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Biomedical subjects

C J Tsai

Publications and source records attributed to C J Tsai.

At least 19 recordsLinked to original sources
PubMed2002

Transmission electron microscopy investigation of the formation of C54-TiSi(2) phase on stressed (001)Si.

The effects of stress on the formation of C54-TiSi2 phase in Ti/(001)Si samples have been investigated by high-resolution transmission electron microscopy in conjunction with auto-correlation function (ACF) analysis. The C54-TiSi2 phase transformation temperature in tensily stressed samples was found to be lowered by about 100 degrees C than that in compressively stressed samples. The thickness of amorphous interlayers (a-interlayers) between Ti metal thin films and Si substrates was found to be thicker and thinner in the tensily and compressively stressed Si samples, respectively. Furthermore, the thicker a-interlayer was found to consist of a higher density of crystallites from the ACF analysis. With a higher density of crystallites in the a-interlayer, the grain size of C49-TiSi2 was reduced since more nucleation sites are available for the formation of C49-TiSi2. The small grain size of C49-TiSi2 in turn enhances the formation of C54-TiSi2. As a result, the phase transformation of C49- to C54-TiSi2 is enhanced by the tensile stress present in silicon substrates.

Journal Article↗
PubMed2001

Thermodynamic differences among homologous thermophilic and mesophilic proteins.

Here, we analyze the thermodynamic parameters and their correlations in families containing homologous thermophilic and mesophilic proteins which show reversible two-state folding <--> unfolding transitions between the native and the denatured states. For the proteins in these families, the melting temperatures correlate with the maximal protein stability change (between the native and the denatured states) as well as with the enthalpic and entropic changes at the melting temperature. In contrast, the heat capacity change is uncorrelated with the melting temperature. These and additional results illustrate that higher melting temperatures are largely obtained via an upshift and broadening of the protein stability curves. Both thermophilic and mesophilic proteins are maximally stable around room temperature. However, the maximal stabilities of thermophilic proteins are considerably greater than those of their mesophilic homologues. At the living temperatures of their respective source organisms, homologous thermophilic and mesophilic proteins have similar stabilities. The protein stability at the living temperature of the source organism does not correlate with the living temperature of the protein. We tie thermodynamic observations to microscopics via the hydrophobic effect and a two-state model of the water structure. We conclude that, to achieve higher stability and greater resistance to high and low temperatures, specific interactions, particularly electrostatic, should be engineered into the protein. The effect of these specific interactions is largely reflected in an increased enthalpy change at the melting temperature.

Acid Anhydride Hydrolases↗
PubMed2001

Structured disorder and conformational selection.

Traditionally, molecular disorder has been viewed as local or global instability. Molecules or regions displaying disorder have been considered inherently unstructured. The term has been routinely applied to cases for which no atomic coordinates can be derived from crystallized molecules. Yet, even when it appears that the molecules are disordered, prevailing conformations exist, with population times higher than those of all alternate conformations. Disordered molecules are the outcome of rugged energy landscapes away from the native state around the bottom of the funnel. Ruggedness has a biological function, creating a distribution of structured conformers that bind via conformational selection, driving association and multimolecular complex formation, whether chain-linked in folding or unlinked in binding. We classify disordered molecules into two types. The first type possesses a hydrophobic core. Here, even if the native conformation is unstable, it still has a large enough population time, enabling its experimental detection. In the second type, no such hydrophobic core exists. Hence, the native conformations of molecules belonging to this category have shorter population times, hindering their experimental detection. Although there is a continuum of distribution of hydrophobic cores in proteins, an empirical, statistically based hydrophobicity function may be used as a guideline for distinguishing the two disordered molecule types. Furthermore, the two types relate to steps in the protein folding reaction. With respect to protein design, this leads us to propose that engineering-optimized specific electrostatic interactions to avoid electrostatic repulsion would reduce the type I disordered state, driving the molten globule (MG) --> native (N) state. In contrast, for overcoming the type II disordered state, in addition to specific interactions, a stronger hydrophobic core is also indicated, leading to the denatured --> MG --> N state.

Enzyme Stability↗
PubMed2001

Collection efficiency and capacity of three samplers for acidic and basic gases.

This paper compares the collection efficiency and capacity of a coated porous metal disk with those of a silica gel tube and an impinger at the acid or basic aerosol concentration of two times the permissible exposure limitation (Taiwan IOSH, Institute of Occupational Safety and Health), relative humidity of 80 +/- 5%, and temperature of 30 +/- 3 degrees C in the laboratory. Using 5% sodium carbonate/glycerin coating, the collection efficiencies of the porous metal disk for nitric acid, hydrogen chloride, and hydrogen fluoride are found to be higher than those of the silica gel tube and the impinger when the sampling time is less than 3.0 h. The gas collection capacity of the porous metal disk coated with 5% sodium carbonate is calculated to be 3.9, 5.8, and >2.5 mg for nitric acid, hydrogen chloride, and hydrogen fluoride, respectively. For ammonia, the porous metal disk coated using 4% citric acid performs slightly better than the impinger, and the corresponding capacity is slightly greater than 33.6 mg.

Aerosols↗
PubMed2001

Protein folding and function: the N-terminal fragment in adenylate kinase.

Three-dimensional protein folds range from simple to highly complex architectures. In complex folds, some building block fragments are more important for correct protein folding than others. Such fragments are typically buried in the protein core and mediate interactions between other fragments. Here we present an automated, surface area-based algorithm that is able to indicate which, among all local elements of the structure, is critical for the formation of the native fold, and apply it to structurally well-characterized proteins. In particular, we focus on adenylate kinase. The fragment containing the phosphate binding, P-loop (the "giant anion hole") flanked by a beta-strand and an alpha-helix near the N-terminus, is identified as a critical building block. This building block shows a high degree of sequence and structural conservation in all adenylate kinases. The results of our molecular dynamics simulations are consistent with this identification. In its absence, the protein flips to a stable, non-native state. In this misfolded conformation, the other local elements of the structure are in their native-like conformations; however, their association is non-native. Furthermore, this element is critically important for the function of the enzyme, coupling folding, and function.

Adenylate Kinase↗
PubMed2001

The building block folding model and the kinetics of protein folding.

Here we show that qualitatively, the building blocks folding model accounts for three-state versus the two-state protein folding. Additionally, it is consistent with the faster versus slower folding rates of the two-state proteins. Specifically, we illustrate that the building blocks size, their mode of associations in the native structure, the number of ways they can combinatorially assemble, their population times and the way they are split in the iterative, step-by-step structural dissection which yields the anatomy trees, explain a broad range of folding rates. We further show that proteins with similar general topologies may have different folding pathways, and hence different folding rates. On the other hand, the effect of mutations resembles that of changes in conditions, shifting the population times and hence the energy landscapes. Hence, together with the secondary structure type and the extent of local versus non-local interactions, a coherent, consistent rationale for folding kinetics can be outlined, in agreement with experimental results. Given the native structure of a protein, these guidelines enable a qualitative prediction of the folding kinetics. We further describe these in the context of the protein folding energy landscape. Quantitatively, in principle, the diffusion-collision model for the building block association can be used. However, the folding rates of the building blocks and traps in their formation and association, need to be considered.

Adenylate Kinase↗
PubMed2001

A proposed structural model for amyloid fibril elongation: domain swapping forms an interdigitating beta-structure polymer.

We propose a model illustrating how proteins, which differ in their overall sequences and structures, can form the propagating, twisted beta-sheet conformations, characteristic of amyloids. Some cases of amyloid formation can be explained through a "domain swapping" event, where the swapped segment is either a beta-hairpin or an unstable conformation which can partially unfold and assume a beta-hairpin structure. As in domain swapping, here the swapped beta-hairpin is at the edge of the structure, has few (if any) salt bridges and hydrogen bonds connecting it to the remainder of the structure and variable extents of buried non-polar surface areas. Additionally, in both cases the swapped piece constitutes a transient "building block" of the structure, with a high population time. Whereas in domain swapping the swapped fragment has been shown to be an alpha-helix, loop, strand or an entire domain, but so far not a beta-hairpin, despite the large number of cases in which it was already detected, here swapping may involve such a structural motif. We show how the swapping of beta-hairpins would form an interdigitated, twisted beta-sheet conformation, explaining the remarkable high stability of the protofibril in vitro. Such a swapping mechanism is attractive as it involves a universal mechanism in proteins, critical for their function, namely hinge-bending motions. Our proposal is consistent with structural superpositioning of mutational variants. While the overall r.m.s.d.s of the wild-type and mutants are small, the proposed hinge-bending region consistently shows larger deviations. These larger deviations illustrate that this region is more prone to respond to the mutational changes, regardless of their location in the sequence or in the structure. Nevertheless, above all, we stress that this proposition is hypothetical, since it is based on assumptions lacking definitive experimental support.

Amyloid↗
PubMed2001

Molecular dynamics simulation of Escherichia coli dihydrofolate reductase and its protein fragments: relative stabilities in experiment and simulations.

We have carried out molecular dynamics simulations of the native dihydrofolate reductase from Escherichia coli and several of its folded protein fragments at standard temperature. The simulations have shown fragments 1--36, 37--88, and 89--159 to be unstable, with a C(alpha)RMSD (C(alpha) root mean squared deviation) >5 A after 3.0 nsec of simulation. The unfolding of fragment 1--36 was immediate, whereas fragments 37--88 and 89--159 gradually unfolded because of the presence of the beta-sheet core structure. In the absence of residues 1--36, the two distinct domains comprising fragment 39--159 associated with each other, resulting in a stable conformation. This conformation retained most of its native structural elements. We have further simulated fragments derived from computational protein cutting. These were also found to be unstable, with the exception of fragment 104--159. In the absence of alpha(4), the loose loop region of residues 120--127 exhibited a beta-strand-like behavior, associating itself with the beta-sheet core of the protein fragment. The current study suggests that the folding of dihydrofolate reductase involves cooperative folding of distinct domains which otherwise would have been unstable as independent folded units in solution. Finally, the critical role of residues 1--36 in allowing the two distinct domains of fragment 104--159 to fold into the final native conformation is discussed.

Computer Simulation↗
PubMed2001

Fluconazole disk diffusion test with methylene blue- and glucose-enriched Mueller-Hinton agar for determining susceptibility of Candida species.

A 25-microg fluconazole disk diffusion test using a Mueller-Hinton agar plate containing 2% glucose and 5 microg of methylene blue/ml (GM-MH) was compared to the macrodilution reference method for 210 Candida species. The GM-MH agar plate was read at 24 h. The predictive values of disks with susceptible, intermediate, and resistant results on the GM-MH agar plate at 24 h were 97.1, 56.3, and 76.5%, respectively.

Antifungal Agents↗
PubMed2001

Transient, highly populated, building blocks folding model.

Protein folding is a hierarchical event, in which transiently formed local structural elements assemble to yield the native conformation. In principle, multiple paths glide down the energy landscape, but, in practice, only a few of the paths are highly traveled. Here, the literature is reviewed in this light, and, particularly, a hierarchical, building block protein-folding model is presented, putting it in the context of a broad range of experimental and theoretical results published over the past few years. The model is based on two premises: First, although the local building block elements may be unstable, they nevertheless have higher population times than all alternate conformations; and, second, protein folding progresses through a combinatorial assembly of these elements. Through the binding of the most favorable building block conformers, there is a redistribution of the conformers in solution, propagating the protein-folding reaction. We describe the algorithm, and illustrate its usefulness, then we focus on its utility in assigning simple vs complex folding pathways, on chaperonin-assisted folding, on its relevance to domain-swapping processes, and on its relevance and relationship to disconnectivity graphs and tree diagrams. Considering protein folding as initiating from local transient structural elements is consistent with available experimental and theoretical results. Here, we have shown that, early in the folding process, sequential interactions are likely to take place, even if the final native fold is a complex, nonsequential one. Such a route is favorable kinetically and entropically. Through the construction of anatomy trees, the model enables derivation of the major folding pathways and their bumps, and qualitatively explains the kinetics of protein folding.

Biophysics↗
PubMed2000

Anatomy of protein structures: visualizing how a one-dimensional protein chain folds into a three-dimensional shape.

Here, we depict the anatomy of protein structures in terms of the protein folding process. Via an iterative, top-down dissecting procedure, tertiary structures are spliced down to reveal their anatomy: first, to produce domains (defined by visual three-dimensional inspection criteria); then, hydrophobic folding units (HFU); and, at the end of a multilevel process, a set of building blocks. The resulting anatomy tree organization not only clearly depicts the organization of a one-dimensional polypeptide chain in three-dimensional space but also straightforwardly describes the most likely folding pathway(s). Comparison of the tree with the formation of the hydrophobic folding units through combinatorial assembly of the building blocks illustrates how the chain folds in a sequential or a complex folding pathway. Further, the tree points to the kinetics of the folding, whether the chain is a fast or a slow folder, and the probability of misfolding. Our ability to successfully dissect the protein into an anatomy tree illustrates that protein folding is a hierarchical process and further validates a building blocks protein folding model.

Actins↗
PubMed2000

Transition-state ensemble in enzyme catalysis: possibility, reality, or necessity?

Proteins are not rigid structures; they are dynamic entities, with numerous conformational isomers (substates). The dynamic nature of protein structures amplifies the structural variation of the transition state for chemical reactions performed by proteins. This suggests that utilizing a transition state ensemble to describe chemical reactions involving proteins may be a useful representation. Here we re-examine the nature of the transition state of protein chemical reactions (enzyme catalysis), considering both recent developments in chemical reaction theory (Marcus theory for SN2 reactions), and protein dynamics effects. The classical theory of chemical reactions relies on the assumption that a reaction must pass through an obligatory transition-state structure. The widely accepted view of enzymatic catalysis holds that there is tight binding of the substrate to the transition-state structure, lowering the activation energy. This picture, may, however, be oversimplified. The real meaning of a transition state is a surface, not a single saddle point on the potential energy surface. In a reaction with a "loose" transition-state structure, the entire transition-state region, rather than a single saddle point, contributes to reaction kinetics. Consequently, here we explore the validity of such a model, namely, the enzymatic modulation of the transition-state surface. We examine its utility in explaining enzyme catalysis. We analyse the possibility that instead of optimizing binding to a well-defined transition-state structure, enzymes are optimized by evolution to bind efficiently with a transition-state ensemble, with a broad range of activated conformations. For enzyme catalysis, the key issue is still transition state (ensemble) stabilization. The source of the catalytic power is the modulation of the transition state. However, our definition of the transition state is the entire transition-state surface rather just than a single well-defined structure. This view of the transition-state ensemble is consistent with the nature of the protein molecule, as embodied and depicted in the protein energy landscape of folding, and binding, funnels.

Animals↗
PubMed2000

Electrostatic strengths of salt bridges in thermophilic and mesophilic glutamate dehydrogenase monomers.

Here we seek to understand the higher frequency of occurrence of salt bridges in proteins from thermophiles as compared to their mesophile homologs. We focus on glutamate dehydrogenase, owing to the availability of high resolution thermophilic (from Pyrococcus furiosus) and mesophilic (from Clostridium symbiosum) protein structures, the large protein size and the large difference in melting temperatures. We investigate the location, statistics and electrostatic strengths of salt bridges and of their networks within corresponding monomers of the thermophilic and mesophilic enzymes. We find that many of the extra salt bridges which are present in the thermophilic glutamate dehydrogenase monomer but absent in the mesophilic enzyme, form around the active site of the protein. Furthermore, salt bridges in the thermostable glutamate dehydrogenase cluster within the hydrophobic folding units of the monomer, rather than between them. Computation of the electrostatic contribution of salt bridge energies by solving the Poisson equation in a continuum solvent medium, shows that the salt bridges in Pyrococcus furiosus glutamate dehydrogenase are highly stabilizing. In contrast, the salt bridges in the mesophilic Clostridium symbiosum glutamate dehydrogenase are only marginally stabilizing. This is largely the outcome of the difference in the protein environment around the salt bridges in the two proteins. The presence of a larger number of charges, and hence, of salt bridges contributes to an electrostatically more favorable protein energy term. Our results indicate that salt bridges and their networks may have an important role in resisting deformation/unfolding of the protein structure at high temperatures, particularly in critical regions such as around the active site.

Clostridium↗
PubMed2000

The value of gallium-67 and thallium-201 whole-body and single-photon emission tomography images in dialysis-related beta 2-microglobulin amyloid.

The aim of this study was to investigate the value of gallium-67 and thallium-201 whole-body and single-photon emission tomography (SPET) images in long-term dialysis patients in whom dialysis-related beta 2-microglobulin amyloid (beta 2-MA) was clinically suspected. Twenty-three patients who had received dialysis for at least 10 years were included in the study. A technetium-99m methylene diphosphonate (MDP) whole-body scan was performed in all of the patients. If there was any MDP accumulation in the articular and/or peri-articular region, 67Ga and 201Tl whole-body and SPET images were then acquired. If any 67Ga and/or 201Tl uptake was observed, a CT-guided biopsy was done. In those patients who had articular and/or peri-articular uptake of 99mTc MDP, 67Ga and/or 201Tl and who were pathologically proven to have beta 2-MA, 99mTc MDP, 67Ga and 201Tl whole-body scans and SPET were carried out again, both 3 months and 1 year after initiation of treatment. This served to evaluate the therapeutic effect and allowed comparison with the clinical findings. Of the 23 patients, eight had abnormal 99mTc MDP uptake. Among these eight, six had intense 99mTc MDP, 67Ga and 201Tl uptake in the articular and peri-articular regions before medication. Three months after the start of treatment, there were very marked decreases in uptake on both the 67Ga and 201Tl scans but less obvious changes in uptake of 99mTc-MDP. In comparison with the other clinical manifestations such as limitation in range of motion, the more the painful disability improved, the less was the uptake on both 67Ga and 201Tl scans. There were virtually no differences in uptake pattern between the three scans of each radiopharmaceutical obtained for each patient in both 3 months and 1 year after initial of treatment. It is concluded that 99mTc-MDP whole-body bone scan can both detect active and pre-existing inactive deposits of beta 2-MA. 67Ga and 201Tl scans are helpful to differentiate active from inactive deposits of beta 2-MA and to evaluate the therapeutic effect on these patients. SPET images are usually needed to distinguish articular and periarticular lesions from bone lesions.

Amyloid↗
PubMed2000

Identification of the spinocerebellar ataxia type 7 mutation in Taiwan: application of PCR-based Southern blot.

Spinocerebellar ataxia (SCA) type 7 is an autosomal dominant disorder characterized by neural loss, mainly in the cerebellum and regions of the brainstem and particularly the inferior olivary complex. This neurodegeneration disease is associated with expansion of unstable CAG repeats within the 5'-translated region of the SCA7 gene, located on chromosome 3p. We conducted a local survey of the normal population and candidate patients for the analysis of the CAG repeats in the SCA7 gene. The distributions of the CAG repeat units of SCA7 gene in the normal population in Taiwan were established in this study by using the radioactive genomic polymerase chain reaction (PCR). The normal range of CAG repeats is from 6 to 17 repeats, with the more common being around 8-13 repeats. The range is narrower than that reported for other ethnic groups (7-35 CAGs). Meanwhile, by the use of a combination of PCR and Southern blot analysis, one SCA7 family was identified and is reported here. A marked instability of the CAG repeat number during transmission from father to son (41 vs. 100) was observed in the SCA7 family. Clinical anticipation is significant in this family including an infantile case, who was found to have nystagmus from the age of 1 month. To date, the SCA7 mutation has been detected in one of 73 families with autosomal dominant cerebellar ataxia phenotypes, which is about 1.4% of the ataxia families referred to us, compared to 1.4% SCA1, 9.6% SCA2, and 27.3% SCA3/Machado-Joseph disease in our collection. In addition, we demonstrate that the PCR-based Southern blot analysis, with the advantages of sensitivity of PCR and specificity of Southern blot, is a reliable diagnostic method for SCA7 mutation screening. The molecular analysis technique makes possible the quick and accurate diagnosis of SCA7 patients and in the future will hopefully be applied to prenatal screening for SCA7 families.

Adolescent↗
PubMed2000

A systematic study of the vibrational free energies of polypeptides in folded and random states.

Molecular vibrations, especially low frequency motions, may be used as an indication of the rigidity or the flatness of the protein folding energy landscape. We have studied the vibrational properties of native folded as well as random coil structures of more than 60 polypeptides. The picture we obtain allows us to perceive how and why the energy landscape progressively rigidifies while still allowing potential flexibility. Compared with random coil structures, both alpha-helices and beta-hairpins are vibrationally more flexible. The vibrational properties of loop structures are similar to those of the corresponding random coil structures. Inclusion of an alpha-helix tends to rigidify peptides and so-called building blocks of the structure, whereas the addition of a beta-structure has less effect. When small building blocks coalesce to form larger domains, the protein rigidifies. However, some folded native conformations are still found to be vibrationally more flexible than random coil structures, for example, beta(2)-microglobulin and the SH3 domain. Vibrational free energy contributes significantly to the thermodynamics of protein folding and affects the distribution of the conformational substates. We found a weak correlation between the vibrational folding energy and the protein size, consistent with both previous experimental estimates and theoretical partition of the heat capacity change in protein folding.

Amino Acid Sequence↗
PubMed2000

Relation of Helicobacter pylori infection and angiographically demonstrated coronary artery disease.

Conventional coronary risk factors explain only part of the variation in the incidence of cases of coronary heart disease. Recently H. pylori genomic material has been demonstrated in the coronary arteries of myocardial infarct. In searching for additional coronary risk factors, the potential role of H. pylori infection deserves to be investigated. To clarify if H. pylori infection is associated with an increased risk of coronary heart disease, a series of patients admitted to the Cardiac Catheterization Laboratory for coronary angiography were recruited prospectively. Cases (N = 165) were defined as those who had at least one coronary artery lesion occupying at least 50% of the luminal diameter on coronary angiography. Patients who had normal coronary angiography were selected as controls (N = 127). Demographic data, cardiovascular risk factors, and socioeconomic status were measured in both of the patients and controls. Stored serum specimens from both groups were tested for the presence of serum IgG antibody to H. pylori using enzyme-linked immunosorbent assay; 69.1% of the cases and 77.2% of the controls were seropositive for H. pylori (odds ratio 0.66, 95% CI 0.38-1.16, P = 0.12). After adjustment for age, gender, cardiovascular risk factors, and socioeconomic class, this remained nonsignificant (odds ratio 0.59, 95% CI 0.32-1.09, P = 0.09). H. pylori seropositivity was not associated with several coronary risk factors in either cases or controls. The proportion of H. pylori-positive patients was higher among the cases with triple vessel disease (77.5%) than those with double vessel disease (67.3%) and single vessel disease (65.7%); however, the differences were not statistically significant (odds ratio 0.57, 95% CI 0.23-1.4, P = 0.19). In this study no increase was found in H. pylori seropositivity in subjects with coronary artery disease. This minor association suggests that previous H. pylori infection, reflecting the early childhood environment, may not be important in determining the risk of coronary heart disease.

Aged↗