Effect of age and body weight on neurohumoral variables in healthy Cavalier King Charles spaniels.

OBJECTIVE: To evaluate the effect of age and body weight on several neurohumoral variables that are commonly altered in heart failure in Cavalier King Charles Spaniels. ANIMALS: 17 healthy privately owned Cavalier King Charles Spaniels, 10 males and 7 females, ranging in age from 0.4 to 9.7 years, and ranging in body weight from 6.6 to 12.2 kg. PROCEDURE: The clinical condition of the dogs was evaluated by physical examination, thoracic radiography, and echocardiography. Plasma nitrate and nitrite (P-NN), N-terminal atrial natriuretic and brain natriuretic peptides (NT-ANP and BNP, respectively), endothelin (ET-1), urine cyclic guanosine monophosphate (U-cGMP), and urine nitrate and nitrite (U-NN) concentrations were analyzed. RESULTS: Plasma concentrations of NT-ANP and P-NN increased significantly with age, but plasma NT-ANP and P-NN also correlated significantly, irrespective of age. A modest increase of left atrial size did not explain the increase of NT-ANP and P-NN with age. Concentration of ET-1 correlated positively with heart rate; heart rate did not change with age. Weight had a negative impact on NT-ANP, P-NN, and U-cGMP concentrations and left atrial relative size. CONCLUSIONS AND CLINICAL RELEVANCE: Age-matched controls are essential for evaluation of NT-ANP and P-NN concentrations and left atrial size. Weight may alter reference values of plasma NT-ANP, P-NN, and urine cGMP concentrations. Natriuretic peptides can be used as further evidence that heart failure exists. The increased plasma concentrations of NT-ANP (but not BNP) and P-NN with aging reflect neurohumoral physiologic changes that must be distinguished from pathologic changes in patients with heart failure.

Conversion of xylose to ethanol by recombinant Saccharomyces cerevisiae: importance of xylulokinase (XKS1) and oxygen availability.

The yeast Saccharomyces cerevisiae efficiently ferments hexose sugars to ethanol, but it is unable to utilize xylose, a pentose sugar abundant in lignocellulosic materials. Recombinant strains containing genes coding for xylose reductase (XR) and xylitol dehydrogenase (XDH) from the xylose-utilizing yeast Pichia stipitis have been reported; however, such strains ferment xylose to ethanol poorly. One reason for this may be the low capacity of xylulokinase, the third enzyme in the xylose pathway. To investigate the potential limitation of the xylulokinase step, we have overexpressed the endogenous gene for this enzyme (XKS1) in S. cerevisiae that also expresses the P. stipitis genes for XR and XDH. The metabolism of this recombinant yeast was further investigated in pure xylose bioreactor cultivation at various oxygen levels. The results clearly indicated that overexpression of XKS1 significantly enhances the specific rate of xylose utilization. In addition, the XK-overexpressing strain can more efficiently convert xylose to ethanol under all aeration conditions studied. One of the important illustrations is the significant anaerobic and aerobic xylose conversion to ethanol by the recombinant Saccharomyces; moreover, this was achieved on pure xylose as a carbon. Under microaerobic conditions, 5.4 g L(-1) ethanol was produced from 47 g L(-1) xylose during 100 h. In fed-batch cultivations using a mixture of xylose and glucose as carbon sources, the specific ethanol production rate was highest at the highest aeration rate tested and declined by almost one order of magnitude at lower aeration levels. Intracellular metabolite analyses and in vitro enzyme activities suggest the following: the control of flux in a strain that overexpresses XKS1 has shifted to the nonoxidative steps of the pentose phosphate pathway (i.e., downstream of xylose 5-phosphate), and enzymatic steps in the lower part of glycolysis and ethanol formation pathways (pyruvate kinase, pyruvate decarboxylase, and alcohol dehydrogenase) do not have a high flux control in this recombinant strain. Furthermore, the intracellular ATP levels were found to be significantly lower for the XK strain compared with either the control strain under similar conditions or glucose-grown Saccharomyces. The ATP : ADP ratios were also lower for the XK strain, especially under microaerobic conditions (0.9 vs 6.4).

The role of xylulokinase in Saccharomyces cerevisiae xylulose catabolism.

Many yeast species have growth rates on D-xylulose of 25-130% of those on glucose, but for Saccharomyces cerevisiae this ratio is only about 6%. The xylulokinase reaction has been proposed to be the rate-limiting step in the D-xylulose fermentation with S. cerevisiae. Over-expression of xylulokinase encoding XKS1 stimulated growth on D-xylulose in a S. cerevisiae strain to about 20% of the growth rate on glucose and deletion of the gene prevented growth on D-xylulose and D-xylulose metabolism. We have partially purified the xylulokinase and characterised its kinetic properties. It is reversible and will also accept D-ribulose as a substrate.

Evidence that the gene YLR070c of Saccharomyces cerevisiae encodes a xylitol dehydrogenase.

The open reading frame YLR070c of Saccharomyces cerevisiae has high sequence similarity to S. cerevisiae sorbitol dehydrogenase and to xylitol dehydrogenase of Pichia stipitis. Overexpression of this open reading frame in S. cerevisiae resulted in xylitol dehydrogenase activity. The enzyme is specific for NADH. The following Michaelis constants were estimated: D-xylulose, 1.1 mM; NADH, 240 microM (at pH 7.0); xylitol, 25 mM; NAD, 100 microM (at pH 9.0). Xylitol dehydrogenase activity with the same kinetic properties can also be induced by xylose in wild type S. cerevisiae cells.