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PubMed2001

Evolution of alpha-amylases: architectural features and key residues in the stabilization of the (beta/alpha)(8) scaffold.

We provide a comprehensive analysis of the current enzymes with alpha-amylase activity (AAMYs) that belong to family 13 glycoside hydrolase (GH-13; 144 Archaea, Bacteria, and Eukaryota sequences from 87 different species). This study aims to further knowledge of the evolutionary molecular relationships among the sequences of their A and B domains with special emphasis on the correlation between what is observed in the structures and protein evolution. Multialignments for the A domain distinguish two clusters for sequences from Archaea organisms, eight for sequences from Bacteria organisms, and three for sequences from Eukaryota organisms. The clusters for Bacteria do not follow any strict taxonomic pathway; in fact, they are rather scattered. When we compared the A domains of sequences belonging to different kingdoms, we found that various pairs of clusters were significantly similar. Using either sequence similarity with crystallized structures or secondary-structure prediction methods, we identified in all AAMYs the eight putative beta-strands that constitute the beta-sheet in the TIM barrel of the A domain and studied the packing in its interior. We also discovered a "hidden homology" in the TIM barrel, an invariant Gly located upstream in the sequence before the conserved Asp in beta-strand 3. This Gly precedes an alpha-helix and is actively involved in capping its N-terminal end with a capping box. In all cases, a Schellman motif caps the C-terminal end of this helix.

Cluster Analysis↗
PubMed2005

Analyzing the degradation of PERIOD protein by the ubiquitin-proteasome pathway in cultured Drosophila cells.

Time-of-day specific changes in the levels of key clock proteins are critical for the normal progression of circadian pacemakers. Evidence indicates a major role for the ubiquitin-proteasome pathway (UPP) in the temporal control of clock protein stability. A conserved feature of animal clocks is that PERIOD (PER) proteins undergo daily rhythms in abundance. The stability of PER proteins is regulated by differential phosphorylation, whereby hyperphosphorylated isoforms are selectively degraded by the UPP. The use of transformed stable cell lines has been instrumental in advancing our understanding of the mechanisms underlying the intersection of the UPP and clock protein metabolism. This article describes several standard methodologies used to analyze the UPP-mediated degradation of Drosophila PER (dPER) expressed in cultured Drosophila cells (Ko et al., 2002). Although this article focuses on dPER as a case study, general issues are discussed that should have broad application to other cell culture-based systems and clock proteins. For example, we discuss (i) advantages?disadvantages of cultured cells, (ii) types of expression vectors and "peptide tags" for recombinant protein production and surveillance, and (iii) standard approaches to determine whether a protein of interest is modified by ubiquitin and degraded by the proteasome. Prior to the discussion on methodologies, the article provides a brief overview of diverse strategies by which clock proteins in a variety of systems are regulated by the UPP.

Animals↗
PubMed2002

Characterization of a Vero cell-adapted virulent strain of enterovirus 71 suitable for use as a vaccine candidate.

Enterovirus 71 (EV71) is a neurotrophic virus that causes seasonal morbidity and mortality in children throughout the world with increasing frequency in recent years. Because of the lack of an effective antiviral agent, primary prevention, including the development of effective vaccines, is a top priority in terms of control strategies. Poliovirus vaccine technology, both live attenuated and inactivated, killed virus vaccines, can be adopted for use with EV71 because of their relatedness. In this study, we have characterized a laboratory-adapted EV71 strain, YN3-4a, which exhibits different characteristics from those of its parent isolate, neu, in having a rapid growth rate in Vero cells, a larger plaque size, and a lower LD(50) in newborn mice. The YN3-4a can be produced at a high viral titer of up to 10(10) tissue culture infective dose (TCID(50)) when grown in Vero cells, an approved substrate for virus vaccine production. Mouse antiserum raised against YN3-4a can neutralize a broad range of strains of EV71 isolated at different times from a variety of geographic regions. On passage in Vero cells, YN3-4a remained genetically and phenotypically stable. Many of the above-described features, such as high viral yield, strong immunogenicity, broad-based antigenic coverage, and passage stability, are desirable features in a prototype virus for the development of an inactivated viral vaccine.

Animals↗
PubMed2005

Structural properties of promoters: similarities and differences between prokaryotes and eukaryotes.

During the process of transcription, RNA polymerase can exactly locate a promoter sequence in the complex maze of a genome. Several experimental studies and computational analyses have shown that the promoter sequences apparently possess some special properties, such as unusual DNA structures and low stability, which make them distinct from the rest of the genome. But most of these studies have been carried out on a particular set of promoter sequences or on promoter sequences from similar organisms. To examine whether the promoters from a wide variety of organisms share these special properties, we have carried out an analysis of sets of promoters from bacteria, vertebrates and plants. These promoters were analyzed with respect to the prediction of three different properties, such as DNA curvature, bendability and stability, which are relevant to transcription. All the promoter sequences are predicted to share certain features, such as stability and bendability profiles, but there are significant differences in DNA curvature profiles and nucleotide composition between the different organisms. These similarities and differences are correlated with some of the known facts about transcription process in the promoters from the three groups of organisms.

Animals↗
PubMed1999

Footwear and postural stability in older people.

Accidental falls in the older population are common and often result in serious injury. Although a number of factors have been recognized as risk factors for falling, the effect of footwear on postural stability is often overlooked. This article reviews the literature on the effect of various footwear features on postural stability and suggests areas that warrant further investigation.

Accidental Falls↗
PubMed1998

Two-year stability of borderline personality measures.

Two-year stability coefficients were computed for several measures of borderline personality disorder within a nonclinical sample (n = 65) that included individuals with significant borderline features. Overall, the stability coefficients were modest (r ranging from .28 to .62; intraclass correlations ranging from .26 to .62). Stability values for each of the self-report measures under study were higher than those for the interview-based measure of BPD features, and, in some cases, these values varied as a function of the prototypicality of the subsamples examined. Analyses conducted to identify moderator effects provided no evidence that the stability of BPD scores was moderated by change in personal distress level; however, changes in BPD self-report scores were related to changes in level of negative affectivity.

Adult↗
PubMed2005

Stability of microfabricated high aspect ratio structures in poly(dimethylsiloxane).

The stability of structures microfabricated in soft elastomeric polymers is an important concern in most applications that use these structures. Although relevant for several applications, the collapse to the ground of high aspect ratio structures (ground collapse) is still poorly understood. The stability of soft microfabricated high aspect ratio structures versus ground collapse was experimentally assessed, and a new model of ground collapse involving adhesion was developed. Sets of posts with diameters from 0.36 to 2.29 microm were fabricated in poly(dimethylsiloxane) and tested in air or immersed in water and ethanol to change the work of adhesion. The critical aspect ratio (the highest length-to-width ratio for which a post is not at risk of collapsing) was determined as a function of the diameter. The critical aspect ratio in air ranged from 2 to 4 and increased with the diameter. Work of adhesion was found to be determinant for and inversely correlated to stability. These results highlight the role played by adhesion and offer the possibility of improving stability by reducing the work of adhesion. The ground collapse model developed accounted for the main features of structure stability. The results indicate that ground collapse can be a limiting factor in the design of soft polymer structures.

Journal Article↗
PubMed2001

Contribution of engineered electrostatic interactions to the stability of cytosolic malate dehydrogenase.

Protein engineering is a promising tool to obtain stable proteins. Comparison between homologous thermophilic and mesophilic enzymes from a given structural family can reveal structural features responsible for the enhanced stability of thermophilic proteins. Structures from pig heart cytosolic and Thermus flavus malate dehydrogenases (cMDH, Tf MDH), two proteins showing a 55% sequence homology, were compared with the aim of increasing cMDH stability using features from the Thermus flavus enzyme. Three potential salt bridges from Tf MDH were selected on the basis of their location in the protein (surface R176-D200, inter-subunit E57-K168 and intrasubunit R149-E275) and implemented on cMDH using site-directed mutagenesis. Mutants containing E275 were not produced in any detectable amount, which shows that the energy penalty of introducing a charge imbalance in a region that was not exposed to solvent was too unfavourable to allow proper folding of the protein. The salt bridge R149-E275, if formed, would not enhance stability enough to overcome this effect. The remaining mutants were expressed and active and no differences from wild-type other than stability were found. Of the mutants assayed, Q57E/L168K led to a stability increase of 0.4 kcal/mol, as determined by either guanidinium chloride denaturalization or thermal inactivation experiments. This results in a 15 degrees C shift in the optimal temperature, thus confirming that the inter-subunit salt bridge initially present in the T.flavus enzyme was formed in the cMDH structure and that the extra energy obtained is transformed into an increase in protein stability. These results indicate that the use of structural features of thermophilic enzymes, revealed by a detailed comparison of three-dimensional structures, is a valid strategy to improve the stability of mesophilic malate dehydrogenases.

Catalysis↗
PubMed1989

mRNA stabilizing signals encoded in the genome of the bacteriophage phi x174.

In Escherichia coli cells infected with bacteriophage phi x174, mRNAs initiated by promoters PB and PD terminate after genes J, F, G, or H (TJ, TF, TG, or TH). These RNAs are relatively stable and contain mRNA-stabilizing signals at their 3' ends. These signals were cloned after gene D of phi x174 in an expression vector plasmid. The cloned signals stabilize mRNA of the upstream gene D and the stabilized mRNA is translationally functional. When these signals are inserted in reverse, no stabilizing effect on mRNA is observed indicating that the correct sequences at the 3' ends of transcripts determine their stability. When a stabilizing signal (+) and a mutated stabilizing signal (-) which has reduced stabilizing activity are tandemly inserted after gene D, two sets of 3' termini of the transcript are observed indicating that both signals also function as terminators. The amount of gpD synthesized from these constructs varies depending upon the relative positions of the (+) or (-) signals after gene D. The stabilizing function seems to act by preventing mRNA degradation from the 3' to 5' direction. Several common features of these stabilizers are described.

Bacteriophage phi X 174↗
PubMed1998

mRNA stabilization by the ompA 5' untranslated region: two protective elements hinder distinct pathways for mRNA degradation.

The 5' untranslated region (UTR) of the long-lived Escherichia coli ompA transcript functions as an mRNA stabilizer that can prolong the cytoplasmic lifetimes of a variety of messages to which it is fused. Previous studies have identified two domains of this 5' UTR that together are responsible for its stabilizing effect. One is a 5'-terminal stem-loop. The other is a single-stranded RNA segment (ss2) that contains a ribosome binding site highly complementary to 16S ribosomal RNA. Here we report a detailed investigation of the function of these two stabilizing elements. Our data indicate that mRNA protection by a 5' stem-loop requires no sequence features or thermodynamic stability beyond the minimum necessary for stem-loop formation. Stabilization by ss2 appears to result not from a high frequency of translation initiation, but rather from a high degree of occupancy of this 5' UTR segment by bound ribosomes. Although close spacing of translating ribosomes is not critical for message stabilization by the ompA 5' UTR, mRNA longevity does require the periodic passage of ribosomes through the protein-coding region. Unlike bound ribosomes, which hinder mRNA cleavage by RNase E, the 5' stem-loop appears to impede degradation of ompA mRNA via a distinct pathway that is RNase E-independent. These findings imply that the ompA 5' UTR prolongs mRNA longevity by impeding multiple pathways for mRNA degradation.

Bacterial Outer Membrane Proteins↗
PubMedDate not supplied

Inclusion of ionic interactions in force field calculations of charged biomolecules--DNA structural transitions.

The potential of mean force (PMF) approach for treating polyion-diffuse ionic cloud interactions [D. M. Soumpasis (1984) Proceedings of the National Academy of Sciences USA 81, 5116-5120] has been combined with the AMBER force field describing intramolecular interactions. The resultant generalized AMBER-PMF force field enables one to treat the conformational stabilities and structural transitions of charged biomolecules in aqueous electrolytes more realistically. For example, we have used it to calculate the relative stabilities of the B and Z conformations of d(C-G)6, and the B and heteronomous (H) conformations of dA12.dT12, as a function of salt concentration. In the case of d(C-G)6, the predicted B-ZI transition occurs at 2.4M and is essentially driven by the phosphate-diffuse ionic cloud interactions alone as suggested by the results of earlier PMF calculations. The ZII conformer is less stable than the B form under all conditions. It is found that the helical parameters of the refined B and Z structures change with salt concentration. For example, the helical rise of B-DNA increases about 10% and the twist angle decreases by the same amount above 1M NaCl. In the range of 0.01-0.3M NaCl, the H form of dA12.dT12 is found to be more stable than the B form and its stability increases with increasing salt concentration. The computed greater relative stability of the H conformation is likely due to noninclusion of the free energy contribution from the spine of hydration, a feature presumed to stabilize the B form of this sequence.

Biopolymers↗
PubMed1992

A model of glucocorticoid receptor unfolding and stabilization by a heat shock protein complex.

It has recently been reported that incubation of avian progesterone receptors, mouse glucocorticoid receptors, or the viral tyrosine kinase pp60src with rabbit reticulocyte lysate reconstitutes their association with the 90 kDa heat shock protein, hsp90. The reassociation is thought to require unfolding of the steroid receptor or pp60src before hsp90 can bind. The unfoldase activity may be provided by hsp70, which is also present in the reconstituted receptor heterocomplex. In this paper we review evidence that hsp70 and hsp90 are associated in cytosolic heterocomplexes that contain a limited number of other proteins. From an analysis of known receptor-hsp interactions and a predicted direct interaction between hsp90 and hsp70 we have developed an admittedly very speculative model of glucocorticoid receptor unfolding and stabilization. One important feature of the model is that the receptor becomes attached to a heat shock protein heterocomplex rather than undergoing independent unfolding and stabilization events. The model requires that hsp70 and hsp90 bind directly to the receptor at independent sites. Importantly, the model accommodates the stoichiometry of 2 hsp90 per 1 molecule of receptor that has been assayed in the untransformed GR heterocomplex in cytosols prepared from hormone-free cells.

Animals↗
PubMed1994

Improving detection of coronary morphological features from digital angiograms. Effect of stenosis-stabilized display.

BACKGROUND: We have developed a digital display method that stabilizes the motion of a stenosis in sequential frames of a coronary angiogram, allowing it to be scrutinized at high display frame rates. The purpose of this study was to determine whether this technique improves visual detection of low-contrast luminal morphological features. METHODS AND RESULTS: An observer detection study was conducted using computer-simulated arterial segments containing known target features, inserted into clinical digital coronary angiograms. Four observers performed a forced-choice detection of a simulated filling defect in each of 320 angiograms using the conventional and stenosis-stabilized dynamic displays (at 7.5, 15, and 32 frames per second) and a single-frame static display (total of 8960 detections). In a second simulated clinical task, three observers detected a bridging stenotic lumen in 600 angiograms using the two displays (3600 detections). In a third experiment, two angiographers rated the likelihood of intraluminal thrombus in 89 right coronary digital angiograms by consensus reading with both dynamic displays. Detectability of the simulated filling defect was similar for both dynamic display methods at 7.5 frames per second (averaging twice that for static images). As display rate was increased to 32 frames per second, detectability for the conventional display declined, whereas the stabilized display detectability increased for all observers (P < .05). On average, stabilization allowed detection of filling defects equivalent to a 71% increase in feature contrast. Response time for the conventional display averaged 12.9 +/- 4.7 seconds. For the stenosis-stabilized display, response time fell with increased frame rate (P < .05) to 4.9 +/- 1.2 seconds at 32 Hz, similar to the time for static images (4.6 +/- 0.8 seconds). The detectability of the bridging stenotic lumen was increased by 62% with the stabilization compared with conventional dynamic display (P < .00001). Consensus reading of coronary angiograms showed differences between the two dynamic display methods (kappa = 0.11) that may be explained by an improvement in observer uncertainty. A rating of definite for thrombus present or absent was more frequent with the stabilized display (39% versus 15%, P < .0001). CONCLUSIONS: These data suggest that stabilized display of coronary angiograms significantly increases detectability, reduces the time required for detection, and improves observer uncertainty for the presence of small luminal morphological features. The method of angiographic display may thus have an impact on clinical coronary angiographic interpretation.

Coronary Angiography↗
PubMed1995

Understanding and increasing protein stability.

This review surveys the processes leading to loss of protein function and the types of molecular interaction that help stabilize proteins. It considers the effects of organic solvents on stability and the special features of thermophilic proteins. The deliberate manipulation of stability by protein engineering is discussed using the enzyme subtilisin as example. Both random and rational mutations of this protein have led to variants with greatly improved tolerances of high temperatures and organic solvents. One can also use chemical modification to modify protein stability and some of the main approaches are reviewed. The chemical and genetic strategies are complementary and have been combined to stabilize cytochrome c by metal-mediated cross-linking following site-specific mutagenesis. The article concludes by summarizing the beneficial effects of certain additives on protein stability.

Enzyme Stability↗
PubMed1986

Embryonic avian cornea contains layers of collagen with greater than average stability.

A unique morphological feature of the embryonic avian cornea is the uniformity of its complement of striated collagen fibrils, each of which has a diameter of 25 nm. We have asked whether this apparent morphological uniformity also reflects an inherent uniformity of the structural and physical properties of these fibrils. For this we have examined the in situ thermal stability of the type I collagen within these fibrils. Corneal tissue sections were reacted at progressively higher temperatures with conformation-dependent monoclonal antibodies directed against the triple-helical domain of the type I collagen molecule. These studies show that the cornea contains layers of collagen fibrils with greater than average stability. The two most prominent of these extend uninterrupted across the entire width of the cornea, and then appear to insert into thick bundles of scleral collagen, which in turn appear to insert into the scleral ossicles, a ring of bony plates which circumscribe the sclera of the avian eye. Once formed, the bands may act to stabilize the shape of the cornea or, conversely, to alter it during accommodation.

Animals↗
PubMed1992

Breast masses. Mammographic and sonographic evaluation.

Asymmetric breast tissue can nearly always be distinguished from a true mass by means of mammographic evaluation. Stellate masses from early invasive breast cancer are often extremely subtle so that optimal technique and meticulous interpretation are essential. Benign stellate masses such as post-biopsy scarring and fat necrosis frequently have a characteristic appearance. A radial scar is usually indistinguishable from malignancy on the mammogram. Nearly all circumscribed masses are benign and are usually cysts, fibroadenomas, or intramammary lymph nodes. A few circumscribed masses represent in situ or invasive carcinoma or both. Characteristics that may allow a definitively benign diagnosis for a circumscribed mass include the presence of fat and certain calcification patterns on the mammogram and features of a simple cyst on the sonogram. Management decisions for other circumscribed masses will depend on characteristics such as shape, margins, calcification, multiplicity, size, stability, and sonographic features as well as patient age and risk factors. Most nonspecific circumscribed masses should be followed rather than biopsied as they are commonly present on mammograms and have a change of malignancy of less than 5%. Even when biopsied on the basis of interval change, most small circumscribed cancers will not have metastasized to the regional nodes. For palpable breast masses, selection of mammography or ultrasonography as the primary imaging modality will depend on patient's age and risk factors.

Breast Diseases↗
PubMed1998

[Stabilization of 99m Tc-HMPAO with methylene blue: its use in leukocyte labelling)].

A method to stabilize the 99mTc-HMPAO obtained from Ceretec(R) saline fractions using methylene blue is described. The mean radiochemical purity two hours after the stabilized 99mTc-HMPAO preparation was 92.5% (SD 1.8%), the pH ranged from 7.0 to 7.6 and the osmolality ranged from 305 to 309 mOsm/kg. These features made the stabilized 99mTc-HMPAO suitable for leukocyte labelling. In order to evaluate this method, we performed the labelling of two identical leukocyte samples withdrawn from 10 blood donors. The first sample was labeled using 99mTc-HMPAO non-stabilized and the second one was labelled using 99mTc-HMPAO stabilized 2 hours before with methylene blue. The mean labelling efficiencies we obtained were 70.9% (SD 6.9%) and 66.1% (SD 4.2%) respectively. With the conditions described using the 99mTc-HMPAO stabilized with methylene blue, we had no drawbacks from the stabilizing blue colour and we did not found changes in the cellular viability.

Cell Survival↗