Low temperature impact on photosynthetic parameters of coffee genotypes

The objective of this work was to evaluate photoprotective mechanisms related to low positive temperatures in Coffea canephora (Conilon clones 02 and 153) and C. arabica ('Catucaí' IPR 102) genotypes, involved in cold temperature tolerance. To accomplish this, one-year-old plants were successively submitted to: temperature decrease of 0.5ºC day-1, from 25/20ºC to 13/8ºC; a three-day chilling cycle at 13/4ºC; and a recovery period of 14 days (25/20ºC). During the experiment, leaf gas exchange, chlorophyll a fluorescence and leaf photosynthetic pigment content were evaluated. Total activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and ribulose-5-phosphate kinase (Ru5PK) were quantified to measure the activity of photosynthesis key enzymes. All genotypes showed low temperature sensitivity, but displayed diverse cold impact and recovery capabilities regarding the photosynthetic-related parameters studied. Catucaí IPR 102 cultivar showed better ability to cope with cold stress than the Conilon clones, especially Conilon 02, and had full recovery of leaf gas exchange, fluorescence parameters, enzymatic activity, and higher contents of the photoprotective pigments zeaxanthin and lutein.

Identification of new transformation products during enzymatic treatment of tetracycline and erythromycin antibiotics at laboratory scale by an on-line turbulent flow liquid-chromatography coupled to a high resolution mass spectrometer LTQ-Orbitrap

This work describes the formation of transformation products (TPs) by the enzymatic degradation at laboratory scale of two highly consumed antibiotics: tetracycline (Tc) and erythromycin (ERY). The analysis of the samples was carried out by a fast and simple method based on the novel configuration of the on-line turbulent flow system coupled to a hybrid linear ion trap – high resolution mass spectrometer. The method was optimized and validated for the complete analysis of ERY, Tc and their transformation products within 10 min without any other sample manipulation. Furthermore, the applicability of the on-line procedure was evaluated for 25 additional antibiotics, covering a wide range of chemical classes in different environmental waters with satisfactory quality parameters. Degradation rates obtained for Tc by laccase enzyme and ERY by EreB esterase enzyme without the presence of mediators were ∼78% and ∼50%, respectively. Concerning the identification of TPs, three suspected compounds for Tc and five of ERY have been proposed. In the case of Tc, the tentative molecular formulas with errors mass within 2 ppm have been based on the hypothesis of dehydroxylation, (bi)demethylation and oxidation of the rings A and C as major reactions. In contrast, the major TP detected for ERY has been identified as the “dehydration ERY-A”, with the same molecular formula of its parent compound. In addition, the evaluation of the antibiotic activity of the samples along the enzymatic treatments showed a decrease around 100% in both cases

Reference intervals for 24 laboratory parameters determined in 24-hour urine collections.

BACKGROUND: Reference intervals for many laboratory parameters determined in 24-h urine collections are either not publicly available or based on small numbers, not sex specific or not from a representative sample. METHODS: Osmolality and concentrations or enzymatic activities of sodium, potassium, chloride, glucose, creatinine, citrate, cortisol, pancreatic α-amylase, total protein, albumin, transferrin, immunoglobulin G, α1-microglobulin, α2-macroglobulin, as well as porphyrins and their precursors (δ-aminolevulinic acid and porphobilinogen) were determined in 241 24-h urine samples of a population-based cohort of asymptomatic adults (121 men and 120 women). For 16 of these 24 parameters creatinine-normalized ratios were calculated based on 24-h urine creatinine. The reference intervals for these parameters were calculated according to the CLSI C28-A3 statistical guidelines. RESULTS: By contrast to most published reference intervals, which do not stratify for sex, reference intervals of 12 of 24 laboratory parameters in 24-h urine collections and of eight of 16 parameters as creatinine-normalized ratios differed significantly between men and women. For six parameters calculated as 24-h urine excretion and four parameters calculated as creatinine-normalized ratios no reference intervals had been published before. For some parameters we found significant and relevant deviations from previously reported reference intervals, most notably for 24-h urine cortisol in women. Ten 24-h urine parameters showed weak or moderate sex-specific correlations with age. CONCLUSIONS: By applying up-to-date analytical methods and clinical chemistry analyzers to 24-h urine collections from a large population-based cohort we provide as yet the most comprehensive set of sex-specific reference intervals calculated according to CLSI guidelines for parameters determined in 24-h urine collections.

Correlation between total nitrite/nitrate concentrations and monoamine oxidase (types A and B) and semicarbazide-sensitive amine oxidase enzymatic activities in human mesenteric arteries from non-diabetic and type 2 diabetic patients

The aim of this study was to determine the correlation between total nitrite/nitrate concentrations (NOx) and the kinetic parameters of monoamine oxidase enzymes (MAO-A and MAO-B) and semicarbazide-sensitive amine oxidase (SSAO) in human mesenteric arteries. Arteries were from non-diabetic and type 2 diabetic patients with sigmoid or rectum carcinoma for whom surgery was the first option and who were not exposed to neo-adjuvant therapy. Segments of human inferior mesenteric arteries from non-diabetic (61.1 ± 8.9 years old, 7 males and 5 females, N = 12) and type 2 diabetic patients (65.8 ± 6.2 years old, 8 males and 4 females, N = 12) were used to determine NOx concentrations and the kinetic parameters of MAO-A, MAO-B and SSAO by the Griess reaction and by radiochemical assay, respectively. The NOx concentrations in arteries from diabetic patients did not differ significantly from those of the non-diabetic group (10.28 ± 4.61 vs 10.71 ± 4.32 nmol/mg protein, respectively). In the non-diabetic group, there was a positive correlation between NOx concentrations and MAO-B parameters: Km (r = 0.612, P = 0.034) and Vmax (r = 0.593, P = 0.042), and a negative correlation with the SSAO parameters: Km (r = -0.625, P = 0.029) and Vmax (r = -0.754, P = 0.005). However, in the diabetic group no correlation was found between NOx concentrations and the three kinetic parameters of the enzymes. These results suggest an important function of sympathetic nerves and vascular NOx concentrations in arteries of non-diabetic patients. Thus, these results confirm the importance of a balance between oxidants and antioxidants in the maintenance of vascular homeostasis to prevent oxidative stress.

Application of enzymatic preparations to produce araçá pulp and juice

In the present study, the effect of pectinases on the production of pulp and juice of araçá and the presence of bioactive compounds were evaluated. An enzyme extract (EE) produced by Aspergillus niger LB-02-SF in solid state fermentation and the commercial enzyme Ultrazym®AFP-L were used in this study. After enzyme treatment with the EE preparation, the extraction yield increased by 23.1% and viscosity decreased by 42.8%, during pulp maceration. During juice processing, there was an increase of 70.6% in clarification and a decrease of 72.87% in turbidity. Higher values of these parameters, 47.7, 69.0, 80.7, and 79.7%, respectively, were obtained using the Ultrazym®AFP-L, which also led to a significant increase in the polyphenol content, both in the pulp (24%) and in the juice (28%), with a less pronounced effect when the EE was applied (10 and 21%, respectively). The anthocyanins content in the araçá pulp increased after treatment with the commercial preparation (23%), and there was no significant increase with the use of EE. The use of Ultrazym®AFP-L increased the β-carotene content by 29.4% in the fruit pulp, while the treatment with EE did not result in significant changes compared with those of the juice and pulp controls.

Lipase-Mediated Enzymatic Catalysis For The Synthesis of New Chiral Polymers

Immobilized lipase B from Candida antarctica (N435) was investigated as a potential biocatalyst to generate silicone-based chiral polymers from monomers derived from the enzymatic dihydroxylation of bromobenzene. Several conditions and parameters have been investigated for this purpose and lipase transesterification preference to each of the free secondary alcohols in the chiral monomers was documented. The N435 was challenged with a series of substrates where the free alcohol moieties were systematically protected in order to study the substrate preference(s) for the transesterification reactions.

Exploring the enzymatic landscape: distribution and kinetics of hydrolytic enzymes in soil particle-size fractions

The location of extracellular enzymes within the soil architecture and their association with the various soil components affects their catalytic potential. A soil fractionation study was carried out to investigate: (a) the distribution of a range of hydrolytic enzymes involved in C, N and P transformations, (b) the effect of the location on their respective kinetics, (c) the effect of long-term N fertilizer management on enzyme distribution and kinetic parameters. Soil (silty clay loam) from grassland which had received 0 or 200 kg N ha(-1) yr(-1) was fractionated, and four particle-size fractions (> 200, 200-63, 63-2 and 0. 1-2 mum) were obtained by a combination of wet-sieving and centrifugation, after low-energy ultrasonication. All fractions were assayed for four carbohydrases (beta-cellobiohydrolase, N-acetyl-beta-glucosammidase, beta-glucosidase and beta-xylosidase), acid phosphatase and leucine-aminopeptidase using a microplate fluorimetric assay based on MUB-substrates. Enzyme kinetics (V-max and K-m) were estimated in three particle-size fractions and the unfractionated soil. The results showed that not all particle-size fractions were equally enzymatically active and that the distribution of enzymes between fractions depended on the enzyme. Carbohydrases predominated in the coarser fractions while phosphatase and leucine-aminopeptidase were predominant in the clay-size fraction. The Michaelis constant (K.) varied among fractions, indicating that the association of the same enzyme with different particle-size fractions affected its substrate affinity. The same values of Km were found in the same fractions from the soil under two contrasting fertilizer management regimes, indicating that the Michaelis constant was unaffected by soil changes caused by N fertilizer management. (C) 2004 Elsevier Ltd. All rights reserved.

The Physiological, Enzymatic, and Genetic Characterization of Staphylococcus sp. Chromium (VI) Reductase Function

A strain of Staphylococcus isolated by Dr. Fekete at the Sandia National Laboratory toxic metal dumping site in Sandia, New Mexico. has been found to reduce toxic Cr(VI) to the less toxic Cr(IlI) state. We have ascertained the environmental parameters for optimal bacterial growth and Cr(VI) reduction. This knowledge may be employed in a comprehensive bioremediation scheme designed to accelerate natural reparation of that Sandia ecosystem. In addition we have investigated the genetic and enzymatic basis for this Cr(VI) reducing ability. This information may allow us to create more effective bioremediation schemes based on the comprehensive knowledge of enzyme and gene function. Preliminary investigations have been carried out toward this end which may serve as the basis for a more thorough investigation.

Thermal analysis as a screening technique for the characterization of babassu flour and its solid fractions after acid and enzymatic hydrolysis

The babassu (Orbignya phalerata) is a native tree found in northern Brazil. Extracts of the babassu coconut have been widely used in industry. Babassu flour has about 60% starch, thus, besides nourishment it can be used as an alternative biofuel source. However, the properties of this starch lack of study and understanding. The main purpose of this study was to investigate the thermal behavior of raw babassu flour and its solid hydrolyzed fraction. The analyses were carried out using SHIMADZU DSC and TG thermic analyzers. The results demonstrated a reduction in thermal stability of the solid hydrolyzed fraction compared to raw matter. The kinetic parameters were investigated using non-isothermal methods and the parameters obtained for its decomposition process were an E(a) of 166.86 kJ mol(-1) and a frequency factor (beta) of 6.283 x 1014 min(-1); this was determined to be a first order reaction (n = 1). (C) 2011 Elsevier B.V. All rights reserved.

Relationship between global structural parameters and Enzyme Commission hierarchy: Implications for function prediction

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Influence of condensed tannins from Brazilian semi-arid legumes on ruminal degradability, microbial colonization and ruminal enzymatic activity in Saanen goats

The present study aimed at determining the influence of condensed tannins present in the Brazilian legume species Mimosa hostilis, Mimosa caesalpinifolia and Bauhinia cheilantha on ruminal degradability, microbial colonization and enzymatic activity. Polyethylene glycol (PEG) was used to reduce the astringency and concentration of soluble condensed tannins. Four ruminally-cannulated Saanen goats (60 +/- 8 kg BW) were fed, in two experimental periods, with a hay diet based on the studied legumes treated or non-treated with PEG. Voluntary intake, microbial colonization, DM, CP, NDF, and ruminal degradability of PEG treated and non-treated forage leaves, as well as pH, ammonia and 1,4 P-endoglucanase activity of the rumen content were evaluated. Astringency and soluble tannin concentration of the studied legumes were reduced by approximately 70% and 50%, respectively, with PEG treatment. Average DM intake was higher for the treated diet (16.76 g DM/kg BW/day against 13.06 g DM/kg BW/day). Percentile values for degradation parameters and for potential and effective degradabilities of DM, CP and NDF were also affected by the tannins, but at different intensities. Electron microscopic observations of ruminally-incubated legume leaves showed a more effective microbial colonization of PEG-treated leaves for all legume species. A decrease in pH and an increase in ammonia concentration and in endoglucanase activity in the ruminal content was also observed for PEG-treated diets at all sampling periods. Condensed tannins of the studied legume species have influenced the adhesion conditions, colonization and enzymatic activity of the microbial ecosystem, and consequently the ruminal degradation of the different dietary fractions. For this reason, the reduction in condensed tannin would be of great importance to improve the nutrition of ruminant feeding of these species. (c) 2005 Elsevier B.V. All rights reserved.

Use of aspartate aminotransferase in diagnosing periodontal disease: a comparative study of clinical and microbiological parameters

The objective of this study was to assess the association between the levels of enzyme aspartate aminotransferase (AST) in gingival crevicular fluid (GCF) with the BANA hydrolysis microbiological test (Perioscan) and clinical periodontal diagnostic measurements, such as bleeding on probing, plaque index, gingival index, probing depth, and attachment level in patients with chronic periodontitis using an enzymatic test (PocketWatch). One hundred and forty-seven sites were evaluated in 22 patients with a probing depth of > or = 5 mm at selected sites. AST and BANA enzymatic tests were carried out, and clinical parameters recorded. Pearson's chi-square and Fisher's exact tests were used for statistical analysis. There was no statistical correlation between AST levels and any of the analyzed parameters. The lack of any association between the factors studied does not indicate, however, that the latter cannot be used in diagnosing the actual periodontal condition of patients and/or sites. However, more research should be carried out to evaluate the true relationship between AST and periodontal disease.

Systemic use of metronidazole in the treatment of chronic periodontitis: a pilot study using clinical, microbiological, and enzymatic evaluation

The aim of the present parallel, double-blind investigation was to evaluate the effect of using systemic metronidazole alone or associated to scaling and root planing on adult chronic periodontal disease, monitored at baseline, 30, 60 and 90 days. Twelve subjects were divided into three groups: the first group (Group I - 22 sites) was submitted to scaling and root planing (SRP) alone; the second group (Group II - 30 sites) received SRP and 250 mg of metronidazole (3 times a day for 10 days), and the third group (Group III - 31 sites) was treated with metronidazole alone. The clinical parameters evaluated were probing depth (PD), clinical attachment level (CAL), plaque index (PlI), gingival index (GI) and bleeding upon probing (BP). Microbiological (BANA test) and enzymatic (Pocket Watch) tests were also performed. All three proposed treatments produced significant improvements in clinical conditions of subjects, from baseline, 30, 60 and 90-day period, except for clinical attachment level. The results obtained by microbiological and enzymatic tests did not show statistical differences among the groups for the 90-day period (r = 0.7924 and r = 0.7757, respectively). In relation to clinical parameters, statistical differences among groups were observed only for the gingival index (p = 0.0261) between Groups I and II, and probing depth (p = 0.0124) between Group I and the others. We conclude that the use of systemic metronidazole did not produce additional effects on the microbiological conditions of these patients with chronic periodontal disease.

Relationship between gingival clinical parameters and the reactivity of the BANA test in subgingival samples from children.

Gingivitis is the first manifestation of periodontal disease, and is characterized by painless and slow evolution. Early diagnosis and intervention must be done to avoid the possibility of precocious periodontitis during the childhood or teenage years. The enzymatic BANA test (N-benzoyl-DL-arginine-naphthylamide) was used to evaluate subgingival samples from 54 children between 6 and 9 years of age. Plaque index (PI) and gingival index (GI) were assessed according to the criteria recommended by Löe (1967). Subgingival plaque was collected from the region that featured the greatest periodontal alteration, represented by a higher gingival index. Resulting data were grouped individually according to visible and non-visible plaque and bleeding and non-bleeding gingiva. Results showed that there was no statistically significant correlation between the presence of visible plaque and the positivity of the BANA test, nor was there a statistically significant correlation between the presence of bleeding and the positivity of the BANA test in subgingival samples obtained from children. This study concluded that the BANA test is not an ideal diagnostic test to be applied to children.

Effects of hydric deficiency on gas exchange parameters and metabolism of Eucalyptus grandis clones

The metabolic effects caused by hydric deficiency (HD) on Eucalyptus grandis clones were assessed by an experiment where plants were cultivated in four blocks. The first was the control block, normally irrigated, whereas the other three blocks were submitted to cycles of hydric deficiency. Analysis of photosynthetic efficiency, enzymatic activity of antioxidant response system, level of pigments and L-proline concentration were performed to evaluate the HD effects. Results showed that HD altered some parameters related to photosynthetic activity, pigments accumulation, proline and enzymatic activity. Clone 433 of E. grandis presented higher response ability to HD.