Applications of Hydrolytic and Decarboxylating Enzymes in Biotransformations

Peptides, and oligosaccharides and glycosides, can be synthesised by making use of the ‘reverse hydrolytic activity’ of proteases and glycosidases respectively. In applying these enzymes to the practical synthesis of these classes of compound, several factors need to be considered, namely the need to shift the rate-determining step through the use of activated substrates, the need to minimise competing hydrolysis of these and the need to minimise hydrolysis of the products. In spite of these problems, the enzymatic methods have many attractive features, not least amongst which is the absolute control of stereochemistry in acyl transfer and glycosyl transfer respectively.     

Influence of Methanogenic Populations in Holocene Lacustrine Sediments Revealed by Clone Libraries and Fatty Acid Biogeochemistry

Methanogenic populations were investigated in subsaline Laguna Potrok Aike sediments, southern Argentina. Microbial density and activity were assessed via cell count and in situ ATP detection for the last ～11K years. Methanogen phylogenetics highlighted species stratification throughout depth, whereas CO₂ reduction was the major pathway leading to methane production. Organic substrates, characterized using pore water analysis, bulk organic fractions and saturated fatty acids, showed a clear link between sediment colonization and initial organic sources. Concentrations and δ¹³C compositions of methane and fatty acids provided final evidence of a microbial imprint on Holocene organic proxies in the most colonized intervals.     

Heart design: free ADP scales with absolute mitochondrial and myofibrillar volumes from mouse to human

Our aim was to estimate a number of bioenergetic parameters in the beating mouse, rat and guinea pig heart in situ and compare the values to those in hearts of mammals over a 2000-fold range in body mass. For the mouse, rat and guinea pig heart, we report a phosphorylation ratio of 1005±50 (n=16), 460±32 (n=10) and 330±22 (n=5) mM⁻¹ and a free cytosolic [ADP] concentration of 13, 18 and 22 μM, respectively. When each parameter was plotted against body mass, they scaled closely to the quarter power (−0.28, r=0.99 and −0.23, r=0.97). A similar regression slope was found when the inverse of free [ADP] was plotted against absolute mitochondrial (slope=−0.26, r=0.99) and myofibrillar volumes (slope=−0.24, r=0.99). The similar slopes indicate that the ratio of absolute mitochondria and myofibrillar volumes in the healthy mammalian heart is a constant, and independent of body size. In conclusion, our study supports the hypothesis that the mammalian heart has a number of highly conserved thermodynamic and kinetic parameters that obey quarter-power laws linking the phosphorylation ratio, ATP turnover rates, free [ADP] and absolute mitochondrial volumes to body size. The results are discussed in terms of possible mechanisms and potential deviations from these laws in some disease states.     

Total bile acids in hyperlipidaemic serum determined by bioluminescent and spectrophotometric methods

A simple, rapid and sensitive bioluminescent method has been used to measure total bile acids in hyperlipidaemic serum. We found that the levels of total bile acids in hypertriglyceridaemic and hypercholesterolemic sera determined by a spectrophotometric method were four-fold higher than those measured by the bioluminescent method (6.73 ± 4.07 μmol/l (mean ± SD) by bioluminescent and 26.10 ± 13.42 μmol/l by the spectrophotometric method). There was no difference in total bile acid levels between these two methods for normal serum (4.72 ± 3.38 μmol/l by bioluminescence and 4.49 ± 3.27 μmol/l by the spectrophotometric method).     

Conversion of 5′-Methylthioadenosine into S-adenosylmethionine by yeast cells

5′-Methylthio[U-¹⁴C]adenosine was used as a culture supplement for Candida utilitis. The resulting S-adenosylmethionine was hydrolyzed into its structural components. Virtually none of the label of the pentose was found in the carbohydrate part of the intracellular S-adenosylmethionine. Much of it was present in the four-carbon chain of the methionine part of the sulfonium compound. The U-¹⁴C)-labeled adenine of 5′-methylthio[U-¹⁴C]adenosine did not contribute to the labeling of the amino acid component of the sulfonium compound.     

Ethanol Administration in the Rat Decreases Prostacyclin Production by Isolated Brain Microvessels

The effects of short- and long-term ethanol administration to rats on basal levels and formation of prostacyclin (PGI2) measured as 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha), and on lipid class content and fatty acid composition of isolated brain microvessels (BMV) were studied. After acute treatment (2 h, at the peak of plasma ethanol concentration) basal 6-keto-PGF1 alpha levels in BMV and release on incubation were reduced to 50% of control values. After chronic administration (15 days), PGI2 release was reduced to about 40% of control values, without changes in basal levels. Total lipid, phospholipid, and cholesterol levels in BMV, measured after prolonged administration of alcohol, were not modified. Also, only minor changes in the fatty acid composition of individual phospholipid classes were detected. The observed reduction of PGI2 synthesis in BMV thus could not be related to changes of the fatty acid precursor pool in the preparation. Precursor release and/or the biosynthetic pathways may be affected by ethanol administration.     

5S ribosomal gene clusters in wheat: pulsed field gel electrophoresis reveals a high degree of polymorphism

Summary The long-range structure of 5S rRNA gene clusters has been investigated in wheat (Triticum aestivum L.) by means of pulsed field gel electrophoresis. Using aneuploid stocks, 5S rRNA gene clusters were assigned to sites on chromosomes 1B, 1D, 513 and 5D. Cluster sizes were evaluated and the copy number of 5S DNA repeats was estimated at 4700-5200 copies for the short repeating unit (410 bp) and about 3100 copies for the long repeat (500 bp) per haploid genome. A comparison of wheat cultivars revealed extremely high levels of polymorphism in the 5S rRNA gene clusters. With one restriction enzyme digest all varieties tested gave unique banding patterns and, on a per fragment basis, 21-fold more polymorphism was detected among cultivars for 5S DNA compared to standard restriction fragment length polymorphisms (RFLPs) detected with single copy clones. Experiments with aneuploid stocks suggest that the 5S rRNA gene clusters at several chromosomal sites contribute to this polymorphism. A number of previous reports have shown that wheat cultivars are not easily distinguished by isozymes or RFLPs. The high level of variation detected in 5S rRNA gene clusters therefore offers the possibility of a sensitive fingerprinting method for wheat. 5S DNA and other macro-satellite sequences may also serve as hypervariable Mendelian markers for genetic and breeding experiments in wheat.