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Biomedical subjects

G Mei

Publications and source records attributed to G Mei.

At least 19 recordsLinked to original sources
PubMed2006

Detection of quantitative trait loci affecting milk production traits on bovine chromosome 6 in a Chinese Holstein population by the daughter design.

Fourteen microsatellite markers with a coverage of 63.5 cM on bovine chromosome 6 were selected, and 26 sire families with 2,260 daughters were analyzed for mapping quantitative trait loci (QTL) affecting 5 milk production traits in a Chinese Holstein population. In the analyses across 26 families and within the largest significant families with a one-QTL model fitted, a QTL near BMS470 was detected that affected fat yield at the 5% experiment-wide significance level. When a 2-QTL model was fitted in the across-family analysis, it was found that there might exist 2 QTL affecting the 3 yield traits, although the exact or empirical thresholds for the significance testing were unknown. In all analyses, the results for milk yield and protein yield were generally consistent, which might have resulted from the same genetic background for milk and protein yield.

Animals↗
PubMed2001

Loss of apoB-100 secondary structure and conformation in hydroperoxide rich, electronegative LDL(-).

A subpopulation of low-density lipoproteins (LDL) is present in human plasma that contains lipid hydroperoxides and is more negatively charged (LDL(-)) than normal native LDL. By circular dichroism and tryptophan lifetime measurements we found that apoB-100 secondary structure is markedly decreased and its conformation is severely altered in LDL(-). The low tryptophan fluorescence intensity confirms the oxidative degradation of the lipoprotein, and the very long lifetime value of one of its decay components indicates a low polarity environment for the remaining unbleached residues. Either a peculiar folding or, most likely, a sinking of the apoB-100 into the lipid core can account for the observed long lifetime component. Oxidation in vitro produces a similar unfolding of the apolipoprotein but the lifetime of tryptophan fluorescence is shifted to lower values, indicating that the denatured apoprotein remains at the hydrophilic surface of the lipoprotein particle. A disordering and an increased polarity of the LDL(-) surface lipids was demonstrated by measuring the generalized polarization of 2-dimethylamino-6-lauroylnaphthalene (Laurdan). The looser monolayer packing apparently favors the new conformation of apoB-100 and its sinking into a more hydrophobic environment, possibly accounting for it reduced receptor binding properties.

2-Naphthylamine↗
PubMed2001

Cytochrome c reconstituted from two peptide fragments displays native-like redox properties.

Recombination of two fragments of horse cytochrome c (the heme-containing N-fragment, residues 1-56, and the C-fragment, residues 57-104), which are substantially unstructured at neutral pH, gives rise to a 1:1 fragment complex with a compact conformation, in which the alpha helical structure and the native Met80-Fe(III) axial bond are recovered. With respect to the native protein, the ferric complex shows a less rigid atomic packing and a decreased stability [Delta(DeltaG(o))D = 14.7 kJ.mol(-1)], ascribed to perturbations involving the Trp59 microenvironment and, to a lower extent, the heme pocket region. The redox potential, E1/2 = 234 +/- 5 mV vs. normal hydrogen electrode at 25 degrees C, is close to that of the intact protein, consistent with recovery of the native Met80-heme Fe(III) axial bond. Furthermore, the fragment complex shows reactivity similar to intact cytochrome c, in the reaction with cytochrome c oxidase. We conclude that the absence in the complex of some native cross-links and interlocked packing important for protein rigidity and stability is not as relevant for maintaining the native redox properties of the protein, provided that some structural requirements (i.e. recovering of the native-like alpha helical structure) are fulfilled and coordination of Met80 to the heme-iron is restored.

Animals↗
PubMed2000

Estradiol enhances the resistance of LDL to oxidation by stabilizing apoB-100 conformation.

Among different proposed mechanisms to account for the protection exerted by estrogens against cardiovascular diseases, the antioxidant effect has attracted considerable attention. We confirmed that 17-beta-estradiol (E2), when added to human LDL at a 6:1 ratio to apoB-100, markedly delays the phase of massive LDL lipid peroxidation induced by Cu(2+). We also observed an increased oxidative resistance of E2-treated LDL by monitoring the early phase of oxidative degradation on the basis of increased LDL surface polarity by the generalized polarization of the lipophilic fluorescent probe 2-(dimethylamino)-6-lauroylnaphthalene (Laurdan). A scavenging of free radicals by E2 is ruled out since, consistent with its structure, its rate constant for the reduction of peroxy radicals is extremely low, i.e., 0.02% of that of vitamin E. Tryptophan fluorescence lifetime and circular dichroism measurements revealed that (i) apoB-100 undergoes a conformational modification and a progressive loss of secondary structure during lipid peroxidation; (ii) E2 increases apoB-100 secondary structure and modifies its conformation; and (iii) the apoB-100 conformational change induced by E2 makes this protein resistant to modifications brought about by lipid peroxidation. We propose that E2, by affecting apoB-100 secondary structure and conformation, modifies the interaction of this protein with the outer layer of the LDL particle thus increasing its overall oxidative resistance.

2-Naphthylamine↗
PubMed2000

Anion size modulates the structure of the A state of cytochrome c.

Several studies have shown that anions induce collapse of acid-denatured cytochrome c into the compact A state having the properties of the molten globule and that the anion charge is the main determinant for the A state stabilization. The results here reported show that the anion size plays a role in determining the overall structure of the A state. In particular, small anions induce formation of an A state in which the native Met80-Fe(III) axial bond is recovered and the nativelike redox properties restored. On the other hand, the A state stabilized by large anions shows a histidine (His26 or His33) as the sixth ligand of the heme-iron, a very weak interaction between Trp59 and the heme propionate, and lacks nativelike redox properties. The two anion-stabilized states show similar stability, indicating that (i) the hydrophobic core (which is equally stabilized by all the anions investigated, independently of their size) is the region that mainly contributes to the macromolecule stabilization, and (ii) the flexible loops are responsible for the spectroscopic (and, thus, structural) and redox differences observed.

Animals↗
PubMed2000

Role of the tertiary and quaternary structures in the stability of dimeric copper, zinc superoxide dismutases.

The equilibrium unfolding process of human Cu,Zn superoxide dismutase has been quantitatively monitored through circular dichroism and fluorescence spectroscopy as a function of increasing guanidinium hydrochloride concentration. The process occurs through the formation of a monomeric intermediate species following a three-state transition equilibrium. Comparison with the stability of the prokaryotic Cu,Zn SOD from P. leiognathi shows that the eukaryotic enzyme is more stable than the prokaryotic enzyme by approximately 3 kcal/mol. This difference is due to the monomer-to-unfolded equilibrium, while the dimer-to-monomer equilibrium is comparable for the two enzymes despite their different intersubunit interactions. These results are confirmed by the unfolding of the copper-depleted derivatives. The Cu,Zn superoxide dismutase represents a good example of how evolution has found two independent quaternary assemblies maintaining the same dimer stability.

Circular Dichroism↗
PubMed2000

Octamer-primed sequencing technology: development of primer identification software.

Octamer sequencing technology (OST) is a primer-directed sequencing strategy in which an individual octamer primer is selected from a pre-synthesized octamer primer library and used to sequence a DNA fragment. However, selecting candidate primers from such a library is time consuming and can be a bottleneck in the sequencing process. To accelerate the sequencing process and to obtain high quality sequencing data, a computer program, electronic OST or eOST, was developed to automatically identify candidate primers from an octamer primer library. eOST integrates the base calling software PHRED to provide a quality assessment for target sequences and identifies potential primer binding sites located within a high quality target region. To increase the sequencing success rate, eOST includes a simple dynamic folding algorithm to automatically calculate the free energy and predict the secondary structure within the template in the vicinity of the octamer-binding site. Several parameters were found to be important, including base quality threshold, the window size of the template sequence segment, and the threshold [Delta] G value. OST, coupled with the eOST software, can be used to sequence short DNA fragments or in the finishing assembly stage of large-scale sequencing of genomic DNA.

Algorithms↗
PubMed2000

Opposite effects of Ca(2+) and GTP binding on tissue transglutaminase tertiary structure.

Tissue transglutaminase (tTG) belongs to a class of enzymes that catalyze a cross-linking reaction between proteins or peptides. The protein activity is known to be finely tuned by Ca(2+) and GTP binding. In this study we report the effects of these ligands on the enzyme structure, as revealed by circular dichroism, and steady-state and dynamic fluorescence measurements. We have found that calcium and GTP induced opposite conformational changes at the level of the protein tertiary structure. In particular the metal ions were responsible for a small widening of the protein molecule, as indicated by anisotropy decay measurements and by the binding of a hydrophobic probe such as 1-anilino-8-naphthalenesulfonic acid (ANS). Unlike Ca(2+), the nucleotide binding increased the protein dynamics, reducing its rotational correlation lifetime from 32 to 25 ns, preventing also the binding of ANS into the protein matrix. Unfolding of tTG by guanidinium hydrochloride yielded a three-state denaturation mechanism, involving an intermediate species with the characteristics of the so-called "molten globule" state. The effect of GTP binding (but not that of Ca(2+)) had an important consequence on the stability of tissue transglutaminase, increasing the free energy change from the native to the intermediate species by at least approximately 0.7 kcal/mol. Also a greater stability of tTG to high hydrostatic pressure was obtained in presence of GTP. These findings suggest that the molecular mechanism by which tTG activity is inhibited by GTP is essentially due to a protein conformational change which, decreasing the accessibility of the protein matrix to the solvent, renders more difficult the exposure of the active site.

Anilino Naphthalenesulfonates↗
PubMed1999

Evidence of stable monomeric species in the unfolding of Cu,Zn superoxide dismutase from Photobacterium leiognathi.

The equilibrium unfolding process of Photobacterium leiognathi Cu,Zn superoxide dismutase has been quantitatively monitored through circular dichroism (CD) and fluorescence spectroscopy, upon increasing the guanidinium hydrochloride concentration. The study has been undertaken for both the holo- and the copper-free derivative to work out the role of copper in protein stability. In both cases the unfolding was reversible. The denaturation curve derived from CD and fluorescence spectroscopy was not coincident, suggesting that the denaturation process occurs through a three-state model with formation of an intermediate monomeric species. The occurrence of an intermediate species has been unambiguously demonstrated following CD and steady-state fluorescence spectra of the enzyme at various concentrations in presence of a fixed amounts of guanidinium hydrochloride.

Circular Dichroism↗
PubMed1999

Endothelin antagonist treatment for successful liver transplantation from non-heart-beating donors.

BACKGROUND: With the shortage of cadaveric donors, non-heart-beating donors (NHBDs) are a potential source of liver allografts. However, warm ischemic injury in NHBDs seriously affects the viability of graft liver. Endothelin (ET)-1 has been reported to be involved in the hepatic microcirculatory disturbances after ischemia-reperfusion. METHODS: In a porcine orthotopic liver transplantation model, changes in the serum and liver tissue ET-1 concentration were measured and the effects of an ET receptor antagonist, TAK-044, were evaluated. After cardiac arrest of the donors, liver allografts were subjected to 90 min of warm ischemia, flushed, and preserved for 4 hr at 4 degrees C. The pigs were divided into two groups: a control group (no drug treatment) and a drug-treated group, in which donors and recipients were treated with TAK-044 (10 mg/kg body, drip intravenous injection). Both groups had six donor/recipient pairs. RESULTS: -The ET-1 concentration in the hepatic venous blood increased after reperfusion of the graft in the control group recipients. ET-1 in the graft liver significantly increased during the cold preservation period. TAK-044 treatment significantly increased recipient 7-day survival rate. After reperfusion of the graft, the concentrations of serum liver enzymes and arterial lactate in the drug-treated group were significantly lower than in the control group. The postoperative increase in portal venous pressure was significantly reduced in the drug-treated group. Measurements of liver enzymes in the washed-out preservation fluid at the time of graft rinsing indicated that TAK-044 treatment of the donors significantly suppressed liver enzyme release during ischemia. CONCLUSIONS: These findings indicate TAK-044 treatment has protective effects on postoperative function of hepatic allografts procured from NHBDs.

Alanine Transaminase↗
PubMed1999

An endothelin receptor antagonist ameliorates injuries of sinusoid lining cells in porcine liver transplantation.

BACKGROUND: TAK-044 is an endothelin receptor antagonist. Whether the agent has protective effects on liver graft injuries from non-heart-beating donors is unknown. METHODS: In donor pigs, cardiac arrest was induced by stopping the respirator. Forty-five minutes after cessation of the respirator, the liver was flushed with University of Wisconsin (UW) solution, preserved for 8 hours at 4 degrees C, and transplanted orthotopically. The pigs were divided into two groups: a control group and a drug-treated group in which TAK-044 was given in the UW solutions (10 mg/L) and was administered to recipients (10 mg/kg body weight). RESULTS: TAK-044 treatment significantly increased recipient survival rate. After reperfusion of the graft, portal venous pressure and 15-minute retention rate of indocyanine green were significantly reduced in the drug-treated group. Electron microscopic findings indicated that TAK-044 attenuated endothelial cell injuries. CONCLUSION: TAK-044 treatment improves the viability of livers harvested from non-heart-beating donors. The main effect of the agent is protection of endothelial cells from ischemia/reperfusion injuries.

Animals↗
PubMed1999

The effect of pressure and guanidine hydrochloride on azurins mutated in the hydrophobic core.

The unfolding of the blue-copper protein azurin from Pseudomonas aeruginosa by guanidine hydrochloride, under nonreducing conditions, has been studied by fluorescence techniques and circular dichroism. The denaturation transition may be fitted by a simple two-state model. The total free energy change from the native to the unfolded state was 9.4 +/- 0.4 kcal.mol-1, while a lower value (6.4 +/- 0.4 kcal.mol-1) was obtained for the metal depleted enzyme (apo-azurin) suggesting that the copper atom plays an important stabilization role. Azurin and apo-azurin were practically unaffected by hydrostatic pressure up to 3000 bar. Site-directed mutagenesis has been used to destabilize the hydrophobic core of azurin. In particular either hydrophobic residue Ile7 or Phe110 has been substituted with a serine. The free energy change of unfolding by guanidinium hydrochloride, resulted to be 5.8 +/- 0.3 kcal.mol-1 and 4.8 +/- 0.3 kcal.mol-1 for Ile7Ser and Phe110Ser, respectively, showing that both mutants are much less stable than the wild-type protein. The mutated apoproteins could be reversible denatured even by high pressure, as demonstrated by steady-state fluorescence measurements. The change in volume associated to the pressure-induced unfolding was estimated to be -24 mL.mol-1 for Ile7Ser and -55 mL.mol-1 for Phe110Ser. These results show that the tight packing of the hydrophobic residues that characterize the inner structure of azurin is fundamental for the protein stability. This suggests that the proper assembly of the hydrophobic core is one of the earliest and most crucial event in the folding process, bearing important implication for de novo design of proteins.

Azurin↗
PubMed1998

Resolution of the heterogeneous fluorescence in multi-tryptophan proteins: ascorbate oxidase.

Ascorbate oxidase is a copper-containing enzyme which catalyzes a redox reaction between vitamin C and molecular oxygen. The protein, which shows a complex tertiary structure, is an homodimer of monomers, each containing three domains and 14 tryptophan residues. Recently, we have demonstrated by spectroscopic and ultracentrifugation techniques the existence of a stable dimeric intermediate along the unfolding pathway of this enzyme [Mei, G., Di Venere, A., Buganza, M., Vecchini, P., Rosato, N. & Finazzi Agrò, A. (1997) Biochemistry 36, 10917-10922]. In this study, the steady-state and dynamic fluorescence features of ascorbate oxidase have been exploited in order to find a way of monitoring the individual subsystems of the protein. The fluorescence intensity and anisotropy upon excitation at 295 nm are extremely sensitive functions of the emission wavelength, indicating a great heterogeneity of the system. The emission decay collected through a cut-off filter can be analyzed in terms of two continuous distributions of lifetimes. Using a monochromator in emission or an optical multichannel analyzer, the two distributions may be attributed to distinct components of the fluorescence spectrum. Differential quenching by cesium chloride also confirmed that the several tryptophan residues present in the protein structure may be grouped into two main classes, each with a different environment. Once the complex fluorescence decay of ascorbate oxidase was analyzed and resolved, a comparison with the crystallographic data allowed a first, approximate attribution of the protein spectroscopic properties to some of the tryptophan residues. This might provide a powerful tool of investigation about the role of definite segments of the protein in its three-dimensional structure and catalytic activity. Furthermore, the methodology set up for ascorbate oxidase can be usefully extended to other multitryptophan proteins.

Ascorbate Oxidase↗
PubMed1998

New stable folding of beta-lactoglobulin induced by 2-propanol.

beta-lactoglobulin A has been studied in 2-propanol-water mixtures by means of circular dichroism, fluorescence and small angle X-ray scattering. At a low ionic strength, 2-propanol induces an increase in alpha-helix structure followed by a further transformation which gives rise to a new feature, rich of beta-sheet fragments. The second step of the secondary structure transformation is time-dependent and depressed at high ionic strength. As a consequence, the tertiary structure is completely modified and a new stable protein folding may be hypothesized. Small angle X-ray scattering measurements reveal that 2-propanol induces a diffuse protein aggregation, but the complex equilibria among intra- and inter-molecular hydrophobic and electrostatic interactions may be modulated by balancing the ionic strength and/or alcohol percentage.

2-Propanol↗
PubMed1998

Formation and movement of Fe(III) in horse spleen, H- and L-recombinant ferritins. A fluorescence study.

Iron oxidation and incorporation into apoferritins of different subunit composition, namely the recombinant H and L homopolymers and the natural horse spleen heteropolymer (10-15% H), have been followed by steady-state and time-resolved fluorescence. After aerobic addition of 100 Fe(II) atoms/polymer, markedly different kinetic profiles are observed. In the rL-homopolymer a slow monotonic fluorescence quenching is observed which reflects binding, slow oxidation at the threefold apoferritin channels, and diffusion into the protein cavity. In the rH-homopolymer a fast fluorescence quenching is followed by a partial, slow recovery. The two processes have been attributed to Fe(II) binding and oxidation at the ferroxidase centers and to Fe(III) released into the cavity, respectively. The fluorescence kinetics of horse spleen apoferritin is dominated by the H chain contribution and resembles that of the H homopolymer. It brings out clearly that the rate of the overall process is limited by the rate at which Fe(III) leaves the ferroxidase centers of the H chains where binding of incoming Fe(II) and its oxidation take place. The data obtained upon stepwise addition of iron and the results of optical absorption measurements confirm this picture. The correspondence between steady-state and time-resolved data is remarkably good; this is manifest when the latter are used to calculate the change in fluorescence intensity as apparent in the steady-state measurements.

Animals↗
PubMed1997

Role of quaternary structure in the stability of dimeric proteins: the case of ascorbate oxidase.

Equilibrium denaturation experiments have been performed in order to study the dissociation into monomers and unfolding of the dimeric copper-containing enzyme ascorbate oxidase by urea and guanidine hydrochloride. The process has been followed by fluorescence intensity and anisotropy, by optical density, and by circular dichroism as a function of denaturant concentration. The noncoincidence of the unfolding curves obtained by different techniques suggests that a multiphasic process is occurring. The study of enzymatic activity and aromatic circular dichroism as a function of denaturant concentration shows that the first transition involves a change in the protein tertiary structure which is accompanied by the loss of biological function. Gel electrophoresis, ultracentrifugation, and protein dilution experiments demonstrate that a large fraction of protein molecules is still dimeric during this first transition with a stability which is strictly dependent on the denaturant used. The free energy change from the native form to this intermediate species was estimated to be approximately 3.5 kcal/mol. The binding of 1-anilino-8-naphthalenesulfonic acid to the partially unfolded, inactive ascorbate oxidase dimer also suggests a large conformational change accompanied by copper release, allowing the probe to penetrate deep inside the protein structure. Further denaturation to give a fully unfolded form is protein concentration dependent, suggesting that dissociation into monomers is occurring. The monomers appear to be very unstable. No evidence for structured intermediates was in fact detected in the last step of the denaturation process. A three-state model has been used to fit the fluorescence data, and the fractions of different species have been calculated as a function of denaturant concentration. The total free energy change of the unfolding transition using either urea or guanidine hydrochloride is rather small ( approximately 15-16 kcal/mol) and quite comparable to the value found for smaller proteins. The loss of secondary structure which occurs in the second part of the unfolding transition may be described by a simple two-state process which is characterized by a free energy change of 12-13 kcal/mol. These results suggest that the folding process of ascorbate oxidase follows a hierarchical model (Jaenicke, 1991). In this context, the assembly of monomers in a dimeric molecule plays a fundamental role by enhancing the protein stability and driving the final organization of the tertiary structure.

Ascorbate Oxidase↗
PubMed1997

Spectroscopic properties of an engineered maltose binding protein.

The maltose binding protein (MBP) has been site specifically labelled with a nitrobenzoxadiazole (NBD) group following mutation of a serine to a cysteine residue at position 337. The resulting protein shows a large ligand (maltose or beta-cyclodextrin) dependent increase in its steady-state fluorescence intensity. Analysis of the static (intensity and anisotropy) and dynamic (lifetime distributions) fluorescence of the NBD label as well as the tryptophan residues in both ligand-bound and ligand-free states of this molecule reveals complex multi-component decays that are interpreted in terms of a ligand-induced solvent shielding mechanism. In the context of the known crystal structures of the various forms of the maltose-binding protein (MBP), ligand-dependent changes in both the fluorescence parameters as well as the circular dichroism spectra of the NBD group are interpreted by a twisted intramolecular charge-transfer (TICT) mechanism, wherein ligand binding locks the NBD group into a conformation that prevents efficient relaxation of the excited state.

4-Chloro-7-nitrobenzofurazan↗