On the flexibility and the dynamics of DNA

We describe and discuss the stability conditions of a naked double stranded DNA molecule starting from the evaluation of condensation or compactness fluctuation of this molecule embedded in pure water.     

Overexpression of alcohol dehydrogenase exacerbates ethanol-induced contractile defect in cardiac myocytes

Alcoholic cardiomyopathy is characterized by impaired ventricular function although its toxic mechanism is unclear. This study examined the impact of cardiac overexpression of alcohol dehydrogenase (ADH), which oxidizes ethanol into acetaldehyde (ACA), on ethanol-induced cardiac contractile defect. Mechanical and intracellular Ca(2+) properties were evaluated in ventricular myocytes from ADH transgenic and wild-type (FVB) mice. ACA production was assessed by gas chromatography. ADH myocytes exhibited similar mechanical properties but a higher efficiency to convert ACA compared with FVB myocytes. Acute exposure to ethanol depressed cell shortening and intracellular Ca(2+) in the FVB group with maximal inhibitions of 23.3% and 23.4%, respectively. Strikingly, the ethanol-induced depression on cell shortening and intracellular Ca(2+) was significantly augmented in the ADH group, with maximal inhibitions of 43.7% and 40.6%, respectively. Pretreatment with the ADH inhibitor 4-methylpyrazole (4-MP) or the aldehyde dehydrogenase inhibitor cyanamide prevented or augmented the ethanol-induced inhibition, respectively, in the ADH but not the FVB group. The ADH transgene also substantiated the ethanol-induced inhibition of maximal velocity of shortening/relengthening and unmasked an ethanol-induced prolongation of the duration of shortening/relengthening, which was abolished by 4-MP. These data suggest that elevated cardiac ACA exposure due to enhanced ADH expression may play an important role in the development of alcoholic cardiomyopathy.     

Metameric organisation of the nervous system in developmental stages of Urechis caupo (Echiura) and its phylogenetic implications

The phylogenetic position of Echiura is still in continuous debate. The commonly accepted view regards Echiura as a distinct taxon, often classified as phylum, which forms the sister group of the Articulata. The alternative view considers Echiura to be a subtaxon of Annelida, which is supported by numerous shared characters. The correct systematic position of Echiura is inevitably linked to the presence or absence of true segmentation. The apparent lack of segmentation in Echiura is considered to be either primary, thereby supporting their exclusion from Annelida, or alternatively to be the result of reduction. The latter would clearly substantiate their classification as a subtaxon of Annelida. Immunohistochemical methods and confocal laser-scanning microscopy clearly demonstrate a metameric organisation of the nervous system in different larval stages of Urechis caupo, which corresponds to the segmental arrangement of ganglia in "typical" Annelida. This segmental pattern is reflected in the serially repetitive distribution of neurons containing the neurotransmitter serotonin (5-hydroxytryptamine) and also in the corresponding distribution of strictly paired peripheral nerves. Precisely two pairs of peripheral nerves are associated with each of the repetitive units. This metameric pattern also corresponds to the transient annulation of the trunk, which is found in late larval stages. Other characters of the nervous system including the paired origin of the ventral nerve cord, the anterior-posterior development gradient and the presence of a distinct suboesophageal ganglion are also found accordingly in typical Annelida. These results are interpreted as an indication that Echiura are derived from formerly segmented ancestors, and thus support their systematic inclusion within Annelida.     

Versatile macrolide-responsive mammalian expression vectors for multiregulated multigene metabolic engineering

The novel macrolide-inducible and -repressible mammalian gene regulation systems (E.REX) have been cloned into a variety of sophisticated expression configurations including (1) multi-purpose expression vectors, (2) pTRIDENT-based artificial operons, (3) dual-regulated expression strategies for independent control of two different transgenes, (4) autoregulated vectors for one-step installation of adjustable multigene expression, and (5) oncoretroviral and lentiviral plasmids for transduction of macrolide-, streptogramin- and tetracycline-dependent transactivators and production of cell lines supporting independent control of three different transgenes. This vector portfolio represents a construction kit-like toolbox for efficient installation of adjustable gene expression responsive to clinically licensed antibiotics and enables the design of multiregulated multigene metabolic engineering strategies required for biopharmaceutical manufacturing, gene therapy, and tissue engineering.     

Identification of essential residues in the type II Hsp40 Sis1 that function in polypeptide binding

Sis1 is an essential yeast Type II Hsp40 protein that assists cytosolic Hsp70 Ssa1 in the facilitation of processes that include translation initiation, the prevention of protein aggregation, and proteasomal protein degradation. An essential function of Sis1 and other Hsp40 proteins is the binding and delivery of non-native polypeptides to Hsp70. How Hsp40s function as molecular chaperones is unknown. The crystal structure of a Sis1 fragment that retains peptide-binding activity suggests that Type II Hsp40s utilize hydrophobic residues located in a solvent-exposed patch on carboxyl-terminal domain I to bind non-native polypeptides. To test this model, amino acid residues Val-184, Leu-186, Lys-199, Phe-201, Ile-203, and Phe-251, which form a depression in carboxyl-terminal domain I, were mutated, and the ability of Sis1 mutants to support cell viability and function as molecular chaperones was examined. We report that Lys-199, Phe-201, and Phe-251 are essential for cell viability and required for Sis1 polypeptide binding activity. Sis1 I203T could support normal cell growth, but when purified it exhibited severe defects in chaperone function. These data identify essential residues in Sis1 that function in polypeptide binding and help define the nature of the polypeptide-binding site in Type II Hsp40 proteins.     

Design and synthesis of 4,5-disubstituted-thiophene-2-amidines as potent urokinase inhibitors

A study of the S1 binding of lead 5-methylthiothiophene amidine 3, an inhibitor of urokinase-type plasminogen activator, was undertaken by the introduction of a variety of substituents at the thiophene 5-position. The 5-alkyl substituted and unsubstituted thiophenes were prepared using organolithium chemistry. Heteroatom substituents were introduced at the 5-position using a novel displacement reaction of 5-methylsulfonylthiophenes and the corresponding oxygen or sulfur anions. Small alkyl group substitution at the 5-position provided inhibitors equipotent with but possessing improved solubility.     

The murine macrophage apoB-48 receptor gene (Apob-48r): homology to the human receptor

Previously we cloned the human macrophage apolipoprotein B-48 receptor (ApoB-48R) and documented its expression in human atherosclerotic foam cells (1). Now we have identified and characterized the murine macrophage apob-48r cDNA gene sequence and its chromosomal location. The cDNA (3,615 bp) -deduced amino acid (aa) sequence (942 aa) is approximately 45% identical to the human macrophage APOB-48R, but not to other known gene families. The murine Apob-48r gene, like the human APOB-48R gene, consists of four exons interrupted by three small introns and is syntenically located on chromosome 7. Functionally significant conserved domains include an N-terminal hydrophobic domain, a glycosaminoglycan attachment site, an N-glycosylation site, and an ExxxLL internalization motif C-terminal to the putative internal transmembrane domain. Two conserved coiled-coil domains are likely involved in the spontaneous homodimerization that generates the active dimeric ligand binding species (mouse, approximately 190 kDa; human, approximately 200 kDa). Transfection of the murine apoB-48R into Chinese hamster ovary cells (CHOs) confers apoB-48R function: rapid, high-affinity, specific uptake of known triglyceride-rich lipoprotein ligands of the apoB-48R and, of note, uptake of the cholesteryl ester-rich apoB-48-containing very low density lipoproteins that accumulate in atherosclerosis-prone apoE-deficient mice. Uptake of these ligands by murine apoB-48R-transfected CHOs causes saturable, visible cellular triglyceride and cholesterol accumulation in vitro that resemble foam cells of atherosclerotic lesions. In aggregate, the data presented here and that previously published suggest that the apoE-independent murine apoB-48R pathway may contribute to the spontaneous development of atherosclerotic lesions rich in macrophage-derived foam cells observed in apoE-deficient mice, a murine model of human atherosclerosis.     

The anti-anginal drug fendiline elevates cytosolic Ca(2+) in rabbit corneal epithelial cells

The effect of the anti-anginal drug fendiline on intracellular free Ca(2+) levels ([Ca(2+)](i)) in a rabbit corneal epithelial cell line (SIRC) was explored using fura-2 as a fluorescent Ca(2+) indicator. At a concentration above 1 microM, fendiline increased [Ca(2+)](i) in a concentration-dependent manner with an EC(50) value of 7 microM. The [Ca(2+)](i) response consisted of an immediate rise and an elevated phase. Extracellular Ca(2+) removal decreased half of the [Ca(2+)](i )signal. Fendiline induced quench of fura-2 fluorescence by Mn(2+) (50 microM), suggesting the presence of Ca(2+) influx across the plasma membrane. This Ca(2+) influx was abolished by La(3+) (50 microM), but was insensitive to dihydropyridines, verapamil and diltiazem. Fendiline (10 microM)-induced store Ca(2+) release was largely reduced by pretreatment with thapsigargin (1 microM) (an endoplasmic reticulum Ca(2+) pump inhibitor) to deplete the endoplasmic reticulum Ca(2+). Conversely, pretreatment with 10 microM fendiline abolished thapsigargin-induced Ca(2+) release. Fendiline (10 microM)-induced Ca(2+) release was not altered by inhibiting phospholipase C with 2 microM 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122). Cumulatively, this study shows that fendiline induced concentration-dependent [Ca(2+)](i )increases in corneal epithelial cells by releasing the endoplasmic reticulum Ca(2+) in a phospholipase C-independent manner, and by causing Ca(2+) influx.     

Mos is not required for the initiation of meiotic maturation in Xenopus oocytes

In Xenopus oocytes, the c-mos proto-oncogene product has been proposed to act downstream of progesterone to control the entry into meiosis I, the transition from meiosis I to meiosis II, which is characterized by the absence of S phase, and the metaphase II arrest seen prior to fertilization. Here, we report that inhibition of Mos synthesis by morpholino antisense oligonucleotides does not prevent the progesterone-induced initiation of Xenopus oocyte meiotic maturation, as previously thought. Mos-depleted oocytes complete meiosis I but fail to arrest at metaphase II, entering a series of embryonic-like cell cycles accompanied by oscillations of Cdc2 activity and DNA replication. We propose that the unique and conserved role of Mos is to prevent mitotic cell cycles of the female gamete until the fertilization in Xenopus, starfish and mouse oocytes.