kcat=k2 with a long acyl chain substrate (p-nitrophenyl dodecanoate) was validated. The inhibition afforded by an irreversible inhibitor (1-hexadecanesulfonyl chloride), structurally related to p-nitrophenyl dodecanoate, was studied by kinetic analysis. Moreover the three-dimensional structure of the double mutant bound to this inhibitor was determined, providing essential information on the enzyme mechanism. In fact, structural analysis explained the observed substrate-induced switch because of an inversion in the binding mode of the long acyl chain derivatives with respect to the acyl- and alcohol-binding sites

The Dark Internet: An Exploration of Culture and User Experience

Our research sought to investigate the culture of the Dark Internet through a combination of cultural analysis and experiential learning. We split our research into three major portions: analysis of culture of the Dark Internet through the way it is viewed by various media outlets and on the Dark Internet itself; how the culture of the Dark Internet reacts in times of crises; and a comparison of the experience of the users of Dark Internet marketplaces versus users of traditional internet marketplaces. Our cultural analysis was accomplished through the use of textual coding; by coding the articles, forums, and pages that we were gathering, we were able to find and observe key commonalities in behavior and communication among the sources. We also went through the process of purchasing goods from both Dark Internet marketplaces and traditional internet marketplaces allowing us to compare the experiences in a variety of ways including: ease of access, ease of purchase, delivery time, etc. This research aims to provide further insight into the nature of the Dark Internet and open the way for future research into this ever-changing culture

Prots: A fragment based protein thermo‐stability potential

Designing proteins with enhanced thermo‐stability has been a main focus of protein engineering because of its theoretical and practical significance. Despite extensive studies in the past years, a general strategy for stabilizing proteins still remains elusive. Thus effective and robust computational algorithms for designing thermo‐stable proteins are in critical demand. Here we report PROTS, a sequential and structural four‐residue fragment based protein thermo‐stability potential. PROTS is derived from a nonredundant representative collection of thousands of thermophilic and mesophilic protein structures and a large set of point mutations with experimentally determined changes of melting temperatures. To the best of our knowledge, PROTS is the first protein stability predictor based on integrated analysis and mining of these two types of data. Besides conventional cross validation and blind testing, we introduce hypothetical reverse mutations as a means of testing the robustness of protein thermo‐stability predictors. In all tests, PROTS demonstrates the ability to reliably predict mutation induced thermo‐stability changes as well as classify thermophilic and mesophilic proteins. In addition, this white‐box predictor allows easy interpretation of the factors that influence mutation induced protein stability changes at the residue level. Proteins 2012; © 2011 Wiley Periodicals, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/89526/1/23163_ftp.pd

Carboxylic ester hydrolases from hyperthermophiles

Carboxylic ester hydrolyzing enzymes constitute a large group of enzymes that are able to catalyze the hydrolysis, synthesis or transesterification of an ester bond. They can be found in all three domains of life, including the group of hyperthermophilic bacteria and archaea. Esterases from the latter group often exhibit a high intrinsic stability, which makes them of interest them for various biotechnological applications. In this review, we aim to give an overview of all characterized carboxylic ester hydrolases from hyperthermophilic microorganisms and provide details on their substrate specificity, kinetics, optimal catalytic conditions, and stability. Approaches for the discovery of new carboxylic ester hydrolases are described. Special attention is given to the currently characterized hyperthermophilic enzymes with respect to their biochemical properties, 3D structure, and classification