Cytochrome c transcriptional activation and mRNA stability during contractile activity in skeletal muscle. (49/4528)

We evaluated contractile activity-induced alterations in cytochrome c transcriptional activation and mRNA stability with unilateral chronic stimulation (10 Hz, 3 h/day) of the rat tibialis anterior (TA) muscle for 1, 2, 3, 4, 5, and 7 days (n = 3-11/group). Transcriptional activation was assessed by direct plasmid DNA injection into the TA with a chloramphenicol acetyltransferase (CAT) reporter gene linked to 326 bp of the cytochrome c promoter. Cytochrome c mRNA in stimulated muscles increased by 1.3- to 1. 7-fold above control between 1 and 7 days. Cytochrome c protein was increased after 5 days of stimulation to reach levels that were 1. 9-fold higher than control by 7 days. Cytochrome c mRNA stability, determined with an in vitro decay assay, was greater in stimulated TA than in control between 2 and 4 days, likely mediated by the induction of a cytosolic factor. In contrast, cytochrome c transcriptional activation was elevated only after 5 days of stimulation when mRNA stability had returned to control levels. Thus the contractile activity-induced increase in cytochrome c mRNA was due to an early increase in mRNA stability, followed by an elevation in transcriptional activation, leading to an eventual increase in cytochrome c protein levels.  (+info)

Autoregulatory sequences are revealed by complex stability screening of the mouse brn-3.0 locus. (50/4528)

The POU-IV or Brn-3 class of transcription factors exhibit conserved structure, DNA-binding properties, and expression in specific subclasses of neurons across widely diverged species. In the mouse CNS, Brn-3.0 expression characterizes specific neurons from neurogenesis through the life of the cell. This irreversible activation of expression suggests positive autoregulation. To search for cis-acting elements that could mediate autoregulation we used a novel method, complex stability screening, which we applied to rapidly identify functional Brn-3.0 recognition sites within a large genomic region encompassing the mouse brn-3.0 locus. This method is based on the observation that the kinetic stability of Brn-3.0 complexes with specific DNA sequences, as measured by their dissociation half-lives, is highly correlated with the ability of those sequences to mediate transcriptional activation by Brn-3.0. The principal Brn-3.0 autoregulatory region lies approximately 5 kb upstream from the Brn-3.0 transcription start site and contains multiple Brn-3.0-binding sites that strongly resemble the optimal binding site for this protein class. This region also mediates transactivation by the closely related protein Brn-3.2, suggesting a regulatory cascade of POU proteins in specific neurons in which Brn-3.2 expression precedes Brn-3.0.  (+info)

Phase diagram, stability, and overcharging of lamellar cationic lipid-DNA self-assembled complexes. (51/4528)

Cationic lipid-DNA (CL-DNA) complexes comprise a promising new class of synthetic nonviral gene delivery systems. When positively charged, they attach to the anionic cell surface and transfer DNA into the cell cytoplasm. We report a comprehensive x-ray diffraction study of the lamellar CL-DNA self-assemblies as a function of lipid composition and lipid/DNA ratio, aimed at elucidating the interactions determining their structure, charge, and thermodynamic stability. The driving force for the formation of charge-neutral complexes is the release of DNA and lipid counterions. Negatively charged complexes have a higher DNA packing density than isoelectric complexes, whereas positively charged ones have a lower packing density. This indicates that the overcharging of the complex away from its isoelectric point is caused by changes of the bulk structure with absorption of excess DNA or cationic lipid. The degree of overcharging is dependent on the membrane charge density, which is controlled by the ratio of neutral to cationic lipid in the bilayers. Importantly, overcharged complexes are observed to move toward their isoelectric charge-neutral point at higher concentration of salt co-ions, with positively overcharged complexes expelling cationic lipid and negatively overcharged complexes expelling DNA. Our observations should apply universally to the formation and structure of self-assemblies between oppositely charged macromolecules.  (+info)

A spectroscopic and calorimetric investigation on the thermal stability of the Cys3Ala/Cys26Ala azurin mutant. (52/4528)

The disulfide bond connecting Cys-3 and Cys-26 in wild type azurin has been removed to study the contribution of the -SS- bond to the high thermal resistance previously registered for this protein (. J. Phys. Chem. 99:14864-14870). Site-directed mutagenesis was used to replace both cysteines for alanines. The characterization of the Cys-3Ala/Cys-26Ala azurin mutant has been carried out by means of electron paramagnetic resonance spectroscopy at 77 K, UV-VIS optical absorption, fluorescence emission and circular dichroism at room temperature. The results show that the spectral features of the Cys-3Ala/Cys-26Ala azurin resemble those of the wild type azurin, indicating that the double mutation does not affect either the formation of the protein's overall structure or the assembly of the metal-binding site. The thermal unfolding of the Cys-3Ala/Cys-26Ala azurin has been followed by differential scanning calorimetry, optical absorption variation at lambda(max) = 625 nm, and fluorescence emission using 295 nm as excitation wavelength. The analysis of the data shows that the thermal transition from the native to the denaturated state of the modified azurin follows the same multistep unfolding pathway as observed in wild type azurin. However, the removal of the disulfide bridge results in a dramatic reduction of the thermodynamic stability of the protein. In fact, the transition temperatures registered by the different techniques are down-shifted by about 20 degrees C with respect to wild type azurin. Moreover, the Gibbs free energy value is about half of that found for the native azurin. These results suggest that the disulfide bridge is a structural element that significantly contributes to the high stability of wild type azurin.  (+info)

Characterization of glucokinase mutations associated with maturity-onset diabetes of the young type 2 (MODY-2): different glucokinase defects lead to a common phenotype. (53/4528)

Glucokinase (GK) is expressed in the pancreatic beta-cells and liver, and plays a key role in the regulation of glucose homeostasis. The enzymatic activity and thermal stability of wild-type (WT) GK and several mutant forms associated with maturity-onset diabetes of the young type 2 (MODY-2) were determined by a steady-state kinetic analysis of the purified expressed proteins. The eight MODY-2 mutations studied were Ala53Ser, Val367Met, Gly80Ala, Thr168Pro, Arg36Trp, Thr209Met, Cys213Arg, and Val226Met. These missense mutations were shown to have variable effects on GK kinetic activity. The Gly80Ala and Thr168Pro mutations resulted in a large decrease in Vmax and a complete loss of the cooperative behavior associated with glucose binding. In addition, the Gly80Ala mutation resulted in a sixfold increase in the half-saturating substrate concentration (S0.5) for ATP, and Thr168Pro resulted in eight- and sixfold increases in the S0.5 values for ATP and glucose, respectively. The Thr209Met and Val226Met mutations exhibited three- and fivefold increases, respectively, in the S0.5 for ATP, whereas the Cys213Arg mutation resulted in a fivefold increase in the S0.5 for glucose. These mutations also led to a small yet significant reduction in Vmax. Of all the mutations studied, only the Cys213Arg mutation had reduced enzymatic activity and decreased thermal stability. Two mutants, Ala53Ser and Val367Met, showed kinetic and thermal stability properties similar to those of WT. These mutants had increased sensitivities to the known negative effectors of GK activity, palmitoyl-CoA, and GK regulatory protein. Taken together, these results illustrate that the MODY-2 phenotype may be linked not only to kinetic alterations but also to the regulation of GK activity.  (+info)

In vitro antibacterial activity of LJC 11,036, an active metabolite of L-084, a new oral carbapenem antibiotic with potent antipneumococcal activity. (54/4528)

LJC 11,036 is the active metabolite of L-084, a novel oral carbapenem that exhibits potent broad-spectrum activity. Antibacterial activities of LJC 11,036 against clinical isolates from respiratory infections, such as Streptococcus pneumoniae (n = 52), Streptococcus pyogenes (n = 19), Haemophilus influenzae (n = 50), Klebsiella pneumoniae (n = 53), and Moraxella catarrhalis (n = 53), and from urinary-tract infections, such as Escherichia coli (n = 53) (MICs at which 90% of the isolates were inhibited [MIC(90)s], 0.1, +info)

Stability of antibiotics used for antibiotic-lock treatment of infections of implantable venous devices (ports). (55/4528)

Antibiotic-lock is a treatment for catheter-related bloodstream infections in which a solution containing heparin and an antibiotic dwells in the lumen of the catheter or port. We tested the stability of vancomycin, cefazolin, ticarcillin-clavulanic acid, ceftazidime, or ciprofloxacin combined with heparin after incubation in vitro at 25 or 37 degrees C for intervals of up to 10 days by bioassay. All the antibiotic solutions except ceftazidime retained >/=90% activity at both 25 and 37 degrees C. Thus, studies of antibiotic-heparin lock solutions with dwell times of up to 10 days are feasible.  (+info)

A thermodynamic study of the 434-repressor N-terminal domain and of its covalently linked dimers. (56/4528)

The isolated N-terminal 1-69 domain of the 434-phage repressor, R69, and its covalently linked (head-to-tail and tail-to-tail) dimers have been studied by differential scanning microcalorimetry (DSC) and CD. At neutral solvent conditions the R69 domain maintains its native structure, both in isolated form and within the dimers. The stability of the domain depends highly upon pH within the acidic range, thus at pH 2 and low ionic strength R69 is already partially unfolded at room temperature. The thermodynamic parameters of unfolding calculated from the DSC data are typical for small globular proteins. At neutral pH and moderate ionic strength, the domains of the dimers behave as two independent units with unfolding parameters similar to those of the isolated domain, which means that linking two R69 domains, either by a long peptide linker or by a designed C-terminal disulfide bridge, does not induce any cooperation between them.  (+info)