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IL13 polymorphism is associated with chronic obstructive pulmonary disease (COPD). Patients with COPD have smaller numbers of mitochondria deoxyribonucleic acid copies (mtDNA-CN) than people without COPD do. However, whether IL13 polymorphism affects the mutation and recombination of mitochondria remains unclear. Data for patients with COPD and non-COPD were collected from Kaohsiung Chang Gung Memorial Hospital to enable a comparison of their leukocyte mtDNA-CN and the association of this information with IL-13 promoter (−1055) polymorphism. This study included 99 patients with COPD and 117 individuals without COPD. The non-COPD individuals included 77 healthy individuals that never smoked and 40 healthy smokers. The patients with COPD exhibited significantly lower mtDNA-CN than non-COPD did (250.34 vs. 440.03; p < 0.001); mtDNA-CN was particularly pronounced in individuals with the IL13 CC and CT genotypes compared with individuals with the TT genotype. When only individuals without COPD were considered and when all participants were considered, the differences in the mtDNA-CNs in individuals with the CC and CT genotypes were more significant than those in individuals with the TT genotype (448.4 and 533.6 vs. 282.8; p < 0.05 in non-COPD group); (368.8 and 362.6 vs. 249.6, p < 0.05 in all participants). The increase mtDNA-CN in the CC and CT genotypes was also more than that in the TT genotype in COPD patients, but showed no significance (260.1 and 230.5 vs. 149.9; p = 0.343). The finding shows that COPD is a mitochondria regulatory disorder and IL-13 promoter (−1055) polymorphism is associated with leukocyte mtDNA-CN. Developing COPD control methods based on mitochondrial regulation will be possible.

Background: Coronavirus disease (COVID-19) impairs the free movement of human beings. The study aims to determine how the COVID-19 pandemic affected population mobility. Methods: The study obtained Google COVID-19 population mobility report and e Taiwan COVID-19 pandemic information from Our World in Data. Results: During the Alpha wave, transit decreased the most, with an average difference of >50%, followed by parks, workplaces, groceries, and pharmacies. During the Omicron wave, the average population flow in parks and transit decreased by about 20%. During the pre-existing wave, the average population visits of transit decreased by 10% at the most, followed by parks and workplaces. The peak of daily new confirmed cases per million (7-day rolling average) was 25.02, 6.39, and 0.81 for Alpha, Omicron, and the pre-existing wave, respectively. Daily new confirmed cases per million people correlated with the change in population visits of various places (all p < 0.001). The reproduction rate (7-day rolling average) correlated with the change of population visits of most places, except retail and recreation. We conclude the Alpha variant affected more individuals than Omicron and pre-existing type. Furthermore, changes in population visits in transit were most impacted. This change was consistent with daily new confirmed cases per million people and reproduction rate (7-day rolling average). Conclusion: The Alpha variant affected more individuals than the Omicron and pre-existing types. Furthermore, changes in population visits in transit locations were most impacted. This change was consistent with the daily new number of confirmed cases per million people and the 7-day rolling average reproduction rate.

The severe acute respiratory syndrome (SARS) coronavirus (CoV) main protease represents an attractive target for the development of novel anti-SARS agents. The tertiary structure of the protease consists of two distinct folds. One is the N-terminal chymotrypsin-like fold that consists of two structural domains and constitutes the catalytic machinery; the other is the C-terminal helical domain, which has an unclear function and is not found in other RNA virus main proteases. To understand the functional roles of the two structural parts of the SARS-CoV main protease, we generated the full-length of this enzyme as well as several terminally truncated forms, different from each other only by the number of amino acid residues at the C- or N-terminal regions. The quaternary structure and K(d) value of the protease were analyzed by analytical ultracentrifugation. The results showed that the N-terminal 1-3 amino acid-truncated protease maintains 76% of enzyme activity and that the major form is a dimer, as in the wild type. However, the amino acids 1-4-truncated protease showed the major form to be a monomer and had little enzyme activity. As a result, the fourth amino acid seemed to have a powerful effect on the quaternary structure and activity of this protease. The last C-terminal helically truncated protease also exhibited a greater tendency to form monomer and showed little activity. We concluded that both the C- and the N-terminal regions influence the dimerization and enzyme activity of the SARS-CoV main protease.

This retrospective study included COPD patients who attended our medical center between January and October 2018, and analyzed the outcomes of their influenza vaccination, including medical visits, hospitalization, medical expenses, and the incidence of respiratory failure. Airflow limitation was stratified according to GOLD guidelines. Overall, 543 COPD patients were enrolled, including 197, 113, 126, and 107 mild, moderate, severe, and very severe patients, respectively. Of all the participants, 238 received an influenza vaccination (43.8%), which significantly reduced hospital utilization for moderate (odds ratio [OR] 0.22, 95%CI 0.09–0.51), severe (OR 0.19, 95%CI 0.08–0.44), and very severe patients (OR 0.15, 95%CI 0.05–0.5) compared to mild patients (OR 0.51, 95%CI 0.2–1.26), reduced emergency department utilization for moderate (OR 0.33, 95%CI 0.14–0.77), severe (OR 0.22, 95%CI 0.10–0.52), and very severe patients (OR 0.30, 95%CI 0.10–0.88) compared to mild patients (OR 0.64, 95%CI 0.30–1.37), and reduced the occurrence of respiratory failure for moderate (OR 0.20, 95%CI 0.06–0.68), severe (OR 0.40, 95%CI 0.16–0.98), and very severe patients (OR 0.36, 95%CI 0.15–0.82) compared to mild patients (OR 0% CI 0.14–3.20). Influenza vaccination is more effective in COPD patients with moderate, severe, and very severe airflow obstruction than in those with mild obstruction with respect to hospital utilization, emergency department utilization, and respiratory failure.

Background The use of electronic medical record (EMR) is anticipated to bring benefits for patients, physicians, and organizations. But limited physicians' acceptance of EMR presents a serious threat to its effective implementation. Objectives The current study incorporates technology acceptance model (TAM) with two antecedents, gender, and clinical specialty and one context-specific factor, financial incentives, to identify the factors that influence physicians' intention to use EMR in Taiwan. Methods The survey methodology was used to collect data from the physicians, working in the regional hospital that had implemented EMR system. A total of 119 out of 213 questionnaires returned in a response rate of 56%. But four responses were considered ineffective due to missing values. The structural equation modeling (SEM) technique was employed to analyze the research framework. Results The partial least squares (PLS) regression indicated that three factors perceived usefulness, financial incentives, and attitude toward using EMR significantly affect physicians' intention. But concerning perceived ease of use (PEOU), an insignificant path coefficient was reported. Additionally, regression analysis showed gender, and clinical specialty positively influenced physicians' intention to use EMR. Discussion and Conclusions The proposed research framework contributes to the conclusive explanation for interpreting physicians' intention to use EMR. Physicians generally have a higher level of computer literacy. Therefore, the factor of PEOU could not be critical regarding adopting new health information technology (HIT). This study also brings perspectives from the gender, and clinical differences have primarily been missing in the literature of the physicians' intention to use HIT. In doing so, it infers how gender, and clinical specialty, may complement (and in some instances, reinforce) the influence of technological and attitudinal factors of HIT use. Thus, health care providers must take these factors into consideration in the development and validation of the theories regarding the intention to use EMR.

Food detergents are commonly used in households. The main components of detergents include surfactants and water. It has been found that certain compounds, which may cause health concerns, appear in food detergents, such as alkylphenols, arsenic, and lead. After applying food detergents is when people may be exposed to various levels of these chemicals when there are residues. In this study, the Taguchi experimental design was performed to determine the possible factors that might affect the residual characteristics of food detergent on dishware, fruits, and vegetables. The results showed that the variety of detergent was found to be the most significant factor affecting the residue amounts of arsenic (62.9%) and lead (71.6%) on fruits and vegetables, whereas the concentration of detergent used affected the amount of lead residue only (10.5%). On the other hand, dishware material, the concentration of analytes, immersion time, and type of surfactant contributed to arsenic residues on dishware, whereas technical nonylphenol isomer residues on dishware increased as the concentration of spiked analyte increased. In addition, the occurrence of 1,4-dioxane, a possible human carcinogen, in household food detergents in Taiwan was also determined in this research by solid-phase microextraction and GC-MS. Among the 80 detergent samples, 71 contained different concentrations ranges of 1,4-dioxane, from 0.03 to ~3.73 µg/g. In the exposure assessment, it was estimated that the maximum amounts of 1,4-dioxane in contact with the skin from the use of household food detergent in Taiwan was 0.015 µg/kg/day.

Aminopeptidase P, a metalloprotease, targets Xaa-Proline peptides for cleavage [1-4]. There are two forms of human AMPP, a membrane-bound form (hmAMPP) and a soluble cytosolic form (hcAMPP)[5]. Similar to the angiotensin-I-converting enzyme, AMPP plays an important role in the catabolism of inflammatory and vasoactive peptides, known as kinins. The plasma kinin, bradykinin, was used as the substrate to conduct enzymatic activity analyses and to determine the Michaelis constant (Km) of 174 μM and the catalytic rate constant (kcat) of 10.8 s-1 for hcAMPP. Significant differences were observed in the activities of Y527F and R535A hcAMPP mutants, which displayed a 6-fold and 13.5-fold for decrease in turnover rate, respectively. Guanidine hydrochloride restored the activity of R535A hcAMPP, increasing the kcat/Km 20-fold, yet it had no impact on the activities of the wild-type or Y527F mutant hcAMPPs. Activity restoration by guanidine derivatives followed the order guanidine hydrochloride >> methyl-guanidine > amino-guanidine > N-ethyl-guanidine. Overall, the results indicate the participation of R535 in the hydrogen bond network that forms a proton relay system. The quaternary structure of hcAMPP was determined by using analytical ultracentrifugation (AUC). The results show that alanine replacement of Arg535 destabilizes the hcAMPP dimer and that guanidine hydrochloride restores the native monomer-dimer equilibrium. It is proposed that Arg535 plays an important role in hcAMMP catalysis and in stabilization of the catalytically active dimeric state.

E-learning is thought of as an innovative approach to enhance nurses' care service knowledge. Extensive research has provided rich information toward system development, courses design, and nurses' satisfaction with an e-learning system. However, a comprehensive view in understanding nursing e-learning system success is an important but less focused-on topic. The purpose of this research was to explore net benefits of nursing e-learning systems based on the updated DeLone and McLean's Information System Success Model. The study used a self-administered questionnaire to collected 208 valid nurses' responses from 21 of Taiwan's medium- and large-scale hospitals that have implemented nursing e-learning systems. The result confirms that the model is sufficient to explore the nurses' use of e-learning systems in terms of intention to use, user satisfaction, and net benefits. However, while the three exogenous quality factors (system quality, information quality, and service quality) were all found to be critical factors affecting user satisfaction, only information quality showed a direct effect on the intention to use. This study provides useful insights for evaluating nursing e-learning system qualities as well as an understanding of nurses' intentions and satisfaction related to performance benefits.

A new class of human GST inhibitors has been identified via rational design approach; we report their discovery, synthesis, inhibitory activity, and synergetic effect in combination with cisplatin against A549 lung cancer cell line. The results of this effort show that the lead 4-O-decyl-gabosine D (24) has optimum synergetic effect in A549 human lung adenocarcinoma epithelial cell and that this activity involves inhibition of glutathione S-transferase M1, apparently consistent with siRNA-mediated knockdown of GSTM1 gene.

The purpose of this study was to explore the status of e-healthcare maturity in Taiwan following a nationwide investigation sponsored by the Department of Health. Based on Nolan's stage model and related studies, we propose a multidimensional model to gain a better understanding of the current status of e-healthcare maturity in hospitals. The target subjects included every hospital in Taiwan. A total of 538 hospitals were successfully interviewed, showing a high response rate of 94.4%. The results indicate that the overall e-healthcare maturity of Taiwanese hospitals is fairly high. Such a high degree of maturity is critical for formulating e-healthcare policy to stimulate the exchange of electronic medical record. This article provides a brief comparison of the situation in other countries and posits that Taiwan holds a position of relative maturity in the world of e-healthcare development.

Transition metal complexes bearing amino linked N-heterocyclic carbenes (NHC) were prepared and evaluated for their antiproliferative activities in human cancer cells. The optimum antiproliferative activity, observed for the gold complex 3 in U-87 MG cells, was found to involve S-phase arrest of the cell cycle. The results indicate that 3 induces apoptosis through a p53-bak pathway, a finding that could serve as a new strategy to reduce the resistance of cancer cells to p53-induced apoptosis.

The dimeric interface of severe acute respiratory syndrome coronavirus main protease is a potential target for the anti-SARS drug development. We have generated C-terminal truncated mutants by serial truncations. The quaternary structure of the enzyme was analyzed using both sedimentation velocity and sedimentation equilibrium analytical ultracentrifugation. Global analysis of the combined results showed that truncation of C-terminus from 306 to 300 had no appreciable effect on the quaternary structure, and the enzyme remained catalytically active. However, further deletion of Gln-299 or Arg-298 drastically decreased the enzyme activity to 1–2% of wild type (WT), and the major form was a monomeric one. Detailed analysis of the point mutants of these two amino acid residues and their nearby hydrogen bond partner Ser-123 and Ser-139 revealed a strong correlation between the enzyme activity loss and dimer dissociation.

Severe acute respiratory syndrome (SARS) is an emerging infectious disease caused by a novel human coronavirus. Viral maturation requires a main protease (3CL(pro)) to cleave the virus-encoded polyproteins. We report here that the 3CL(pro) containing additional N- and/or C-terminal segments of the polyprotein sequences undergoes autoprocessing and yields the mature protease in vitro. The dimeric three-dimensional structure of the C145A mutant protease shows that the active site of one protomer binds with the C-terminal six amino acids of the protomer from another asymmetric unit, mimicking the product-bound form and suggesting a possible mechanism for maturation. The P1 pocket of the active site binds the Gln side chain specifically, and the P2 and P4 sites are clustered together to accommodate large hydrophobic side chains. The tagged C145A mutant protein served as a substrate for the wild-type protease, and the N terminus was first digested (55-fold faster) at the Gln(-1)-Ser1 site followed by the C-terminal cleavage at the Gln306-Gly307 site. Analytical ultracentrifuge of the quaternary structures of the tagged and mature proteases reveals the remarkably tighter dimer formation for the mature enzyme (K(d) = 0.35 nm) than for the mutant (C145A) containing 10 extra N-terminal (K(d) = 17.2 nM) or C-terminal amino acids (K(d) = 5.6 nM). The data indicate that immature 3CL(pro) can form dimer enabling it to undergo autoprocessing to yield the mature enzyme, which further serves as a seed for facilitated maturation. Taken together, this study provides insights into the maturation process of the SARS 3CL(pro) from the polyprotein and design of new structure-based inhibitors.

Guanine deaminase, a key enzyme in the nucleotide metabolism, catalyzes the hydrolytic deamination of guanine into xanthine. The crystal structure of the 156-residue guanine deaminase from Bacillus subtilis has been solved at 1.17-A resolution. Unexpectedly, the C-terminal segment is swapped to form an intersubunit active site and an intertwined dimer with an extensive interface of 3900 A2 per monomer. The essential zinc ion is ligated by a water molecule together with His53, Cys83, and Cys86. A transition state analog was modeled into the active site cavity based on the tightly bound imidazole and water molecules, allowing identification of the conserved deamination mechanism and specific substrate recognition by Asp114 and Tyr156′. The closed conformation also reveals that substrate binding seals the active site entrance, which is controlled by the C-terminal tail. Therefore, the domain swapping has not only facilitated the dimerization but has also ensured specific substrate recognition. Finally, a detailed structural comparison of the cytidine deaminase superfamily illustrates the functional versatility of the divergent active sites found in the guanine, cytosine, and cytidine deaminases and suggests putative specific substrate-interacting residues for other members such as dCMP deaminases.

Pigeon cytosolic malic enzyme has a double dimer quaternary structure with three tryptophanyl residues in each monomer distributed in different structural domains. The enzyme showed a three-state unfolding phenomenon upon increasing the urea concentration (Chang, H. C., Chou, W. Y., and Chang, G. G. (2002) J. Biol. Chem. 277, 4663-4671). At urea concentration of 4-4.5 m, where the intermediate form was detected, the enzyme existed as partially unfolded dimers, which were easily polymerized. Mn2+ provided full protection against the polymerization. To further characterize this phenomenon, three mutants of the enzyme (W129, W321, and W548), each with only one tryptophanyl residue left, were constructed. All these mutants were successfully overexpressed in Escherichia coli cells and purified to homogeneity. Changes in the circular dichroism spectra of all mutants revealed a three-state urea-unfolding process in the absence of Mn2+. In the presence of 4 mm Mn2+, W548 and wild type (WT) enzymes shifted to monophasic, while W129 and W321 were still biphasic. Similar results were obtained from the fluorescence spectral changes, except for W321, which showed monophasic denaturation curve with or without Mn2+. Analytical ultracentrifugation analysis indicated that the mutant enzymes were polymerized at 4.5 m urea, and Mn2+ provided protective effect on W548 and WT enzymes only. Other mutants with mutated Trp-548 polymerized at 4.5 m urea in the absence or presence of 4 mm Mn2+. The above results indicate that a single residue, Trp-548, in the subunit interface region, is responsible for the integrity of the quaternary structure of the pigeon cytosolic malic enzyme.

S-Crystallin from octopus lens has a tertiary structure similar to sigma-class glutathione transferase (GST). However, after isolation from the lenses, S-crystallin was found to aggregate more easily than sigma-GST. In vitro experiments showed that the lens S-crystallin can be polymerized and finally denatured at increasing concentration of urea or guanidinium chloride (GdmCl). In the intermediate concentrations of urea or GdmCl, the polymerized form of S-crystallin is aggregated, as manifested by the increase in light scattering and precipitation of the protein. There is a delay time for the initiation of polymerization. Both the delay time and rate of polymerization depend on the protein concentration. The native protein showed a maximum fluorescence emission spectrum at 341nm. The GdmCl-denatured protein exhibited two fluorescence maxima at 310nm and 358nm, respectively, whereas the urea-denatured protein showed a fluorescence peak at 358nm with a small peak at 310nm. The fluorescence intensity was quenched. Monomers, dimers, trimers, and polymers of the native protein were observed by negative-stain electron microscopic analysis. The aggregated form, however, showed irregular structure. The aggregate was solubilized in high concentrations of urea or GdmCl. The redissolved denatured protein showed an identical fluorescence spectrum to the protein solution that was directly denatured with high concentrations of urea or GdmCl. The denatured protein was readily refolded to its native state by diluting with buffer solution. The fluorescence spectrum of the renatured protein solution was similar to that of the native form. The phase diagrams for the S-crystallin in urea and GdmCl were constructed. Both salt concentration and pH value of the solution affect the polymerization rate, suggesting the participation of ionic interactions in the polymerization. Comparison of the molecular models of the S-crystallin and sigma-GST suggests that an extra ion-pair between Asp-101 and Arg-14 in S-crystallin contributes to stabilizing the protomer. Furthermore, the molecular surface of S-crystallin has a protruding Lys-208 on one side and a complementary patch of aspartate residues (Asp-90, Asp-94, Asp-101, Asp-102, Asp-179, and Asp-180) on the other side. We propose a molecular model for the S-crystallin polymer in vivo, which involves side-by-side associations of Lys-208 from one protomer and the aspartate patch from another protomer that allows the formation of a polymeric structure spontaneously into a liquid crystal structure in the lens.

Background: Hospital information systems (HISs) are widely used in Taiwan, and HIS performance must be carefully evaluated. Nursing personnel are the largest group of staff in a hospital and are the center of care delivery; thus, they play an important role in the adoption and evaluation of HISs. Purpose: The primary objective of this study was to explore the critical factors affecting the acceptance of HISs in Taiwan from a nursing perspective. On the basis of the technology acceptance model, we used six exogenous variables (system quality, information quality, user self-efficacy, compatibility, top management support, and project team competency) as investigation factors. Methods: Survey research targeted nursing personnel in the selected case hospital as participants. A total of 545 questionnaires were sent out, and 501 were returned, indicating a valid response rate of 91.9%. Collected data were analyzed using multiple regression analysis. Results: Results indicate that user self-efficacy, top management support, compatibility, and information quality have significant impacts on perceived ease of use. In addition, top management support, compatibility, and information quality were identified as having significant impacts on perceived usefulness. Furthermore, nurses' perceived ease of use and perceived usefulness on HISs was found to impact significantly on system acceptance, with 45.1% of the total explained variance. Conclusions/Implications for Practice: Results can help managers understand key considerations affecting HIS development and use and may be applied as a reference for system development and improvement.

A new type of competitive human GST inhibitors has been developed via the bioisostere and structure activity profile strategies; we report their discovery, preparation, inhibitory activity, and synergetic effect in combination with chemotherapy drugs against breast cancer cells.

Malic enzyme is a tetrameric protein with double dimer quaternary structure. In 3-5 M urea, the pigeon cytosolic NADP(+)-dependent malic enzyme unfolded and aggregated into various forms with dimers as the basic unit. Under the same denaturing conditions but in the presence of 4 mM Mn(2+), the enzyme existed exclusively as a molten globule dimer in solution. Similar to pigeon enzyme (Chang, G. G., T. M. Huang, and T. C. Chang. 1988. Biochem. J. 254:123-130), the human mitochondrial NAD(+)-dependent malic enzyme also underwent a reversible tetramer-dimer-monomer quaternary structural change in an acidic pH environment, which resulted in a molten globule state that is also prone to aggregate. The aggregation of pigeon enzyme was attributable to Trp-572 side chain. Mutation of Trp-572 to Phe, His, Ile, Ser, or Ala abolished the protective effect of the metal ions. The cytosolic malic enzyme was completely digested within 2 h by trypsin. In the presence of Mn(2+), a specific cutting site in the Lys-352-Gly-Arg-354 region was able to generate a unique polypeptide with M(r) of 37 kDa, and this polypeptide was resistant to further digestion. These results indicate that, during the catalytic process of malic enzyme, binding metal ion induces a conformational change within the enzyme from the open form to an intermediate form, which upon binding of L-malate, transforms further into a catalytically competent closed form.

SARS (severe acute respiratory syndrome) has been one of the most severe viral infectious diseases last year and still remains as a highly risky public health problem around the world. Exploring the types of interactions responsible for structural stabilities of its component protein molecules constitutes one of the approaches to find a destabilization method for the virion particle. In this study, we performed a series of experiments to characterize the quaternary structure of the dimeric coronavirus main protease (M(pro), 3CL(pro)). By using the analytical ultracentrifuge, we demonstrated that the dimeric SARS coronavirus main protease exists as the major form in solution at protein concentration as low as 0.10 mg/mL at neutral pH. The enzyme started to dissociate at acidic and alkali pH values. Ionic strength has profound effect on the dimer stability indicating that the major force involved in the subunit association is ionic interactions. The effect of ionic strength on the protease molecule was reflected by the drastic change of electrostatic potential contour of the enzyme in the presence of NaCl. Analysis of the crystal structures indicated that the interfacial ionic interaction was attributed to the Arg-4...Glu-290 ion pair between the subunits. Detailed examination of the dimer-monomer equilibrium at different pH values reveals apparent pK(a) values of 8.0 +/- 0.2 and 5.0 +/- 0.1 for the Arg-4 and Glu-290, respectively. Mutation at these two positions reduces the association affinity between subunits, and the Glu-290 mutants had diminished enzyme activity. This information is useful in searching for substances that can intervene in the subunit association, which is attractive as a target to neutralize the virulence of SARS coronavirus.

Guanine deaminase, a key enzyme in the nucleotide metabolism, catalyzes the hydrolytic deamination of guanine into xanthine. The crystal structure of the 156-residue guanine deaminase from Bacillus subtilis has been solved at 1.17-A resolution. Unexpectedly, the C-terminal segment is swapped to form an intersubunit active site and an intertwined dimer with an extensive interface of 3900 A(2) per monomer. The essential zinc ion is ligated by a water molecule together with His(53), Cys(83), and Cys(86). A transition state analog was modeled into the active site cavity based on the tightly bound imidazole and water molecules, allowing identification of the conserved deamination mechanism and specific substrate recognition by Asp(114) and Tyr(156'). The closed conformation also reveals that substrate binding seals the active site entrance, which is controlled by the C-terminal tail. Therefore, the domain swapping has not only facilitated the dimerization but has also ensured specific substrate recognition. Finally, a detailed structural comparison of the cytidine deaminase superfamily illustrates the functional versatility of the divergent active sites found in the guanine, cytosine, and cytidine deaminases and suggests putative specific substrate-interacting residues for other members such as dCMP deaminases.

We have cloned, expressed and characterized catfish alphaB-crystallin (FalphaB). Genomic sequence comparison has revealed conservation of intron splicing sites and coding regions, however, the two intron sequences, 5'- and 3'-untranslated regions of FalphaB gene are shorter than those reported for other vertebrates. In contrast to mammalian homologues with a subunit association ratio (alphaA-crystallin/alphaB-crystallin) of 3:1, alpha-crystallin from catfish lens showed a ratio of 19:1. The biophysical properties and chaperone-like activity of recombinant FalphaB and porcine alphaB-crystallin (PalphaB) were studied and compared by heat denaturation, circular dichroism, intrinsic and dye-binding fluorescence, gel-filtration, and analytical ultracentrifugation. FalphaB shows 50% precipitation occurring at 72 degrees C that is higher than PalphaB at 66 degrees C. Even though FalphaB also possesses more surface hydrophilic regions than PalphaB, FalphaB still possesses higher chaperone activity to prevent aggregation of alcohol dehydrogenase at 60 degrees C. The molecular mass of FalphaB showed a smaller size (450 kDa) than PalphaB (550 kDa), which is also confirmed by analytical ultracentrifugation. In addition, FalphaB possesses better refolding potential after preheating treatment than PalphaB. FalphaB also exhibits higher chaperone-like activity than PalphaB to prevent insulin aggregation induced by dithiothreitol. In contrast to the prevalent notion that fish crystallins generally denature easily, FalphaB with chaperone-like activity appears to be more stable than mammalian homologues towards thermal and chemical denaturation.

The cytosolic malic enzyme from the pigeon liver is sensitive to chemical denaturant urea. When monitored by protein intrinsic fluorescence or circular dichroism spectral changes, an unfolding of the enzyme in urea at 25 degrees C and pH 7.4 revealed a biphasic phenomenon with an intermediate state detected at 4-5 m urea. The enzyme activity was activated by urea up to 1 m but was completely lost before the intermediate state was detected. This suggests that the active site region of the enzyme was more sensitive to chemical denaturant than other structural scaffolds. In the presence of 4 mm Mn(2+), the urea denaturation pattern of malic enzyme changed to monophasic. Mn(2+) helped the enzyme to resist phase I urea denaturation. The [urea](0.5) for the enzyme inactivation shifted from 2.2 to 3.8 m. Molecular weight determined by the analytical ultracentrifuge indicated that the tetrameric enzyme was dissociated to dimers in the early stage of phase I denaturation. In the intermediate state at 4-5 m urea, the enzyme showed polymerization. However, the polymer forms were dissociated to unfolded monomers at a urea concentration greater than 6 m. Mn(2+) retarded the polymerization of the malic enzyme. Three mutants of the enzyme with a defective metal ligand (E234Q, D235N, E234Q/D235N) were cloned and purified to homogeneity. These mutant malic enzymes showed a biphasic urea denaturation pattern in the absence or presence of Mn(2+). These results indicate that the Mn(2+) has dual roles in the malic enzyme. The metal ion not only plays a catalytic role in stabilization of the reaction intermediate, enol-pyruvate, but also stabilizes the overall tetrameric protein architecture.