Implication of guanosine 3′,5′-cyclic monophosphate,adenosine 3′,5′-cyclic monophosphate,adenosine 5′-mono-, di- and triphosphate and fructose-2,6-bisphosphate in the regulation of the glycolytic pathway in hypoxic/anoxic mussel,Mytilus galloprovincialis

The change in the content of cyclic GMP, cyclic AMP, ATP, ADP, AMP and fructose-2,6-bisphosphate that occurred in the mantle of the mussel Mytilus galloprovincialis Lmk when specimens of this mollusk were subjected to a hypoxia/anoxia situation were assessed. After the early 24 h in anaerobiosis, a clear decrease was observed in the ATP content, which remained close to that value for the rest of the time. AMP content doubled during the early 24 h in anaerobiosis and, from that time on, it remained close to that value. Fructose-2,6-bisphoshate and cyclic GMP showed a similar behavior. The levels of these compounds rose significantly during the early hours in anaerobiosis, and then fell to values similar to those of aerobiosis, remaining constant for the rest of the time. Neither ADP nor cAMP showed significant variations.     

Covalent immobilization of cyclodextrin glucosyltransferase (CGTase) in activated silica and Sepharose

Cyclodextrin glucanotransferase is a non-Leloir glycosyltransferase that directly employs the free energy of cleavage of starch to produce cyclodextrins. In presence of appropriate acceptors, this enzyme synthesizes oligosaccharides containing alpha(1-->4) bonds. We have investigated the covalent immobilization of CGTase onto different activated supports. Silica was aminated and further activated with glutaraldehyde. The maximum amount of bound protein was about 4 mg CGTase per gram of support; however, the catalytic efficiency of the immobilized enzyme was lower than 6%. Sepharose 4B activated with cyanogen bromide (CNBr-activated Sepharose) and Sepharose 4B with a spacer arm of 1,6-diaminohexane (EAH Sepharose) were also assayed. These gels react with the amino and carboxylic groups of CGTase, respectively. With CNBr-activated Sepharose, a low percentage of enzyme was bound to the support but with a significant catalytic efficiency (29%). A higher recovery of protein was obtained with EAH Sepharose (62%), but only 2.4% of the initial activity was present in the immobilized biocatalyst. The results were discussed in terms of CGTase structure and mechanism. In addition, the solvent accessibility of amino or carboxylic groups, calculated using the NACCESS software, was considered.     

Calculation of hydrodynamic properties of small nucleic acids from their atomic structure

Hydrodynamic properties (translational diffusion, sedimentation coefficients and correlation times) of short B-DNA oligonucleotides are calculated from the atomic-level structure using a bead modeling procedure in which each non-hydrogen atom is represented by a bead. Using available experimental data of hydrodynamic properties for several oligonucleotides, the best fit for the hydrodynamic radius of the atoms is found to be ~2.8 Å. Using this value, the predictions for the properties corresponding to translational motion and end-over-end rotation are accurate to within a few percent error. Analysis of NMR correlation times requires accounting for the internal flexibility of the double helix, and allows an estimation of ~0.85 for the Lipari–Szabo generalized order parameter. Also, the degree of hydration can be determined from hydrodynamics, with a result of ~0.3 g (water)/g (DNA). These numerical results are quite similar to those found for globular proteins. If the hydrodynamic model for the short DNA is simply a cylindrical rod, the predictions for overall translation and rotation are slightly worse, but the NMR correlation times and the degree of hydration, which depend more on the cross-sectional structure, are more severely affected.     

Hydrogen peroxide inhibits cell cycle progression by inhibition of the spreading of mitotic CHO cells

Hydrogen peroxide (H(2)O(2)) induces a number of events, which are also induced by mitogens. Since the progression through the G1 phase of the cell cycle is dependent on mitogen stimulation, we were interested to study the effect of H(2)O(2) on the cell cycle progression. This study demonstrates that H(2)O(2) inhibits DNA synthesis in a dose-dependent manner when given to cells in mitosis or at different points in the G1 phase. Interestingly, mitotic cells treated immediately after synchronization are significantly more sensitive to H(2)O(2) than cells treated in the G1, and this is due to the inhibition of the cell spreading after mitosis by H(2)O(2). H(2)O(2) reversibly inhibits focal adhesion activation and stress fiber formation of mitotic cells, but not those of G1 cells. The phosphorylation of MAPK is also reversibly inhibited in both mitotic and G1 cells. Taken together, H(2)O(2) is probably responsible for the inhibition of the expression of cyclin D1 and cyclin A observed in cells in both phases. In conclusion, H(2)O(2) inhibits cell cycle progression by inhibition of the spreading of mitotic CHO cells. This may play a role in pathological processes in which H(2)O(2) is generated.     

Glutaredoxins catalyze the reduction of glutathione by dihydrolipoamide with high efficiency

Glutaredoxins (Grx) are small (approximately 12kDa) proteins which catalyze thiol disulfide oxidoreductions involving glutathione (GSH) and disulfides in proteins or small molecules. Here, we present data which demonstrate the ability of glutaredoxins to catalyze the reduction of oxidized glutathione (GSSG) by dihydrolipoamide (DHL), an important biological redox catalyst and synthetic antioxidant. We have designed a new assay method to quantify the rate of reduction of GSSG and other disulfides by reduced lipoamide and have tested a set of eight recombinant Grx from human, rat, yeast, and E. coli. Lipoamide dependent activity is highest with the large atypical E. coli Grx2 (k(cat)=3.235 min(-1)) and lowest for human mitochondrial Grx2a (k(cat)=96 min(-1)) covering a wider range than k(cat) for the standard reduction of hydroxyethyldisulfide (HED) by GSH (290-2.851 min(-1)). The lipoamide/HED activity ratio was highest for yeast Grx2 (1.25) and E. coli Grx2 and lowest for E. coli Grx1 (0.13). These results suggest a new role for Grxs as ancillary proteins that could shunt reducing equivalents from main catabolic pathways to recycling of GSSG via a lipoyl group, thus serving biochemical functions which involve GSH but without NAD(P)H consumption.     

L-phenylalanine binding and domain organization in human phenylalanine hydroxylase: a differential scanning calorimetry study

Human phenylalanine hydroxylase (hPAH) is a tetrameric enzyme that catalyzes the hydroxylation of L-phenylalanine (L-Phe) to L-tyrosine; a dysfunction of this enzyme causes phenylketonuria. Each subunit in hPAH contains an N-terminal regulatory domain (Ser2-Ser110), a catalytic domain (Asp112-Arg410), and an oligomerization domain (Ser411-Lys452) including dimerization and tetramerization motifs. Two partially overlapping transitions are seen in differential scanning calorimetry (DSC) thermograms for wild-type hPAH in 0.1 M Na-Hepes buffer, 0.1 M NaCl, pH 7.0. Although these transitions are irreversible, studies on their scan-rate dependence support that the equilibrium thermodynamics analysis is permissible in this case. Comparison with the DSC thermograms for truncated forms of the enzyme, studies on the protein and L-Phe concentration effects on the transitions, and structure-energetic calculations based on a modeled structure support that the thermal denaturation of hPAH occurs in three stages: (i) unfolding of the four regulatory domains, which is responsible for the low-temperature calorimetric transition; (ii) unfolding of two (out of the four) catalytic domains, which is responsible for the high-temperature transition; and (iii) irreversible protein denaturation, which is likely responsible for the observed exothermic distortion in the high-temperature side of the high-temperature transition. Stages 1 and 2 do not appear to be two-state processes. We present an approach to the analysis of ligand effects on DSC transition temperatures, which is based on the general binding polynomial formalism and is not restricted to two-state transitions. Application of this approach to the L-Phe effect on the DSC thermograms for hPAH suggests that (i) there are no binding sites for L-Phe in the regulatory domains; therefore, contrary to the common belief, the activation of PAH by L-Phe seems to be the result of its homotropic cooperative binding to the active sites. (ii) The regulatory domain appears to be involved in cooperativity through its interactions with the catalytic and oligomerization domains; thus, upon regulatory domain unfolding, the cooperativity in the binding of L-Phe to the catalytic domains seems to be lost and the value of the L-Phe concentration corresponding to half-saturation is increased. Overall, our results contribute to the understanding of the conformational stability and the substrate-induced cooperative activation of this important enzyme.     

3,4-Methylenedioxymethamphetamine ("Ecstasy") induces apoptosis of cultured rat liver cells

"Ecstasy" (3,4-methylenedioxymethamphetamine, MDMA) has been shown to be hepatotoxic for human users, but molecular mechanisms involved in this effect remained poorly understood. MDMA-induced cell damage is related to programmed cell death in serotonergic and dopaminergic neurons. However, until now there has been no evidence of apoptosis induced by MDMA in liver cells. Here we demonstrate that exposure to MDMA caused apoptosis of freshly isolated rat hepatocytes and of a cell line of hepatic stellate cells (HSC), as shown by chromatin condensation of the nuclei and accumulation of oligonucleosomal fragments in the cytoplasm. In both cell types, apoptosis correlated with decreased levels of bcl-x(L), release of cytochrome c from the mitochondria and activation of caspase 3. In HSC, but not in hepatocytes, MDMA induced poly(ADP-ribose)polymerase (PARP) proteolysis. These results suggest that apoptosis of liver cells could be involved in the hepatotoxicity of MDMA.     

Stimulation of voltage-dependent calcium channels during capacitation and by progesterone in human sperm

To fertilize, mammalian sperm must undergo two sequential steps that require activation of calcium entry mechanisms, capacitation and acrosomal exocytosis, induced in the latter case by the egg zona pellucida glycoprotein ZP3 or by progesterone. Voltage-dependent calcium channels (VDCC) could participate in these processes. Since patch clamp recordings are extremely difficult in mature sperm, the activity of VDCC has been alternatively analyzed with optical detectors of membrane potential and intracellular calcium in sperm populations. Using this approach, we previously reported that in human sperm there is a voltage-dependent calcium influx system that strongly indicates that human sperm are endowed with functional VDCC. In this study we developed evidence indicating that calcium influx through VDCC is significantly stimulated during sperm in vitro capacitation and by progesterone action, which is present in the follicular fluid that surrounds the egg. The observed effects of capacitation and progesterone on VDCC may be physiologically significant for sperm-egg interaction.