Amino acid catabolism in cheese-related bacteria: selection and study of the effects of pH, temperature and NaCl by quadratic response surface methodology

AIMS: To screen the cystathionine lyase and L-methionine aminotransferase activities of cheese-related bacteria (lactococci, non-starter lactobacilli and smear bacteria) and to determine the individual and interactive effects of temperature, pH and NaCl concentration on selected enzyme activities. METHODS AND RESULTS: A subcellular fractionation protocol and specific enzyme assays were used, and a quadratic response surface methodology was applied. The majority of the strains, 21 of 33, had detectable cystathionine lyase activity which differed in the specificity. Aminotransferase activity on L-methionine was observed in only three strains. The cystathionine lyase activities of Lactobacillus reuteri DSM20016, Lactococcus lactis subsp. cremoris MG1363, Brevibacterium linens 10 and Corynebacterium ammoniagenes 8 and the L-methionine aminotransferase activity of Lact. reuteri DSM20016 had temperature and pH optima of 30-45 degrees C, and 7.5-8.0, respectively. As shown by the quadratic response surface methodology these enzymes retained activities in the range of temperature, pH and NaCl concentration which characterized the cheeses from which the bacteria originated. CONCLUSION: The enzyme activities may have a role in flavour development during cheese ripening. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of this work contribute to the knowledge about the amino acid catabolic enzymes in order to improve cheese ripening.     

Trace mineral source influences digestion,ruminal fermentation,and ruminal copper,zinc, and manganese distribution in steers fed a diet suitable for lactating dairy cows

High solubility of certain trace minerals (TM) in the rumen can alter nutrient digestibility and fermentation. The objectives of the present studies were to determine the effects of TM source on 1) nutrient digestibility and ruminal fermentation, 2) concentrations of soluble Cu, Zn, and Mn in the rumen following a pulse dose of TM, and 3) Cu, Zn, and Mn binding strength on ruminal digesta using dialysis against a chelating agent in steers fed a diet formulated to meet the requirements of a high producing dairy cow. Twelve Angus steers fitted with ruminal cannulae were adapted to a diet balanced with nutrient concentrations similar to a diet for a high producing lactating dairy cow for 21 d. Steers were then randomly assigned to dietary treatments consisting of 10 mg Cu, 40 mg Mn, and 60 mg Zn/kg DM from either sulfate (STM), hydroxychloride (HTM) or complexed trace minerals (CTM). The experimental design did not include a negative control (no supplemental Cu, Mn, or Zn) because the basal diet did not meet the National Research Council requirement for Cu and Zn. Copper, Mn, and Zn are also generally supplemented to lactating dairy cow diets at concentrations approximating those supplied in the present study. Following a 14-d adaptation period, total fecal output was collected for 5-d. Following the fecal collection period, rumen fluid was collected for Volatile fatty acid (VFA) parameters. On the following day, the same diet was provided for 14 d, without supplemental Cu, Zn, and Mn. This period served as a wash-out period. A pulse dose of 100, 400, and 600 mg of Cu, Zn, Mn, respectively, from either STM, HTM, or CTM, was administered via ruminal cannulae to the steers on day 15. Over a 24-h period ruminal samples were obtained every 2-h. Following centrifugation, the supernatant was analyzed for Cu, Mn, and Zn. Ruminal solid digesta samples from times 0, 12, and 24 h after bolus dosing were exposed to dialysis against Tris-EDTA. Digestibility of NDF and ADF were lesser in STM vs. HTM and vs. CTM supplemented steers. Steers receiving HTM and CTM had greater total VFA concentrations than STM, and molar proportions of individual VFA were not affected by treatment. Ruminal soluble Cu and Zn concentrations were greater post dosing in STM and CTM supplemented steers at 2, 4, and 6 h for Cu and 4, 6, 8, 10 and 12 h for Zn when compared to HTM supplemented steers. The release of Cu and Zn from ruminal solid digesta following dialysis against Tris-EDTA at 12 and 24 h postdosing was greater for steers receiving HTM compared to those receiving STM or CTM. Results indicate trace mineral source impacts: 1) how tightly bound Cu and Zn are to ruminal solid digesta; 2) fiber digestion; 3) and ruminal total VFA concentrations.     

Increased in vivo mitochondrial oxygenation with right ventricular failure induced by pulmonary arterial hypertension: mitochondrial inhibition as driver of cardiac failure?

Background

The leading cause of mortality due to pulmonary arterial hypertension (PAH) is failure of the cardiac right ventricle. It has long been hypothesized that during the development of chronic cardiac failure the heart becomes energy deprived, possibly due to shortage of oxygen at the level of cardiomyocyte mitochondria. However, direct evaluation of oxygen tension levels within the in vivo right ventricle during PAH is currently lacking. Here we directly evaluated this hypothesis by using a recently reported technique of oxygen-dependent quenching of delayed fluorescence of mitochondrial protoprophyrin IX, to determine the distribution of mitochondrial oxygen tension (mitoPO₂) within the right ventricle (RV) subjected to progressive PAH.

Methods

PAH was induced through a single injection of monocrotaline (MCT). Control (saline-injected), compensated RV hypertrophy (30 mg/kg MCT; MCT30), and RV failure (60 mg/kg MCT; MCT60) rats were compared 4 wk after treatment. The distribution of mitoPO₂ within the RV was determined in mechanically-ventilated, anaesthetized animals, applying different inspired oxygen (FiO₂) levels and two increment dosages of dobutamine.

Results

MCT60 resulted in RV failure (increased mortality, weight loss, increased lung weight), MCT30 resulted in compensated RV hypertrophy. At 30% or 40% FiO₂, necessary to obtain physiological arterial PO₂ in the diseased animals, RV failure rats had significantly less mitochondria (15% of total mitochondria) in the 0-20 mmHg mitoPO₂ range than hypertrophied RV rats (48%) or control rats (54%). Only when oxygen supply was reduced to 21% FiO_2, resulting in low arterial PO₂ for the MCT60 animals, or when oxygen demand increased with high dose dobutamine, the number of failing RV mitochondria with low oxygen became similar to control RV. In addition, metabolic enzyme analysis revealed similar mitochondrial mass, increased glycolytic hexokinase activity following MCT, with increased lactate dehydrogenase activity only in compensated hypertrophied RV.

Conclusions

Our novel observation of increased mitochondrial oxygenation suggests down-regulation of in vivo mitochondrial oxygen consumption, in the absence of hypoxia, with transition towards right ventricular failure induced by pulmonary arterial hypertension.     

Mapping von Willebrand factor A domain binding sites on a snake venom metalloproteinase cysteine-rich domain

The PIII class of the snake venom metalloproteinases (SVMPS) are acknowledged to be one of the major hemorrhage producing toxins in crotalid venoms. This class of SVMPS are structurally distinguished by the presence of disintegrin-like and cysteine-rich domains carboxy to the metalloproteinase domain and thus share structural homology with many of the ADAMs proteins. It has been suggested that the presence of the carboxy domain are the key structural determinants for potent hemorrhagic activity in that they may serve to target the proteinases to specific key extracellular matrix and cell surface substrates for proteolysis leading to hemorrhage production at the capillaries. Following from previous studies in our laboratory in this investigation we scanned the cysteine-rich domain of the PIII hemorrhagic SVMP jararhagin using synthetic peptides in an attempt to identify regions which could bind to von Willebrand factor (vWF), a known binding partner for jararhagin. From these studies we identified two such peptide, Jar6 and Jar7 that could support binding to vWF as well as block the recombinant cysteine-rich domain of jararhagin binding to vWF. Using the coordinates for the recently solved crystal structure of the PIII SVMP VAP1, we modeled the structure of jararhagin and attempted to dock the modeled cysteine-rich structure of that protein to the A1 domain of vWF. These studies indicated that effective protein-protein interaction between the two ligands was possible and supported the data indicating that the Jar6 peptide was involved, whereas the Jar7 peptide was observed to be sterically blocked from interaction. In summary, our studies have identified a region on the cysteine-rich domain of a PIII SVMP that interacts with vWF and based on molecular modeling could be involving in the interaction of the cysteine-rich domain of the SVMP with the A1 domain of vWF thus serving to target the toxin to the protein for subsequent proteolytic degradation.     

The effect of estrogen on muscle damage biomarkers following prolonged aerobic exercise in eumenorrheic women

This study assessed the influence of estrogen (E₂) on muscle damage biomarkers [skeletal muscle - creatine kinase (CK); cardiac muscle - CK-MB] responses to prolonged aerobic exercise. Eumenorrheic women (n=10) who were physically active completed two 60-minute treadmill running sessions at ∼60-65% maximal intensity during low E₂ (midfollicular menstrual phase) and high E₂ (midluteal menstrual phase) hormonal conditions. Blood samples were collected prior to exercise (following supine rest), immediately post-, 30 min post-, and 24 hours post-exercise to determine changes in muscle biomarkers. Resting blood samples confirmed appropriate E₂ hormonal levels Total CK concentrations increased following exercise and at 24 hours post-exercise were higher in the midfollicular low E₂ phase (p<0.001). However, CK-MB concentrations were unaffected by E₂ level or exercise (p=0.442) resulting in the ratio of CK-MB to total CK being consistently low in subject responses (i.e., indicative of skeletal muscle damage). Elevated E₂ levels reduce the CK responses of skeletal muscle, but had no effect on CK-MB responses following prolonged aerobic exercise. These findings support earlier work showing elevated E₂ is protective of skeletal muscle from exercise-induced damage associated with prolonged aerobic exercise.     

A comparative map viewer integrating genetic maps for <Emphasis Type="Italic">Brassica</Emphasis> and <Emphasis Type="Italic">Arabidopsis</Emphasis>

Background  

Molecular genetic maps provide a means to link heritable traits with underlying genome sequence variation. Several genetic maps have been constructed for Brassica species, yet to date, there has been no simple means to compare this information or to associate mapped traits with the genome sequence of the related model plant, Arabidopsis.     

Deficiency of functional mannose-binding lectin is not associated with infections in patients with systemic lupus erythematosus

Infection imposes a serious burden on patients with systemic lupus erythematosus (SLE). The increased infection rate in SLE patients has been attributed in part to defects of immune defence. Recently, the lectin pathway of complement activation has also been suggested to play a role in the occurrence of infections in SLE. In previous studies, SLE patients homozygous for mannose-binding lectin (MBL) variant alleles were at an increased risk of acquiring serious infections in comparison with patients who were heterozygous or homozygous for the normal allele. This association suggests a correlation between functional MBL level and occurrence of infections in SLE patients. We therefore investigated the biological activity of MBL and its relationship with the occurrence of infections in patients with SLE. Demographic and clinical data were collected in 103 patients with SLE. Functional MBL serum levels and MBL-induced C4 deposition were measured by enzyme-linked immunosorbent assay using mannan as coat and an MBL- or C4b-specific monoclonal antibody. The complete MBL-dependent pathway activity was determined by using an assay that measures the complete MBL pathway activity in serum, starting with binding of MBL to mannan, and was detected with a specific monoclonal antibody against C5b-9. Charts were systematically reviewed to obtain information on documented infections since diagnosis of SLE. Major infections were defined as infections requiring hospital admission and intravenous administration of antibiotics. In total, 115 infections since diagnosis of lupus, including 42 major infections, were documented in the 103 SLE patients (mean age 41 ± 13 years, mean disease duration 7 ± 4 years). The percentage of SLE patients with severe MBL deficiency was similar to that in 100 healthy controls: 13% versus 14%, respectively. Although deposition of C4 to mannan and MBL pathway activity were reduced in 21% and 43% of 103 SLE patients, respectively, neither functional MBL serum levels nor MBL pathway activity was associated with infections or major infections in regression analyses. In conclusion, SLE patients frequently suffer from infections, but deficiency of functional MBL does not confer additional risk.     

Antibodies to mutated citrullinated vimentin and disease activity score in early arthritis: a cohort study

Introduction  

The aim of our study was to investigate the association between arthritic disease activity and antibodies to mutated citrullinated vimentin (anti-MCV), because such a relation has been suggested.     

Are biologics more effective than classical disease-modifying antirheumatic drugs?

Major achievements have been reached in the treatment of rheumatoid arthritis during past decades due to the recognition of methotrexate as an anchor drug for treatment of rheumatoid arthritis, due to the notion of a treatment window of opportunity in patients with recent-onset rheumatoid arthritis necessitating early aggressive therapy, due to the development of biologics and due to remission as a treatment target. Most biologics have a much faster onset of action than synthetic disease-modifying anti-rheumatic drugs, but presently there is no convincing evidence that biologic drugs have a superior clinical efficacy in comparison with the synthetic drugs. Biologics are, however, accompanied by less radiological deterioration.