The O-antigen gene cluster of Escherichia coli O55:H7 and identification of a new UDP-GlcNAc C4 epimerase gene

Escherichia coli O55 is an important antigen which is often associated with enteropathogenic E. coli clones. We sequenced the genes responsible for its synthesis and identified genes for O-antigen polymerase, O-antigen flippase, four enzymes involved in GDP-colitose synthesis, and three glycosyltransferases, all by comparison with known genes. Upstream of the normal O-antigen region there is a gne gene, which encodes a UDP-GlcNAc epimerase for converting UDP-GlcNAc to UDP-GalNAc and is essential for O55 antigen synthesis. The O55 gne product has only 20 and 26% identity to the gne genes of Pseudomonas aeruginosa and E. coli O113, respectively. We also found evidence for the O55 gene cluster's having evolved from another gene cluster by gain and loss of genes. Only three of the GDP-colitose pathway genes are in the usual location, the other two being separated, although nearby. It is thought that the E. coli O157:H7 clone evolved from the O55:H7 clone in part by transfer of the O157 gene cluster into an O55 lineage. Comparison of genes flanking the O-antigen gene clusters of the O55:H7 and O157:H7 clones revealed one recombination site within the galF gene and located the other between the hisG and amn genes. Genes outside the recombination sites are 99.6 to 100% identical in the two clones, while most genes thought to have transferred with the O157 gene cluster are 95 to 98% identical.     

D-003, a potential antithrombotic compound isolated from sugar cane wax with effects on arachidonic acid metabolites

D-003 is a natural mixture of higher primary saturated aliphatic acids purified from sugar cane wax, whose main component is octacosanoic acid followed by triacontanoic, dotriacontanoic, and tetratriacontanoic acids. D-003 inhibits platelet aggregation and arterial thrombosis experimentally induced in a dose-dependent fashion. This study was undertaken to investigate the effects of D-003 (25 and 200 mg/kg) in experimental models of venous thrombosis and on plasma levels of two metabolites from arachidonic acid (AA) : thromboxane A(2) (TxA(2)) and prostacyclin (PgI(2)). D-003 orally administered as single doses of 200 mg/kg, but not at 25 mg/kg, significantly increased plasma levels of 6 keto PgF1alpha levels, a stable metabolite of PgI(2) in PPP obtained from collagen-stimulated blood (4 microg/ml) compared with control group. Nevertheless, levels of 6 keto PgF1alpha levels determined after 10 days of oral treatment with both doses of D-003 were significantly larger than those of the controls. Likewise, single and repeated oral doses of D-003 (25 and 200 g/kg) significantly reduced the TxB(2) and MDA plasma levels obtained from whole blood stimulated by collagen. Hence, TxB(2)/6 keto PgF1alpha ratio significantly decreased in animals treated with D-003. Single and repeated oral doses of D-003 (25 and 200 mg/kg) significantly reduced the weight of venous thrombus experimentally induced in rats. D-003 at single doses (400 mg/kg but not 200 mg/kg) significantly protected from death induced by endovenous infusion of collagen plus epinephrine in mice. The present results support that these effects of D-003 on AA metabolites could explain, at least partially, its antiplatelet and antithrombotic effects.     

Cultivation of explants of the marine sponge Crambe crambe in closed systems

Explants of the sponge Crambe crambe were cultured in natural seawater, with or without marine microalga (Phaeodactylum tricornutum) in discontinuous flow through systems and in continuous flow-through systems (DFTHS and CFTHS, respectively). Growth was measured as the increase in underwater weight. In the experiment carried out in the CFTHS, the explants average underwater weight increased by up to 1380% of the initial weight in 22-45 days. Growth in DFTHS was much slower producing a gain of up to an average value of 322% of the initial weight in 100-210 days. Growth kinetics varied considerably for different explants. Explants grew fastest in the first 10-days of subculture. The sponges grew better in CFTHS compared with the DFTHS. The high growth rates observed in CFTHS suggest that this technique is a promising method for culturing C. crambe in closed systems.     

Lysyl oxidase oxidizes basic fibroblast growth factor and inactivates its mitogenic potential

Lysyl oxidase (LO) plays a central role in the crosslinking of collagen and elastin in the extracellular matrix. Here we demonstrate that basic fibroblast growth factor (bFGF), a polypeptide which regulates proliferation, differentiation, and migration of a variety of cell types, is a substrate of LO. The oxidation of lysine residues in bFGF by LO resulted in the covalent crosslinking of bFGF monomers to form dimers and higher order oligomers and dramatically altered its biological properties. Both the mitogenic potential and the nuclear localization of bFGF were markedly inhibited in the Swiss 3T3 cells upon its oxidation by LO. NIH 3T3 IgBNM 6-1 cells (6-1 cells) overexpress bFGF which participates in an autocrine mechanism accounting for the transformation of these cells into a tumorigenic state. Exposure of the 6-1 cells to nanomolar concentrations of LO in culture oxidized lysine and generated crosslinkages in bFGF within the cell and markedly reduced proliferative rates. The lack of LO expression has been correlated with hyperproliferative cell growth, while this enzyme has been identified as a suppressor of ras-induced tumorigenesis. The present results illustrate a mechanism by which LO can depress normal and transformed cell growth.     

Local expression of apolipoprotein A-I gene and a possible role for HDL in primary defence in the carp skin

Antimicrobial proteins and peptides play an important role in the primary defence of epithelial barriers in vertebrates and invertebrates. Here we report the detection of the apolipoproteins A-I and A-II in the epidermis and epidermal mucus of the carp (Cyprinus carpio L.) by immunohistochemistry and Western blot analysis. Both apolipoproteins are major constituents of high density lipoprotein and have been shown to display antiviral and antimicrobial activity in mammals. Therefore the aim of this study was to evaluate if they could be part of the innate immune system of teleost fish. A cDNA clone containing most of the coding region for carp apoA-I was isolated and used as a probe to demonstrate the expression of apoA-I gene in the skin. In addition, mucus apoA-I was shown to be associated to small particles that could correspond to nascent HDL. Finally, affinity purified plasma HDL displayed bactericidal activity in vitro against a non-pathogenic Escherichia coli strain, suggesting a defensive role for HDL and its associated proteins in the carp epidermis and mucus.     

Individual cartilage aggrecan macromolecules and their constituent glycosaminoglycans visualized via atomic force microscopy

Atomic force microscopy was used in ambient conditions to directly image dense and sparse monolayers of bovine fetal epiphyseal and mature nasal cartilage aggrecan macromolecules adsorbed on mica substrates. Distinct resolution of the non-glycosylated N-terminal region from the glycosaminoglycan (GAG) brush of individual aggrecan monomers was achieved, as well as nanometer-scale resolution of individual GAG chain conformation and spacing. Fetal aggrecan core protein trace length (398+/-57 nm) and end-to-end length (257+/-87 nm) were both larger than that of mature aggrecan (352+/-88 and 226+/-81 nm, respectively). Similarly, fetal aggrecan GAG chain trace length (41+/-7 nm) and end-to-end (32+/-8 nm) length were both larger than that of mature aggrecan GAG (32+/-5 and 26+/-7 nm, respectively). GAG-GAG spacing along the core protein was significantly smaller in fetal compared to mature aggrecan (3.2+/-0.8 and 4.4+/-1.2nm, respectively). Together, these differences between the two aggrecan types were likely responsible for the greater persistence length of the fetal aggrecan (110 nm) compared to mature aggrecan (82 nm) calculated using the worm-like chain model. Measured dimensions and polymer statistical analyses were used in conjunction with the results of Western analyses, chromatographic, and carbohydrate electrophoresis measurements to better understand the dependence of aggrecan structure and properties on its constituent GAG chains.     

Specificity of binding of the plectin actin-binding domain to beta4 integrin

Plectin is a major component of the cytoskeleton and links the intermediate filament system to hemidesmosomes by binding to the integrin beta4 subunit. Previously, a binding site for beta4 was mapped on the actin-binding domain (ABD) of plectin and binding of beta4 and F-actin to plectin was shown to be mutually exclusive. Here we show that only the ABDs of plectin and dystonin bind to beta4, whereas those of other actin-binding proteins do not. Mutations of the ABD of plectin-1C show that Q131, R138, and N149 are critical for tight binding of the ABD to beta4. These residues form a small cavity, occupied by a well-ordered water molecule in the crystal structure. The beta4 binding pocket partly overlaps with the actin-binding sequence 2 (ABS2), previously shown to be essential for actin binding. Therefore, steric interference may render binding of beta4 and F-actin to plectin mutually exclusive. Finally, we provide evidence indicating that the residues preceding the ABD in plectin-1A and -1C, although unable to mediate binding to beta4 themselves, modulate the binding activity of the ABD for beta4. These studies demonstrate the unique property of the plectin-ABD to bind to both F-actin and beta4, and explain why several other ABD-containing proteins that are expressed in basal keratinocytes are not recruited into hemidesmosomes.     

Tolerance is dependent on complement C3 fragment iC3b binding to antigen-presenting cells

Systemic tolerance can be induced by the introduction of antigen into an immune-privileged site. Here we investigated the role of complement in the induction of tolerance after intraocular injection. We found that the development of antigen-specific tolerance is dependent on a complement activation product. The ligation of the complement C3 activation product iC3b to complement receptor type 3 (the iC3b receptor) on antigen-presenting cells resulted in the sequential production of transforming growth factor-beta2 and interleukin-10, which is essential for the induction of tolerance. These observations may extend to the development of both neonatal tolerance and other forms of acquired tolerance.     

Mitochondrial,sarcoplasmic membrane integrity and protein degradation in heart and skeletal muscle in exercised rats

Several different exercise regimens varied in the severity of tissue damage induced. Therefore, this study investigated the effects of a single bout of exercise versus endurance training in heart and skeletal muscles with different predominant fiber types on indices of mitochondrial, endoplasmic reticulum (ER) integrity and protein degradation. Male Wistar rats performed different treadmill exercise protocols: exhaustive, maximal exhaustive, eccentric, training and exhaustive exercise after training. The maximal and eccentric exercises resulted in a significant loss of integrity of the sarcoplasmic and ER muscle, while no changes were observed in cardiac muscle. Mitochondrial membrane fluidity measured by the fluorescence polarization method was significantly increased post-acute exercises in heart and oxidative muscles. Regular exercise can stabilize and preserve the viscoelastic nature of mitochondrial membranes in both tissues. The highest increase in carbonyl content was obtained in heart after exhaustive exercise protocol, from 1+/-0.1 to 3.6+/-0.14 nmol mg protein(-1), such increase were not found after regular exercise with values significantly decreased. Nitrate heart levels showed attenuated generation of nitric oxide after training. Muscle protein oxidation was produced in all exhaustive exercises including eccentric exercise.