Bacillus subtilis genome project: cloning and sequencing of the 97 kb region from 325° to 333deg;

In the framework of the European project aimed at the sequencing of the Bacillus subtilis genome the DNA region located between gerB (314°) and sacXV (333°) was assigned to the Institut Pasteur. In this paper we describe the cloning and sequencing of a segment of 97 kb of contiguous DNA. Ninety-two open reading frames were predicted to encode putative proteins among which only forty-two were found to display significant similarities to known proteins present in databanks, e.g. amino acid permeases, proteins involved in cell wall or antibiotic biosynthesis, various regulatory proteins, proteins of several dehydrogenase families and enzymes II of the phosphotransferase system involved in sugar transport. Additional experiments led to the identification of the products of new B. subtilis genes, e.g. galactokinase and an operon involved in thiamine biosynthesis.     

Potassium and sodium ions in the glycerinated skeletal muscle. Distribution changes induced by adenosine triphosphate and nondissociable anesthetic substances.

Investigation of the ionic behavior of glycerinated muscle fibers showed that the residual structures of this biologic cellular material, lacking functional membranes, are able to discriminate between alkaline ions. The characteristics of the ionic selectivity of the glycerinated fibers change with their functional state and with the presence in the medium of certain nonionic substances. Among the more important features of ionic distribution between the membrane-free fibers and the medium are the following: (1) There is evident adsorption of potassium on the fibers, in the absence of ATP. (2) This adsorption increases in contraction and decreases in relaxation. (3) At high ionic concentrations, in contrast to what occurs at low potassium concentrations, the glycerinated muscle prefers sodium to potassium, but even under these conditions both ions are accumulated in the fibers to far greater levels than in the medium. This strongly suggests a Donnan ionic equilibrium developing parallel to the adsorption process. (4) Nonionic substances of the general anesthetic group markedly alter the ionic selectivity of the glycerinated fibers, probably by their action on the water's physical state. A mechanism is proposed for the observed ionic adsorption specific of the muscle-a mechanism in which actin-myosin coupling plays the cardinal adsorption role. In the general interpretation of the data a synthetic concept is advanced according to which an entire set of processes and factors concurs with the distribution of ions between the muscle and the medium.     

Peptides of type II collagen can induce the cleavage of type II collagen and aggrecan in articular cartilage.

The objective of this study was to determine whether a fragment(s) of type II collagen can induce cartilage degradation. Fragments generated by cyanogen bromide (CB) cleavage of purified bovine type II collagen were separated by HPLC. These fragments together with selected overlapping synthetic peptides were first analysed for their capacity to induce cleavage of type II collagen by collagenases in chondrocyte and explant cultures of healthy adult bovine articular cartilage. Collagen cleavage was measured by immunoassay and degradation of proteoglycan (mainly aggrecan) was determined by analysis of cleavage products of core protein by Western blotting. Gene expression of matrix metalloproteinases MMP-13 and MMP-1 was measured using Real-time PCR. Induction of denaturation of type II collagen in situ in cartilage matrix with exposure of the CB domain was identified with a polyclonal and monoclonal antibodies that only react with this domain in denatured but not native type II collagen. As well as the mixture of CB fragments and peptide CB12, a single synthetic peptide CB12-II (residues 195-218), but not synthetic peptide CB12-IV (residues 231-254), potently and consistently induced in explant cultures at 10 microM and 25 microM, in a time, cell and dose dependent manner, collagenase-induced cleavage of type II collagen accompanied by upregulation of MMP-13 expression but not MMP-1. In isolated chondrocyte cultures CB12-II induced very limited upregulation of MMP-13 as well as MMP-1 expression. Although this was accompanied by concomitant induction of cleavage of type II collagen by collagenases, this was not associated by aggrecan cleavage. Peptide CB12-IV, which had no effect on collagen cleavage, clearly induced aggrecanase specific cleavage of the core protein of this proteoglycan. Thus these events involving matrix molecule cleavage can importantly occur independently of each other, contrary to popular belief. Denaturation of type II collagen with exposure of the CB12-II domain was also shown to be much increased in osteoarthritic human cartilage compared to non-arthritic cartilage. These observations reveal that peptides of type II collagen, to which there is increased exposure in osteoarthritic cartilage, can when present in sufficient concentration induce cleavage of type II collagen (CB12-II) and aggrecan (CB12-IV) accompanied by increased expression of collagenases. Such increased concentrations of denatured collagen are present in adult and osteoarthritic cartilages and the exposure of chondrocytes to the sequences they encode, either in soluble or more likely insoluble form, may therefore play a role in the excessive resorption of matrix molecules that is seen in arthritis and development.     

Modeling interactions between voltage-gated Ca2+ channels and KCa1.1 channels

High voltage-activated (HVA) Cav channels form complexes with KCa1.1 channels, allowing reliable activation of KCa1.1 current through a nanodomain interaction. We recently found that low voltage-activated Cav3 calcium channels also create KCa1.1-Cav3 complexes. While coimmunoprecipitation studies again supported a nanodomain interaction, the sensitivity to calcium chelating agents was instead consistent with a microdomain interaction. A computational model of the KCa1.1-Cav3 complex suggested that multiple Cav3 channels were necessary to activate KCa1.1 channels, potentially causing the KCa1.1-Cav3 complex to be more susceptible to calcium chelators. Here, we expanded the model and compared it to a KCa1.1-Cav2.2 model to examine the role of Cav channel conductance and kinetics on KCa1.1 activation. As found for direct recordings, the voltage-dependent and kinetic properties of Cav3 channels were reflected in the activation of KCa1.1 current, including transient activation from lower voltages than other KCa1.1-Cav complexes. Substantial activation of KCa1.1 channels required the concerted activity of several Cav3.2 channels. Combined with the effect of EGTA, these results suggest that the Ca²⁺ domains of several KCa1.1-Cav3 complexes need to cooperate to generate sufficient [Ca²⁺]_i, despite the physical association between KCa1.1 and Cav3 channels. By comparison, Cav2.2 channels were twice as effective at activating KCa1.1 channels and a single KCa1.1-Cav2.2 complex would be self-sufficient. However, even though Cav3 channels generate small, transient currents, the regulation of KCa1.1 activity by Cav3 channels is possible if multiple complexes cooperate through microdomain interactions.     

Large Scale Fabrication of Gold Nano-Structured Substrates Via High Temperature Annealing and Their Direct Use for the LSPR Detection of Atrazine

The present work is reporting on the fabrication of localized surface plasmonic resonant (LSPR) gold nano-structures on glass substrate by using different high annealing temperatures (500 °C, 550 °C, 600 °C) of initially created semi-continue gold films (2 nm and 5 nm) by the electron beam evaporation technique. Interestingly, well-defined gold nano-structures were also obtained from continuous 8 nm evaporated gold film - known as the value above gold percolated thickness - once exposed to high temperatures. The surface morphology and plasmonic spectroscopy of “annealed” nano-structures were controlled by key experimental parameters such as evaporated film thickness and annealing temperature. By using scanning electron microscopy (SEM) characterization of annealed surface it was noticed that the size and inter-particle distance between nano-structures were highly dependent on the evaporated thin film thickness, while the nanoparticle shape evolution was mainly affected by the employed annealing temperature. Due to the well-controlled morphology of gold nano-particles, prominent and stable LSPR spectra were observed with good plasmon resonance tunability from 546 nm to 780 nm that recommend the developed protocol as a robust alternative to fabricate large scale LSPR surface. An example of a LSPR-immunosensor is reported. Thus, the monoclonal anti-atrazine antibodies immobilizion on the “annealed” gold nano-structures, as well as the specific antigen (atrazine) recognition were monitored as variations of the resonance wavelength shifts and optical density changes in the extinction measurements.     

Topical Reappraisal of Molecular Pharmacological Approaches to Endothelial Dysfunction in Diabetes Mellitus Angiopathy

Diabetes mellitus (DM) is a frequent medical problem, affecting more than 4% of the population in most countries. In the context of diabetes, the vascular endothelium can play a crucial pathophysiological role. If a healthy endothelium—which is a dynamic endocrine organ with autocrine and paracrine activity—regulates vascular tone and permeability and assures a proper balance between coagulation and fibrinolysis, and vasodilation and vasoconstriction, then, in contrast, a dysfunctional endothelium has received increasing attention as a potential contributor to the pathogenesis of vascular disease in diabetes. Hyperglycemia is indicated to be the major causative factor in the development of endothelial dysfunction. Furthermore, many shreds of evidence suggest that the progression of insulin resistance in type 2 diabetes is parallel to the advancement of endothelial dysfunction in atherosclerosis. To present the state-of-the-art data regarding endothelial dysfunction in diabetic micro- and macroangiopathy, we constructed this literature review based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA). We interrogated five medical databases: Elsevier, PubMed, PMC, PEDro, and ISI Web of Science.     

Patterns of sequence variation in the mitochondrial D-loop region of shrews

Direct sequencing of the mitochondrial displacement loop (D-loop) of shrews (genus Sorex) for the region between the tRNA(Pro) and the conserved sequence block-F revealed variable numbers of 79-bp tandem repeats. These repeats were found in all 19 individuals sequenced, representing three subspecies and one closely related species of the masked shrew group (Sorex cinereus cinereus, S. c. miscix, S. c. acadicus, and S. haydeni) and an outgroup, the pygmy shrew (S. hoyi). Each specimen also possessed an adjacent 76-bp imperfect copy of the tandem repeats. One individual was heteroplasmic for length variants consisting of five and seven copies of the 79-bp tandem repeat. The sequence of the repeats is conducive to the formation of secondary structure. A termination-associated sequence is present in each of the repeats and in a unique sequence region 5' to the tandem array as well. Mean genetic distance between the masked shrew taxa and the pygmy shrew was calculated separately for the unique sequence region, one of the tandem repeats, the imperfect repeat, and these three regions combined. The unique sequence region evolved more rapidly than the tandem repeats or the imperfect repeat. The small genetic distance between pairs of tandem repeats within an individual is consistent with a model of concerted evolution. Repeats are apparently duplicated and lost at a high rate, which tends to homogenize the tandem array. The rate of D- loop sequence divergence between the masked and pygmy shrews is estimated to be 15%-20%/Myr, the highest rate observed in D-loops of mammals. Rapid sequence evolution in shrews may be due either to their high metabolic rate and short generation time or to the presence of variable numbers of tandem repeats.