Highly thermostable amylase and pullulanase of the extreme thermophilic eubacterium Rhodothermus marinus: production and partial characterization

Five strains of the extreme thermophilic Rhodothermus marinus were screened for the production of amylolytic and pullulytic activities. The culture medium for the selected strain, R. marinus ITI 990, was optimized using central composite designs for enhanced enzyme production. The optimized medium containing 1.5 gl(-1) of maltose and 8.3 gl(-1) of yeast extract yielded amylase, pullulanase and alpha-glucosidase activities of 45, 33 and 2.1 nkatml(-1), respectively. Among the various carbon sources tested, maltose was most effective for the formation of these enzymes, followed by soluble maize starch, glycogen and pullulan. The crude amylase and pullulanase showed maximum activities at pH 6.5-7.0, and 85 and 80 degrees C, respectively. At 85 degrees C amylase and pullulanase had half lives of 3 h and 30 min, respectively.     

Drug susceptibility of Brazilian strains of Mycobacterium bovis using traditional and molecular techniques

Transmission of Mycobacterium bovis from cattle to humans has been reported and can cause tuberculosis (Tb) and a problem in certain risk populations. Therefore, knowledge of resistance of M. bovis towards antibiotics used for therapy of human Tb could help avoiding cure delay and treatment cost increase when dealing with drug resistant organisms. We therefore evaluated the susceptibility of M. bovis isolates towards streptomycin, isoniazide, rifampicin, ethambutol, and ethionamide, the first line antibiotics for human Tb. Therefore, 185 clinical samples from cattle with clinical signs of tuberculosis were processed and submitted to culturing and bacterial isolates to identification and drug susceptibility testing using the proportion method. Among 89 mycobacterial strains, 65 were identified as M. bovis and none were resistant to any of the antibiotics used. Confirmation of present results by future studies, enrolling a large number of isolates and designed to properly represent Brazilian regions, may favor the idea of using isoniazide preventive therapy as part of a Tb control strategy in special situations. Also, nucleic acids from bacterial isolates were submitted to rifoligotyping, a recently described reverse hybridization assay for detection of mutations causing resistance towards rifampicin. Concordance between the conventional and the molecular test was 100%, demonstrating the use of such methodology for rapid evaluation of drug susceptibility in M. bovis.     

In vitro maturation and transfer of equine oocytes after transport of ovaries at 12 or 22 degrees C

Transportation of equine ovaries would allow shipment of oocytes for research purposes or transfer after the death of a valuable mare. The objective of this study was to compare two temperatures for maintaining ovaries during a transport interval of 18-24 h. The goal was to obtain pregnancies after transport of ovaries, maturation of oocytes in vitro, and transfer of oocytes. Each shipment was composed of ovaries four to seven mares collected from an abattoir. From each mare, one ovary was packaged at approximately 12 degrees C, and the other was packaged at approximately 22 degrees C. Upon arrival at our laboratory, oocytes were collected and cultured for 24 h. For each transfer, between 9 and 15 oocytes from each group were placed into the oviducts of estrous mares through standing flank laparotomies. Recipients received human chorionic gonadotropin (hCG; 2000 IU, i.v.) 30-36 h before transfer (to synchronize ovulation). Recipients were inseminated 18-20 h before transfers with 2 x 10(9) progressively motile sperm. Uteri of recipients were examined with ultrasound to determine the number of developing embryos. On Day 16 ( ovulation = day 0), developing embryos were recovered by uterine lavage. Parentage verification was performed on recovered vesicles. Pregnancy rates were analyzed by Chi-square. The percentage of oocytes that developed into embryonic vesicles on Day 16 was not different between transport temperatures (22 degrees C, 13/73, 18% versus 12 degrees C, 11/73, 15%). In conclusion, pregnancies were obtained from in vitro matured oocytes that were recovered from ovaries transported for 18-24h at 12 or 22 degrees C.     

Anti-inflammatory activity of tea (Camellia sinensis) root extract

Pharmacological studies were carried out with methanol-water (1:1) extract of dried tea (Camellia sinensis) root extract (TRE). TRE was found to possess anti-inflammatory, analgesic and antipyretic activities at 1/10th of its LD50 dose of 100 mg/kg i.p. It was found that TRE inhibited the arachidonic acid-induced paw oedema in rats which indicated that TRE produced the anti-inflammatory activity by inhibiting both the cyclooxygenase and lypooxygenase pathways of arachidonic acid metabolism. TRE also enhanced peritoneal cell count and the number of macrophages in normal mice. It is plausible that the saponins present in TRE may be responsible for these activities of TRE.     

Comparative analysis of amplified and nonamplified RNA for hybridization in cDNA microarray

Limiting amounts of RNA is a major issue in cDNA microarray, especially when one is dealing with fresh tissue samples. Here we describe a protocol based on template switch and T7 amplification that led to efficient and linear amplification of 1300x. Using a glass-array containing 368 genes printed in three or six replicas covering a wide range of expression levels and ratios, we determined quality and reproducibility of the data obtained from one nonamplified and two independently amplified RNAs (aRNA) derived from normal and tumor samples using replicas with dye exchange (dye-swap measurements). Overall, signal-to-noise ratio improved when we used aRNA (1.45-fold for channel 1 and 2.02-fold for channel 2), increasing by 6% the number of spots with meaningful data. Measurements arising from independent aRNA samples showed strong correlation among themselves (r(2)=0.962) and with those from the nonamplified sample (r(2)=0.975), indicating the reproducibility and fidelity of the amplification procedure. Measurement differences, i.e, spots with poor correlation between amplified and nonamplified measurements, did not show association with gene sequence, expression intensity, or expression ratio and can, therefore, be compensated with replication. In conclusion, aRNA can be used routinely in cDNA microarray analysis, leading to improved quality of data with high fidelity and reproducibility.     

A fluorimetric method for the determination of pepsin activity

An intramolecularly quenched fluorogenic peptide containing o-aminobenzoyl (Abz) and ethylenediamine 2,4-dinitrophenyl (Eddnp) groups at amino- and carboxyl-terminal amino acid residues, Abz-Lys-Pro-Ile-Glu-Phe-Phe-Arg-Leu-Eddnp, was hydrolyzed by purified human pepsin, gastricsin, and gastric juice uniquely at the Phe-Phe bond. Kinetic parameters determined for purified pepsin were K(m)=0.68+/-0.11 microM; k(cat)=6.3+/-0.16s(-1); k(cat)/K(m)=9.26s(-1) microM(-1); Gastricsin showed K(m)=2.69+/-0.18 microM; k(cat)=0.03+/-0.005s(-1); k(cat)/K(m)=0.011s(-1) microM(-1). Gastric juice (21 samples) from subjects without gastric disorders at endoscopy examination showed activities varying from 0.0008 to 9.72 micromolml(-1)min(-1). Pepstatin A inhibition of gastric juice enzymatic activity was complete at 3.4x10(-5)M (final concentration) inhibitor. In the proposed method the presence of a unique scissile bond in the synthetic substrate provides a direct ratio between enzymatic activity and amount of substrate hydrolyzed, and a unique step reaction facilitates the use of this assay for the determination of the activity of aspartic proteinases in biological fluids and during enzyme purification procedures.     

FTIR spectroscopic characterization of the cytochrome aa3 from Acidianus ambivalens: evidence for the involvement of acidic residues in redox coupled proton translocation

The aa(3)-type quinol oxidase from Acidianus ambivalens is a divergent member of the heme-copper oxidases superfamily, namely, concerning the putative channels for intraprotein proton conduction. In this study, we used electrochemically induced FTIR difference spectroscopy to identify residues involved in redox-coupled protonation changes. In the spectral region characteristic for the nu(C=O) mode from protonated aspartic or glutamic acid side chains, a number of prominent features can be observed between 1790 and 1710 cm(-)(1), clearly indicating the reorganization or protonation of more than four protonatable residues upon electron transfer. A direct comparison of the Fourier-transform infrared difference spectra at different pH values reveals the noteworthy high pK of >8 for some of these residues, and the protonation of two of them. These acidic residues may play a role in the proton transport to the oxygen reducing site, in proton pumping pathways, or in protonation reactions concomitant with quinone reduction. Whereas the residues contributing between 1790 and 1750 cm(-)(1) have the typical position of an aspartic/glutamic acid side chain buried in the protein, a position closer to the surface is suggested for the residues contributing below approximately 1730 cm(-)(1). The possible involvement of residues contributing between 1750 and 1720 cm(-)(1) in the quinone binding site is demonstrated on the basis of experiments in the presence and absence of ubiquinone-2 and of the native electron carrier of the A. ambivalens respiratory chain, caldariella quinone. Most signals seen here are not observable in comparable spectra of typical members of the heme copper oxidase superfamily and thus reflect unique features of the enzyme from the hyperthermoacidophilic archaeon A. ambivalens.     

Survival of Atta sexdens workers on different food sources

Leaf-cutting ants belonging to the tribe Attini are major herbivores and important agriculture pests in the neotropics, these ants being thought to feed on the sap which exudes from the plant material which they cut and also on the mycelium of a symbiotic fungus that grows on plant material inside their nests in what is called "the fungus garden". However, we have found that the survival of Atta sexdens worker ants on leaves, on mycelium of the ants' symbiotic fungus, Leucoagaricus gongylophorus, or on plant polysaccharides was the same as that of starved A. sexdens, while, conversely, significantly longer survival was achieved by ants fed on the fungus garden material or on some of the products (especially glucose) of the hydrolysis of plant polysaccharides. We found that the fungus garden contained glucose at a higher concentration than that found in leaves or fungal mycelium, and that this glucose was consumed by the ant to the extent that it was probably responsible for up to 50% of the nutritional needs of the workers. The fungus garden contained polysaccharide degrading enzymes (pectinase, amylase, xylanase and cellulase) in proportions similar to that observed in laboratory cultures of L. gongylophorus. It thus appears that A. sexdens workers obtain a significant part of their nutrients from plant polysaccharide hydrolysis products produced by the action of extracellular enzymes released by L. gongylophorus. In this paper we discuss the symbiotic nutrition strategy of A. sexdens workers and brood and the role played by plant polysaccharides in the nutrition of attine ants.     

Identification of a novel mutation in the ARSB gene that is frequent among Brazilian MPSVI patients

Mucopolysaccharidosis type VI, or Maroteaux-Lamy syndrome, is an autosomal recessive disease caused by the deficiency of arylsulfatase B (ARSB; N-acetyl-galactosamine-4-sulfatase, E.C.3.1.6.12), which is involved in the stepwise degradation of dermatan sulfate and chondroitin sulfate. The deficiency of this enzyme causes storage in the lysozomes and excretion in the urine of partially degraded dermatan sulfate. Twenty patients with MPSVI were analyzed, including 2 siblings. Genomic DNA from patients was extracted and amplified by PCR followed by analysis by single-strand conformation polymorphism (SSCP), which detects altered patterns in the single-stranded DNA. Amongst the patients analyzed for exon 8 of the ARSB gene, 5 patients presented an altered band pattern when compared to controls. After sequencing, we have detected a 23-bp deletion, extending from nucleotides 1,533 to 1,555, causing a frameshift and changing 2 amino acids before creating a premature stop codon at amino acid 514.