Localization of mucidin-resistant locus muc3 on mitochondrial DNA with respect to ubiquinol-cytochrome c reductase deficient box loci. Locus muc3 is allelic to box2

Summary Genetic relations between mitochondrial mucidin-resistant locus muc3 and ubiquinol-cytochrome c reductase-deficient box loci have been studied by recombination and petite deletion analysis. It was found that the locus muc3 maps in the segment of mitochondrial DNA corresponding to the locus box2. The results suggest the participation of box2/muc3 locus in the sequences of the structural gene for cytochrome b.     

Development of a physics-based force field for the scoring and refinement of protein models

The minimal requirements of a physics-based potential that can refine protein structures are the existence of a correlation between the energy with native similarity and the scoring of the native structure as the lowest in energy. To develop such a force field, the relative weights of the Amber ff03 all-atom potential supplemented by an explicit hydrogen-bond potential were adjusted by global optimization of energetic and structural criteria for a large set of protein decoys generated for a set of 58 nonhomologous proteins. The average correlation coefficient of the energy with TM-score significantly improved from 0.25 for the original ff03 potential to 0.65 for the optimized force field. The fraction of proteins for which the native structure had lowest energy increased from 0.22 to 0.90. Moreover, use of an explicit hydrogen-bond potential improves scoring performance of the force field. Promising preliminary results were obtained in applying the optimized potentials to refine protein decoys using only an energy criterion to choose the best decoy among sampled structures. For a set of seven proteins, 63% of the decoys improve, 18% get worse, and 19% are not changed.     

Association of protein kinase A type I with detergent-resistant structures of mammalian sperm cells

The finding that flagellar movement in detergent-permeabilized sperm cells is restored when Mg ATP and cAMP are added implicated detergent-resistant protein kinase A (PKA) in the regulation of sperm motility. It is widely believed that only the PKA regulatory subunit RII can associate with the cytoskeleton and/or organelles. In this paper we used monoclonal antibodies against the PKA catalytic subunit and RI subunit and demonstrated that PKA type I is also associated with the sperm cytoskeleton. To our knowledge, this is the first report showing anchored PKA type I. This association was found in sperm of nonrodent mammalian species and, to a lesser extent, also in mouse sperm. The PKA catalytic subunit is bound to the cytoskeleton secondarily via its complex with the regulatory subunit. The detergent-resistant complexes of RI and catalytic subunits localize predominantly to the flagellum. Ultrastructural immunogold labeling revealed the association of detergent-resistant PKA type I with outer dense fibers (ODF) and the fibrous sheath (FS) but not with microtubules. This location is consistent with a proposed role of PKA in regulation of FS sliding on underlying ODF. Mol. Reprod. Dev. 50:79–85, 1998. © 1998 Wiley-Liss, Inc.     

Effect of cyclosporin A on immunological response in lungs of guinea pigs infected with Trichinella spiralis

The effects of cyclosporin A (CsA), a potent immunosuppressive drug with antiparasitic activity, on the innate immunological response in guinea pig lungs during an early period (6th and 14th days) after T. spiralis infection were studied. CsA treatment of T. spiralis-infected guinea pigs caused a significant attenuation of immunological response in lungs by decreasing lymphocyte infiltration into pulmonary alveolar space, inhibiting alveolar macrophage superoxide anion production and lowering both the production of NO metabolites measured in bronchoalveolar lavage fluid and expression of the iNOS protein in lung homogenates, allowing us to speculate that the T. spiralis-dependent immunological response is dependent on lymphocyte T function. Interestingly, CsA itself had a pro-inflammatory effect, promoting leucocyte accumulation and macrophage superoxide production in guinea pig lungs. This observation may have a relevance to the situation in patients undergoing CsA therapy. Macrophage expression of the iNOS protein, evaluated by immunoblotting was not influenced by treatment of animals with CsA or anti-TGF-antibody, indicating different regulation of the guinea pig and murine enzymes.     

The usefulness of calyculin a for cytogenetic prenatal diagnosis.

An increased number of chromosome plates can be obtained by use of calyculin A (CLA). CLA is an inhibitor of protein phosphatases (type 1 and type 2A serine/threonine). Inactivation of these phosphatases leads to premature chromosome condensation (PCC) in all phases of the cell cycle; thus, it is possible to investigate both metaphase and G(2)-PCC chromosomes. Amniotic fluid (AF) cultures were treated with calyculin A (CLA). GTG banding was obtained. Using this method it is possible to investigate all cell cycle phases, GTG banding, chromosomal breaks, and rates of PCD on the same preparation. Analyses of AF cultures treated with CLA allow complex studies on fetal genetic material. This work presents potential usefulness of CLA for cytogenetic prenatal diagnosis.     

Cellular toxicity of oxycholesterols

Oxycholesterols (OS) are formed from cholesterol or its immediate precursors by enzymatic or free radical action in vivo, or they may be derived from food. OS exhibit a wide spectrum of biological activities. In OS cytotoxicity, several mechanisms seem to be involved: e.g. inhibition of HMG-CoA reductase activity, antiproliferative action, apoptosis induction, replacement of cholesterol by OS in membranes followed by changes in cellular membrane structure and functionality, and immune system functions alteration. Furthermore, OS may be mutagenic and carcinogenic and may serve as intracellular signaling or regulatory molecules. Here we review OS cellular activities with special attention to the cytotoxic action in vivo and in vitro using experimental models.     

Disulfide bonding arrangements in active forms of the somatomedin B domain of human vitronectin

The N-terminal cysteine-rich somatomedin B (SMB) domain (residues 1-44) of the human glycoprotein vitronectin contains the high-affinity binding sites for plasminogen activator inhibitor-1 (PAI-1) and the urokinase receptor (uPAR). We previously showed that the eight cysteine residues of recombinant SMB (rSMB) are organized into four disulfide bonds in a linear uncrossed pattern (Cys(5)-Cys(9), Cys(19)-Cys(21), Cys(25)-Cys(31), and Cys(32)-Cys(39)). In the present study, we use an alternative method to show that this disulfide bond arrangement remains a major preferred one in solution, and we determine the solution structure of the domain using NMR analysis. The solution structure shows that the four disulfide bonds are tightly packed in the center of the domain, replacing the traditional hydrophobic core expected for a globular protein. The few noncysteine hydrophobic side chains form a cluster on the outside of the domain, providing a distinctive binding surface for the physiological partners PAI-1 and uPAR. The hydrophobic surface consists mainly of side chains from the loop formed by the Cys(25)-Cys(31) disulfide bond, and is surrounded by conserved acidic and basic side chains, which are likely to contribute to the specificity of the intermolecular interactions of this domain. Interestingly, the overall fold of the molecule is compatible with several arrangements of the disulfide bonds. A number of different disulfide bond arrangements were able to satisfy the NMR restraints, and an extensive series of conformational energy calculations performed in explicit solvent confirmed that several disulfide bond arrangements have comparable stabilization energies. An experimental demonstration of the presence of alternative disulfide conformations in active rSMB is provided by the behavior of a mutant in which Asn(14) is replaced by Met. This mutant has the same PAI-1 binding activity as rVN1-51, but its fragmentation pattern following cyanogen bromide treatment is incompatible with the linear uncrossed disulfide arrangement. These results suggest that active forms of the SMB domain may have a number of allowed disulfide bond arrangements as long as the Cys(25)-Cys(31) disulfide bond is preserved.