Synovial fluid chondroitin sulphate indicates abnormal joint metabolism in asymptomatic osteochondritic horses

Reasons for performing study: Alternative methods to evaluate the joint condition in asymptomatic osteochondrosis dissecans (OCD) and other joint diseases may be useful. Objectives: To investigate possible changes in synovial fluid composition that may lead to joint conditions in asymptomatic OCD, in mature horses. Methods: Animals aged >2 years, of different breeds, with OCD in the intermediate ridge of distal tibia, symptomatic or not, were studied. Synovial fluid samples (10 healthy; 11 asymptomatic OCD; 25 symptomatic OCD) were collected by arthroscopy from 29 horses. Glycosaminoglycans (GAGs) were analysed by a combination of agarose gel electrophoresis and enzymatic degradation with specific GAG lyases. The viscosity, white blood cell (WBC) count, protein concentration and hyaluronic acid (HA) molecular weight were also determined. Results: The method used here to analyse synovial fluid GAGs is reliable, reproducible and specific. The main synovial fluid GAGs are HA and chondroitin sulphate (CS), 93% and 7% respectively in normal horses. In symptomatic OCD, the concentrations of both increased (expressed as GAG/urea ratios), but CS increased more. The CS increased also in asymptomatic OCD. An inflammatory reaction was suggested by the increased WBC counts in OCD. The molecular weight of the synovial fluid HA was reduced in OCD, explaining the lower viscosity observed. Conclusions: The increased CS in synovial fluid of OCD joints in mature horses suggests that the synovial fluid CS and the WBC count are good markers of the joint conditions, allowing the identification of pathological phase in joint diseases. Potential relevance: The analysis of synovial fluid GAGs shows that cartilage damage occurs even in asymptomatic OCD, implying that arthroscopic removal of osteochondral fragments should be performed even in asymptomatic OCD.

Witzkeite: A new rare nitrate-sulphate mineral from a guano deposit at Punta de Lobos, Chile

Witzkeite, ideally Na4K4Ca(NO3)(2)(SO4)(4)center dot 2H(2)O, is a new mineral found in the oxidation zone of the guano mining field at Punta de Lobos, Tarapaca region, Chile. It occurs as colorless, tabular crystals up to 140 mu m in length, associated with dittmanite and nitratine. Witzkeite is colorless and transparent, with a white streak and a vitreous luster. It is brittle, with Molts hardness 2 and distinct cleavage on {001}. Measured density is 2.40(2) g/cm(3), calculated density is 2.403 g/cm(3). Witzkeite is biaxial (-) with refractive indexes alpha = 1.470(5), beta = 1.495(5), gamma = 1.510(5), measured 2V = 50-70 degrees. The empirical composition is (electron microprobe, mean of five analyses, H2O, CO2, and N2O5 by gas chromatography; wt%): Na2O 12.83, K2O 22.64, CaO 7.57, FeO 0.44, SO3 39.96, N2O5 12.7, H2O 4.5, total 100.64; CO2 was not detected. The chemical formula, calculated based on 24 O, is: Na3.40K3.95Ca1.11Fe0.05(NO3)(1.93)(SO4)(4.10)(H4.10O1.81). Witzkeite is monoclinic, space group C2/c, with unit-cell parameters: a = 24.902(2), b = 5.3323(4), c = 17.246(1) angstrom, beta = 94.281(7)degrees, V = 2283.6(3) angstrom(3) (Z = 4). The crystal structure was solved using single-crystal X-ray diffraction data and refined to R-1(F) = 0.043. Witzkeite belongs to a new structure type and is noteworthy for the very rare simultaneous presence of sulfate and nitrate groups. The eight strongest X-ray powder-diffraction lines [d in angstrom (I in %) (h k l)] are: 12.38 (100) (2 0 0), 4.13 (19) (6 0 0), 3.10 (24) (8 0 0), 2.99 (7) ((8) over bar 02), 2.85 (6) (8 02), 2.69 (9) ((7) over bar 1 3), 2.48 (12) (10 0 0), and 2.07 (54) (12 0 0). The IR spectrum of witzkeite was collected in the range 390-4000 cm(-1). The spectrum shows the typical bands of SO42- ions (1192, 1154, 1116, 1101, 1084, 993, 634, and 617 cm(-1)) and of NO3- ions (1385, 1354, 830, 716, and 2775 cm(-1)). Moreover, a complex pattern of bands related to H2O is visible (bands at 3565, 3419, 3260, 2405, 2110, 1638, and 499 cm(-1)). The IR spectrum is discussed in detail.

Digestive peptidases and proteinases in the midgut gland of the pink shrimp Farfantepenaeus paulensis (Crustacea, Decapoda, Penaeidae)

Proteases from the midgut gland of the Farfantepenaeus paulensis juveniles were assessed. Enzyme activity was determined using protease substrates and inhibitors. The effect of pH, temperature and calcium on proteolytic activity was assayed. Caseinolytic activity was analysed in substrate-sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Trypsin, chymotrypsin and leucine aminopeptidase activity was detected. Proteolytic activity was strongly inhibited by the specific trypsin inhibitors. Tosyl-phenylalanine chloromethyl ketone inhibited 59.3% of chymotrypsin activity. The greatest trypsin-like activity occurred at pH 8.0 and 45 degrees C. Chymotrypsin-like activity reached maximal values at alkaline pH (7.2-9.0) and 55 degrees C. CaCl(2) did not increase trypsin-like activity, but rather inhibited it at concentrations of 30 (20%), 50 (30%) and 100 mM (50%). The substrate-SDS-PAGE zymogram revealed eight proteinase bands. Two possibly thermal-resistant (85 degrees C, 30 min) chymotrypsin isoforms were found, which were inhibited by phenyl-methyl-sulphonyl-fluoride. Aminopeptidase activity of enzyme extracts (Arg, Leu, Lys, Phe and Val) and the recommended concentrations of these essential amino acids in penaeid shrimp diets were positively correlated (P < 0.05). Beause protein digestion involves the combined action of different enzymes, adequate knowledge of shrimp digestion and enzyme characteristics is required for the assessment of the digestive potential of different feed sources and development of in vitro digestibility protocols.

End-to-end Distance Distribution in Fluorescent Derivatives of Bradykinin in Interaction with Lipid Vesicles

Cellular membranes have relevant roles in processes related to proteases like human kallikreins and cathepsins. As enzyme and substrate may interact with cell membranes and associated co-factors, it is important to take into account the behavior of peptide substrates in the lipid environment. In this paper we report an study based on energy transfer in two bradykinin derived peptides labeled with the donor-acceptor pair Abz/Eddnp (ortho-aminobenzoic acid/N-[2,4-dinitrophenyl]-ethylenediamine). Time-resolved fluorescence experiments were performed in phosphate buffer and in the presence of large unilamelar vesicles of phospholipids, and of micelles of sodium dodecyl sulphate (SDS). The decay kinetics were analyzed using the program CONTIN to obtain end-to-end distance distribution functions f(r). Despite of the large difference in the number of residues the end-to-end distance of the longer peptide (9 amino acid residues) is only 20 % larger than the values obtained for the shorter peptide (5 amino acid residues). The proline residue, in position 4 of the bradykinin sequence promotes a turn in the longer peptide chain, shortening its end-to-end distance. The surfactant SDS has a strong disorganizing effect, substantially broadening the distance distributions, while temperature increase has mild effects in the flexibility of the chains, causing small increase in the distribution width. The interaction with phospholipid vesicles stabilizes more compact conformations, decreasing end-to-end distances in the peptides. Anisotropy experiments showed that rotational diffusion was not severely affected by the interaction with the vesicles, suggesting a location for the peptides in the surface region of the bilayer, a result consistent with small effect of lipid phase transition on the peptides conformations.

Coffee is highly tolerant to cadmium, nickel and zinc: Plant and soil nutritional status, metal distribution and bean yield

Sewage sludge has been used to fertilize coffee, increasing the risk of metal contamination in this crop. The aim of this work was to study the effects of Cd, Zn and Ni in adult coffee plants growing under field conditions. Seven-year-old coffee plants growing in the field received one of three;loses of Cd, Zn or Ni: 15,45 and 90 g Cd plant(-1); 35, 105 and 210 g Ni plant(-1); and 100, 300 and 600 g Zn plant(-1), with all three metals in the form of sulphate salts. After three months, we noticed good penetration of the three metals into the soil, especially in the first 50 cm, which is the region where most coffee plant roots are concentrated. Leaf concentrations of K, Ca, Mg, S, B, Cu, Fe and Mn were nor affected. N levels did not change with the application of Ni or Zn but were reduced with either 45 or 90 g Cd plant(-1). Foliar P concentrations decreased with the addition of 45 and 90 g Cd plant(-1) and 600 g Zn plant(-1). Zn levels in leaves were not affected by the application of Cd or Ni. The highest concentrations. of Zn were found in branches (30-230 mg kg(-1)), leaves (7-35 mg kg(-1)) and beam (4-6.5 mg kg(-1)); Ni was found in leaves (4-45 mg kg(-1)), branches (3-18 mg kg(-1)) and beans (1-5 mg kg(-1)); and Cd was found in branches (0-6.2 mg kg(-1)) and beans (0-1.5 mg kg(-1)) but was absent in leaves. The mean yield of two harvests was not affected by Ni, but it decreased at the highest dose of Zn (600 g plant(-1)) and the two higher doses of Cd (45 and 90 g plant(-1)). Plants died when treated with the highest dose of Cd and showed symptoms of toxicity with the highest dose of Zn. Nevertheless, based on the amounts of metal used and the results obtained, we conclude that coffee plants are highly tolerant to the three metals tested. Moreover, even at high doses, there was very little transport to the beans, which is the part consumed by humans. (C) 2011 Elsevier B.V. All rights reserved.

High prevalence of OXA-143 and alteration of outer membrane proteins in carbapenem-resistant Acinetobacter spp. isolates in Brazil

Carbapenem resistance amongst Acinetobacter spp. has been increasing in the last decade. This study evaluated the outer membrane protein (OMP) profile and production of carbapenemases in 50 carbapenem-resistant Acinetobacter spp. isolates from bloodstream infections. Isolates were identified by API20NE. Minimum inhibitory concentrations (MICs) for carbapenems were determined by broth microdilution. Carbapenemases were studied by phenotypic tests, detection of their encoding gene by polymerase chain reaction (PCR) amplification, and imipenem hydrolysis. Nucleotide sequencing confirming the enzyme gene type was performed using MegaBACE 1000. The presence of OMPs was studied by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and PCR. Molecular typing was performed using pulsed-field gel electrophoresis (PFGE). All isolates were resistant to carbapenems. Moreover, 98% of the isolates were positive for the gene encoding the enzyme OXA-51-like, 18% were positive for OXA-23-like (only one isolate did not show the presence of the insertion sequence ISAba1 adjacent to this gene) and 76% were positive for OXA-143 enzyme. Five isolates (10%) showed the presence of the IMP-1 gene. Imipenem hydrolysing activity was detected in only three strains containing carbapenemase genes, comprising two isolates containing the bla(IMP) gene and one containing the bla(OXA-51/OXA-23-like) gene. The OMP of 43 kDa was altered in 17 of 25 strains studied, and this alteration was associated with a high meropenem MIC (256 mu g/mL) in 5 of 7 strains without 43 kDa OMP. On the other hand, decreased OMP 33-36 kDa was found in five strains. The high prevalence of OXA-143 and alteration of OMPs might have been associated with a high level of carbapenem resistance. (C) 2012 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

Lyotropic mixture made of potassium laurate/1-undecanol/K2SO4/water presenting high birefringences and large biaxial nematic phase domain: a laser conoscopy study

The lyotropic liquid crystalline quaternary mixture made of potassium laurate (KL), potassium sulphate, 1-undecanol and water was investigated by experimental optical methods (optical microscopy and laser conoscopy). In a particular temperature and relative concentrations range, the three nematic phases (two uniaxial and one biaxial) were identified. The biaxial domain in the temperature/KL concentration surface is larger when compared to other lyotropic mixtures. Moreover, this new mixture gives nematic phases with higher birefringence than similar systems. The behavior of the symmetric tensor order parameter invariants sigma(3) and sigma(2) calculated from the measured optical birefringences supports that the uniaxial-to-biaxial transitions are of second order, described by a mean-field theory.

Effect of alkyl chain length of alcohols on nematic uniaxial-to-biaxial phase transitions in a potassium laurate/alcohol/K2SO4/water lyotropic mixture

Lyotropic liquid crystalline quaternary mixtures of potassium laurate (KL), potassium sulphate (K2SO4)/alcohol (n-OH)/water, with the alcohols having different numbers of carbon atoms in the alkyl chain (n), from 1-octanol to 1-hexadecanol, were investigated by optical techniques (optical microscopy and laser conoscopy). The biaxial nematic phase domain is present in a window of values of n = n(KL) +/- 2, where n(KL) = 11 is the number of carbon atoms in the alkyl chain of KL. The biaxial phase domain became smaller and the uniaxial-to-biaxial phase transition temperatures shifted to relatively higher temperatures upon going from 1-nonanol to 1-tridecanol. Moreover, compared with other lyotropic mixtures these new mixtures present high birefringence values, which we expect to be related to the micellar shape anisotropy. Our results are interpreted assuming that alcohol molecules tend to segregate in the micelles in a way that depends on the relative value of n with respect to nKL. The larger the value of n, the more alcohol molecules tend to be located in the curved parts of the micelle, favoring the uniaxial nematic calamitic phase with respect to the biaxial and uniaxial discotic nematic phases.

Isolation, enzymatic characterization and antiedematogenic activity of the first reported rattlesnake hyaluronidase from Crotalus durissus terrificus venom

A hyaluronidase (CdtHya1) from Crotalus durissus terrificus snake venom (CdtV) was isolated and showed to exhibit a high activity on hyaluronan cleavage. However, surveys on this enzyme are still limited. This study aimed at its isolation, functional/structural characterization and the evaluation of its effect on the spreading of crotoxin and phospholipase A(2) (PLA(2)). The enzyme was purified through cation exchange, gel filtration and hydrophobic chromatography. After that, it was submitted to a reverse-phase fast protein liquid chromatography (RP-FPLC) and Edman degradation sequencing, which showed the first N-terminal 44 amino acid residues whose sequence evidenced identity with other snake venom hyaluronidases. CdtHya1 is a monomeric glycoprotein of 64.5 kDa estimated by SDS-PAGE under reducing conditions. It exhibited maximum activity in the presence of 0.2 M NaCl, at 37 degrees C, pH 5.5 and a specificity to hyaluronan higher than that to chondroitin-4-sulphate, chondroitin-6-sulphate or dermatan. Divalent cations (Ca2+ and Mg2+) and 1 M NaCl significantly reduced the enzyme activity. The specific activity of CdtHya1 was 5066 turbidity reducing units (TRU)/mg, against 145 TRU/mg for the soluble venom, representing a 34.9-fold purification. The pure enzyme increased the diffusion of crotoxin and PLA (2) through mice tissues. CdtHya1 (32 TRU/40 mu L) potentiated crotoxin action, as evidenced by mice death, and it decreased the oedema caused by subplantar injections of buffer, crotoxin or PLA(2), thus evidencing the relevance of hyaluronidase in the crotalic envenoming. This work yielded a highly active antiedematogenic hyaluronidase from CdtV, the first one isolated from rattlesnake venoms. (C) 2012 Elsevier Masson SAS. All rights reserved.

Isotope determination of sulfur by mass spectrometry in soil samples

Sulphur plays an essential role in plants and is one of the main nutrients in several metabolic processes. It has four stable isotopes (32S, 33S, 34S, and 36S) with a natural abundance of 95.00, 0.76, 4.22, and 0.014 in atom %, respectively. A method for isotopic determination of S by isotope-ratio mass spectrometry (IRMS) in soil samples is proposed. The procedure involves the oxidation of organic S to sulphate (S-SO4(2-)), which was determined by dry combustion with alkaline oxidizing agents. The total S-SO4(2-) concentration was determined by turbidimetry and the results showed that the conversion process was adequate. To produce gaseous SO2 gas, BaSO4 was thermally decomposed in a vacuum system at 900 ºC in the presence of NaPO3. The isotope determination of S (atom % 34S atoms) was carried out by isotope ratio mass spectrometry (IRMS). In this work, the labeled material (K2(34)SO4) was used to validate the method of isotopic determination of S; the results were precise and accurate, showing the viability of the proposed method.