The guanylate binding protein-1 GTPase controls the invasive and angiogenic capability of endothelial cells through inhibition of MMP-1 expression

Expression of the large GTPase guanylate binding protein-1 (GBP-1) is induced by inflammatory cytokines (ICs) in endothelial cells (ECs), and the helical domain of the molecule mediates the repression of EC proliferation by ICs. Here we show that the expression of GBP-1 and of the matrix metalloproteinase-1 (MMP-1) are inversely related in vitro and in vivo, and that GBP-1 selectively inhibits the expression of MMP-1 in ECs, but not the expression of other proteases. The GTPase activity of GBP-1 was necessary for this effect, which inhibited invasiveness and tube-forming capability of ECs in three-dimensional collagen-I matrices. A GTPase-deficient mutant (D184N-GBP-1) operated as a transdominant inhibitor of wild-type GBP-1 and rescued MMP-1 expression in the presence of ICs. Expression of D184N-GBP-1, as well as paracrine supplementation of MMP-1, restored the tube-forming capability of ECs in the presence of wild-type GBP-1. The latter finding indicated that the inhibition of capillary formation is specifically due to the repression of MMP-1 expression by GBP-1, and is not affected by the anti-proliferative activity of the helical domain of GBP-1. These findings substantiate the role of GBP-1 as a major regulator of the anti-angiogenic response of ECs to ICs.     

The structure of ActVA-Orf6, a novel type of monooxygenase involved in actinorhodin biosynthesis

ActVA-Orf6 monooxygenase from Streptomyces coelicolor that catalyses the oxidation of an aromatic intermediate of the actinorhodin biosynthetic pathway is a member of a class of small monooxygenases that carry out oxygenation without the assistance of any of the prosthetic groups, metal ions or cofactors normally associated with activation of molecular oxygen. The overall structure is a ferredoxin-like fold with a novel dimeric assembly, indicating that the widely represented ferredoxin fold may sustain yet another functionality. The resolution (1.3 A) of the enzyme structure and its complex with substrate and product analogues allows us to visualize the mechanism of binding and activation of the substrate for attack by molecular oxygen, and utilization of two gates for the reaction components including a proton gate and an O(2)/H(2)O gate with a putative protein channel. This is the first crystal structure of an enzyme involved in the tailoring of a type II aromatic polyketide and illustrates some of the enzyme-substrate recognition features that may apply to a range of other enzymes involved in modifying a polyketide core structure.     

Relationship between the action of reactive oxygen and nitrogen species on bilayer membranes and antioxidants

Membrane lipid peroxidation (LPO) induced by hydroxyl (*OH) and ascorbyl (*Asc) radicals and by peroxynitrite (ONOO-) was investigated in asolectin (ASO), egg phosphatidylcholine (PC) and PC/phosphatidic acid mixtures (PC:PA) liposomes and rat liver microsomes (MC). Enthalpy variation (DeltaH) of PC:PA at different molar ratios were obtained by differential scanning calorimetry. It was also evaluated the LPO inhibition by quercetin, melatonin and Vitamin B6. The oxidant effect power follows the order *OH approximately *Asc > ONOO- on PC and MC; whilst on ASO liposomes, it follows *Asc > *OH approximately ONOO-. Increasing amounts of PA in PC liposomes resulted in lower levels of LPO. The DeltaH values indicate a more ordered membrane arrangement as a function of PA amount. The results were discussed in order to provide a complete view involving the influence of membranes, oxidants and antioxidants intrinsic behavior on the LPO dynamics.     

Effect of lipopolysaccharides on human dendritic cell cultures and its inhibition by polymyxin B

Dendritic cells have been described as effective antigen presenting cells. Human dentritic cells are highly susceptible to lipopolysaccharide (LPS) tolerance, consisting of a differential deactivation state in which some cellular functions are impaired. LPS tolerance can be experimentally induced in vitro, in which the presence of LPS strongly affects the behavior of cultured dendritic cells. Recombinant proteins obtained from bacterial systems or protein extracts of ectoparasites containing LPS can be used as stimuli to enhance maturation processes in these cells. The present study evaluated the effect of LPS in human dendritic cell cultures, and the activity of polymyxin B as an inhibitor of the LPS effect. Dendritic cells were obtained from peripheral blood monocytes in the presence of IL-4 and GM-CSF, followed by exposure with LPS and PGE2/TNFalpha. Surface markers and cytokine levels were evaluated by flow cytometry. The dendritic cells pre-exposed to single doses of endotoxin demonstrated a reduced capacity to mature, reduced CD83 expression, inhibited secretion of IL-12, TNFalpha, IL-10 and diminished secretion of IL-6. Furthermore, polymyxin B at 10 mg/ml inhibits LPS activity at 1 mg/ml. The maximum polymyxin B concentration with no effect on cellular morphology was 50 mg/ml. Consequently, polymyxin B was determined to be an effective LPS inhibitor in dendritic cell cultures.     

Correlation between the physicochemical properties of organic solvents and their biocompatibility toward epoxide hydrolase activity in whole-cells of a yeast, Rhodotorula sp.

Epoxides are often highly hydrophobic substrates and the presence of an organic co-solvent within an aqueous bioreactor is in such cases indicated. The effect of 40 water-miscible and -immiscible organic solvents on epoxide hydrolase activity in whole-cells of the yeast Rhodotorula sp. UOFS Y-0448 was investigated. No formal correlation between solvent biocompatibility and physicochemical properties was deductible, although the introduction of hydroxyl groups increased biocompatibility. 1-Pentanol, 2-methylcyclohexanol and 1-octanol were the most biocompatible resulting in relatively low activity losses when used at up to 20% (v/v).     

Effect of thyroid hormones on the gene expression of calcium transport systems in rat muscles

It has been previously shown that modification of thyroid hormone levels have a profound impact on cardiac function, predominantly through a direct regulation of the sarcoplasmic reticulum protein levels. Nevertheless, little is known about the regulation of calcium transport systems in skeletal muscle due to the altered concentration of thyroid hormones. Thus, the goal of our study was to find out whether altered thyroid status could change the gene expression of the Na(+)/Ca(2+) exchanger (NCX), the inositol 1,4,5-trisphosphate (IP(3)) receptors and ryanodine receptors (RyRs) in slow and fast skeletal muscles of rats. A hyperthyroid state was maintained in rats by triiodothyronine (T(3)) administration, while methimazole was employed for inducing hypothyroidism. After a period of 2-10 months of T(3) treatment we observed a significant increase in mRNA levels of the NCX, RyRs and IP(3) receptors. This increase was more pronounced in the slow soleus than in the fast extensor digitorum longus (EDL) muscle. It is tempting to speculate that thyroid hormones also alter calcium concentration and thus influence the process of excitation-contraction coupling in the skeletal muscle.     

Sensitivity and variability of visual scoring in the comet assay. Results of an inter-laboratory scoring exercise with the use of silver staining

Nineteen scorers from seven Cuban laboratories participated in this slide exercise designed to test the influence of the scorer on the accuracy, sensitivity and variability of the comet assay when a visual method of DNA damage evaluation is used. The assay was performed using human lymphocytes from a single donor exposed in vitro for 5 min at 0 degrees C to doses of 0, 5, 10, 25, 50, 100 and 200 microM of hydrogen peroxide. Each participant scored the same set of 14 coded slides with silver stained comets. The comets were classified visually into five categories according to the appearance resulting from the relative proportion of DNA in the tail. The extent of DNA damage was expressed in arbitrary units. At zero dose the median values of 12 scorers out of 19 were included between the values of the overall 25 and 75 per thousand. This proportion remains practically the same as the dose increases. The lowest dose detected by this method for the majority of scorers (11) was 10 microM. The coefficient of variation at the control dose was the highest (median value 26%), progressively declined to 20%, and starting from 25 microM, values are around 10%. The results of the exercise show the reliability of the silver staining and visual scoring for the comet method.     

Enhanced photosynthesis and redox energy production contribute to salinity tolerance in Dunaliella as revealed by homology-based proteomics

Salinity is a major limiting factor for the proliferation of plants and inhibits central metabolic activities such as photosynthesis. The halotolerant green alga Dunaliella can adapt to hypersaline environments and is considered a model photosynthetic organism for salinity tolerance. To clarify the molecular basis for salinity tolerance, a proteomic approach has been applied for identification of salt-induced proteins in Dunaliella. Seventy-six salt-induced proteins were selected from two-dimensional gel separations of different subcellular fractions and analyzed by mass spectrometry (MS). Application of nanoelectrospray mass spectrometry, combined with sequence-similarity database-searching algorithms, MS BLAST and MultiTag, enabled identification of 80% of the salt-induced proteins. Salinity stress up-regulated key enzymes in the Calvin cycle, starch mobilization, and redox energy production; regulatory factors in protein biosynthesis and degradation; and a homolog of a bacterial Na(+)-redox transporters. The results indicate that Dunaliella responds to high salinity by enhancement of photosynthetic CO(2) assimilation and by diversion of carbon and energy resources for synthesis of glycerol, the osmotic element in Dunaliella. The ability of Dunaliella to enhance photosynthetic activity at high salinity is remarkable because, in most plants and cyanobacteria, salt stress inhibits photosynthesis. The results demonstrated the power of MS BLAST searches for the identification of proteins in organisms whose genomes are not known and paved the way for dissecting molecular mechanisms of salinity tolerance in algae and higher plants.     

Molecular cloning and 3D structure prediction of the first raw-starch-degrading glucoamylase without a separate starch-binding domain

Raw-starch-degrading glucoamylases have been known as multidomain enzymes consisting of a catalytic domain connected to a starch-binding domain (SBD) by an O-glycosylated linker region. A molecular genetics approach has been chosen to find structural differences between two related glucoamylases, raw-starch-degrading Glm and nondegrading Glu, from the yeasts Saccharomycopsis fibuligera IFO 0111 and HUT 7212, respectively. We have found that Glm and Glu show a high primary (77%) and tertiary structure similarity. Glm, although possessing a good ability for raw starch degradation, did not show consensus amino acid residues to any SBD found in glucoamylases or other amylolytic enzymes. Raw starch binding and digestion by Glm must thus depend on the existence of a site(s) lying within the intact protein which lacks a separate SBD. The enzyme represents a structurally new type of raw-starch-degrading glucoamylase.