Expression of cathepsin K is regulated by shear stress in cultured endothelial cells and is increased in endothelium in human atherosclerosis

Cathepsins, the lysosomal cysteine proteases, are involved in vascular remodeling and atherosclerosis. Genetic knockout of cathepsins S and K in mice has shown to reduce atherosclerosis, although the molecular mechanisms remain unclear. Because atherosclerosis preferentially occurs in arteries exposed to disturbed flow conditions, we hypothesized that shear stress would regulate cathepsin K expression and activity in endothelial cells. Mouse aortic endothelial cells (MAEC) exposed to proatherogenic oscillatory shear (OS, +/- 5 dyn/cm(2) for 1 day) showed significantly higher cathepsin K expression and activity than that of atheroprotective, unidirectional laminar shear stress (LS, 15 dyn/cm(2) for 1 day). Western blot and active-site labeling studies showed an active, mature form of cathepsin K in the conditioned medium of MAEC exposed to OS but not in that of LS. Functionally, MAEC exposed to OS significantly increased elastase and gelatinase activity above that of LS. The OS-dependent elastase and gelatinase activities were significantly reduced by knocking down cathepsin K with small-interfering (si) RNA, but not by a nonsilencing siRNA control, suggesting that cathepsin K is a shear-sensitive protease. In addition, immunohistochemical analysis of atherosclerotic human coronary arteries showed a positive correlation between the cathepsin K expression levels in endothelium and elastic lamina integrity. These findings suggest that cathepsin K is a mechanosensitive, extracellular matrix protease that, in turn, may be involved in arterial wall remodeling and atherosclerosis.     

Seasonal variations in the intermediate metabolism of Parastacus varicosus (Crustacea, Decapoda, Parastacidae)

The aim of this study was to analyze the seasonal variations in the metabolism of Parastacus varicosus and examine the possible relationships to its reproduction. Animals were sampled (9 h to 10 h) in each month in the Gravataí River, RS, Brazil. Haemolymph samples were collected from each crayfish in the field for determination of glucose, total proteins, total lipids, and total cholesterol. Hepatopancreas, abdominal muscle, and gonads were removed for determination of glycogen, total proteins, total lipids, and total cholesterol. ANOVA revealed significant seasonal differences in the biochemical composition of all tissues studied; when the sexes were compared these parameters did not show any significant difference in the hepatopancreas and muscle. However, in haemolymph we observed significant variation only in cholesterol and lipid levels. The results suggest that the metabolic variability is related to the stage of maturation of the gonads, in females, where the hepatopancreas and other tissue studies can store and transfer reserves to support maturation to complement the food intake. Variations in the gonadosomatic and hepatosomatic indices suggest that reproduction occurs principally in summer. As in other decapods, abiotic factors such as water temperature, oxygen content, etc. influence the intermediate metabolism.     

Asymmetric biodegradation of the nerve agents Sarin and VX by human dUTPase: chemometrics,molecular docking and hybrid QM/MM calculations

Organophosphorus compounds (OP) nerve agents are among the most toxic chemical substances known. Their toxicity is due to their ability to bind to acetylcholinesterase. Currently, some enzymes, such as phosphotriesterase, human serum paraoxonase 1 and diisopropyl fluorophosphatase, capable of degrading OP, have been characterized. Regarding the importance of bioremediation methods for detoxication of OP, this work aims to study the interaction modes between the human human deoxyuridine triphosphate nucleotidohydrolase (dUTPase) and Sarin and VX, considering their R_p and S_p enantiomers, to evaluate the asymmetric catalysis of those compounds. In previous work, this enzyme has shown good potential to degrade phosphotriesters, and based on this characteristic, we have applied the human dUTPase to the OP degradation. Molecular docking, chemometrics and mixed quantum and molecular mechanics calculations have been employed, showing a good interaction between dUTPase and OP. Two possible reaction mechanisms were tested, and according to our theoretical results, the catalytic degradation of OP by dUTPase can take place via both mechanisms, beyond being stereoselective, that is, dUTPase cleaves one enantiomer preferentially in relation to other. Chemometric techniques provided excellent assistance for performing this theoretical investigation. The dUTPase study shows importance by the fact of it being a human enzyme.

Communicated by Ramaswamy H. Sarma     

Photobiomodulation increases intrusion tooth movement and modulates IL‐6, IL‐8 and IL‐1β expression during orthodontically bone remodeling

This study investigated the effects of photobiomodulation (PBM) on upper molar intrusion movement, regarding acceleration of orthodontic movement and its molecular effects. The sample consisted of 30 patients with indication of tooth intrusion for oral rehabilitation. Teeth were divided into three different groups: G1 (n = 10) pre‐molars without force or laser application (control); G2 (n = 10) upper molar intrusion; and G3 (n = 10) upper molar intrusion and PBM. On PBM treated molars, the teeth were irradiated with a low‐power diode laser (808 nm, 100 mW), receiving 1 J per point, density of 25 J/cm², with application of 10 s per point, 10 points (5 per vestibular and 5 per palatal region). Orthodontic force of intrusion applied every 30 days and PBM was performed immediately, 3 and 7 days after force application for 3 months. Gingival crevicular fluid was collected at the same time periods as the laser applications and interleukins (IL) 1‐β, ‐6 and ‐8 were evaluated by enzyme‐linked immunosorbent assay. Clinical measures were performed monthly to verify the amount of intrusion. The levels of IL‐6, IL‐8 and IL‐1β increased under orthodontic force (G2 and G3) when compared to control group (G1), however, the cytokines levels were significantly higher after PBM (G3). The mean intrusion velocity was 0.26 mm/month in the irradiated group (G3), average duration of 8 months vs 0.17 mm/month for the non‐irradiated group (G2), average duration of 12 months. This study suggests that PBM accelerates tooth movement during molar intrusion, due to modulation of IL‐6, IL‐8 and IL‐1β during bone remodeling.      

Characterization of a Nitrilase and a Nitrile Hydratase from Pseudomonas sp. Strain UW4 That Converts Indole-3-Acetonitrile to Indole-3-Acetic Acid

Indole-3-acetic acid (IAA) is a fundamental phytohormone with the ability to control many aspects of plant growth and development. Pseudomonas sp. strain UW4 is a rhizospheric plant growth-promoting bacterium that produces and secretes IAA. While several putative IAA biosynthetic genes have been reported in this bacterium, the pathways leading to the production of IAA in strain UW4 are unclear. Here, the presence of the indole-3-acetamide (IAM) and indole-3-acetaldoxime/indole-3-acetonitrile (IAOx/IAN) pathways of IAA biosynthesis is described, and the specific role of two of the enzymes (nitrilase and nitrile hydratase) that mediate these pathways is assessed. The genes encoding these two enzymes were expressed in Escherichia coli, and the enzymes were isolated and characterized. Substrate-feeding assays indicate that the nitrilase produces both IAM and IAA from the IAN substrate, while the nitrile hydratase only produces IAM. The two nitrile-hydrolyzing enzymes have very different temperature and pH optimums. Nitrilase prefers a temperature of 50°C and a pH of 6, while nitrile hydratase prefers 4°C and a pH of 7.5. Based on multiple sequence alignments and motif analyses, physicochemical properties and enzyme assays, it is concluded that the UW4 nitrilase has an aromatic substrate specificity. The nitrile hydratase is identified as an iron-type metalloenzyme that does not require the help of a P47K activator protein to be active. These data are interpreted in terms of a preliminary model for the biosynthesis of IAA in this bacterium.     

Gastric pythiosis in a dog

BackgroundPythiosis is caused by the agent Pythium insidiosum, an aquatic oomycete of the kingdom Stramenopila.AimsTo describe the symptoms, pathological changes and diagnosis methods of gastric pythiosis in dogs.MethodsA three-year-old female German shepherd, with access to wetlands, was attended due to vomiting and recurrent diarrhea of 30 days of duration. A palpable mass in the abdomen filling the left epigastric region was identified in the clinical examination. Simple and contrasted radiological examination and ultrasound of abdominal cavity were performed. The animal was referred for exploratory laparotomy for the removal of the mass. The extent of the mass prevented from the excision and the animal was euthanized. Samples of the tumor mass were collected and sent for morphological study and immunohistochemistry.ResultsThe changes observed in imaging studies were consistent with gastric pythiosis. In cytology and histopathology, non-septate hyphae were identified, and in immunohistochemistry a strong positivity of anti-Pythium antibodies was observed, confirming the diagnosis of pythiosis.ConclusionsPythiosis in dogs is diagnosed late and tends to evolve in the animal's death. The definitive diagnosis is by cytology, histology and immunohistochemistry.     

Biologically active abietane and ent-kaurane diterpenoids and other constituents from Erythroxylum suberosum

Bioassay-guided fractionation of the ethanol extract from the branches of Erythroxylum suberosum, which was toxic to brine shrimp larvae, afforded five diterpenes bearing abietane and ent-kaurane-type skeletons from an active fraction. From these, four were new, 7-oxo-16-hydroxy-abiet-15(17)-en-19-al, 16-hydroxyabiet-15(17)-en-7-one, 7α,16-dihydroxy-abiet-15(17)-en-19-al and ent-12α-hydroxy-kaur-16-en-19-al, while methyl ent-7α,15β-dihydroxy-kaur-16-en-19-oate is reported for the first time as a natural product. This is also the first reported occurrence of abietane-type diterpenes in the genus Erythroxylum. The flavonoid ombuin-3-rutinoside was isolated from an inactive fraction, while rutin (quercetin-3-rutinoside) was obtained from the non-toxic ethanol extract of the leaves. The structures of the new and known compounds were established by analyses of 1D- and 2D-NMR and mass spectrometry data.     

Identification of a new quaternary association for legume lectins

Lotus tetragonolobus lectin (LTA) is a fucose-specific legume lectin. Although several studies report a diverse combination of biological activities for LTA, little is known about the mechanisms involved in l-fucosyl oligosaccharide recognition. The crystal structure of LTA at 2.0A resolution reveals a different legume lectin tetramer. Its structure consists of a homotetramer composed of two back-to-back GS4-like dimers arranged in a new mode, resulting in a novel tetramer. The LTA N-linked carbohydrate at Asn4 and the unusual LTA dimer-dimer interaction are related to its particular mode of tetramerization. In addition, we used small angle X-ray scattering to investigate the quaternary structure of LTA in solution and to compare it to the crystalline structure. Although the crystal structure of LTA has revealed a conserved metal-binding site, its l-fucose-binding site presents some punctual differences. Our investigation of the new tetramer of LTA and its fucose-binding site is essential for further studies related to cross-linking between LTA and complex divalent l-fucosyl carbohydrates.     

Methanogenic archaea: ecologically relevant differences in energy conservation

Most methanogenic archaea can reduce CO(2) with H(2) to methane, and it is generally assumed that the reactions and mechanisms of energy conservation that are involved are largely the same in all methanogens. However, this does not take into account the fact that methanogens with cytochromes have considerably higher growth yields and threshold concentrations for H(2) than methanogens without cytochromes. These and other differences can be explained by the proposal outlined in this Review that in methanogens with cytochromes, the first and last steps in methanogenesis from CO(2) are coupled chemiosmotically, whereas in methanogens without cytochromes, these steps are energetically coupled by a cytoplasmic enzyme complex that mediates flavin-based electron bifurcation.     

Length‐weight relationships for freshwater fish species from humid forest enclaves at the Brazilian semiarid

We report the length‐weight relationships (LWR) for 11 fish species inhabiting headwater streams in the Araripe and Ibiapaba plateaus, Brazilian semiarid. Values of r², a and b ranged respectively from .953 to .998, 0.010 to 0.043 and 2.81 to 3.69. Our reports on LWR are pioneer in humid forest enclaves at the Caatinga biome. Such enclaves of forest can be a checkpoint to climate change, but are under strong human pressure and remain under low investigation.