The composition of milk xanthine oxidase

The composition of milk xanthine oxidase has been reinvestigated. When the enzyme is prepared by methods that include a selective denaturation step in the presence of sodium salicylate the product is obtained very conveniently and in high yield, and is homogeneous in the ultracentrifuge and in recycling gel filtration. It has specific activity higher than previously reported preparations of the enzyme and its composition approximates closely to 2mol of FAD, 2g-atoms of Mo and 8g-atoms of Fe/mol of protein (molecular weight about 275000). In contrast, when purely conventional preparative methods are used the product is also homogeneous by the above criteria but has a lower specific activity and is generally comparable to the crystallized enzyme described previously. Such samples also contain 2mol of FAD/mol of protein but they have lower contents of Mo (e.g. 1.2g-atom/mol). Amino acid compositions for the two types of preparation are indistinguishable. These results confirm the previous conclusion that conventional methods give mixtures of xanthine oxidase with an inactive modification of the enzyme now termed ;de-molybdo-xanthine oxidase', and show that salicylate can selectively denature the latter. The origin of de-molybdo-xanthine oxidase was investigated. FAD/Mo ratios show that it is present not only in enzyme purified by conventional methods but also in ;milk microsomes' (Bailie & Morton, 1958) and in enzyme samples prepared without proteolytic digestion. We conclude that it is secreted by cows together with the active enzyme and we discuss its occurrence in the preparations of other workers. Studies on the milks of individual cows show that nutritional rather than genetic factors determine the relative amounts of xanthine oxidase and de-molybdo-xanthine oxidase. A second inactive modification of the enzyme, now termed ;inactivated xanthine oxidase', causes variability in activity relative to E(450) or to Mo content and formation of it decreases these ratios during storage of enzyme samples including samples free from demolybdo-xanthine oxidase. We conclude that even the best purified xanthine oxidase samples described here and by other workers are contaminated by significant amounts of the inactivated form. This may complicate the interpretation of changes in the enzyme taking place during the slow phase of reduction by substrates. Attempts to remove iron from the enzyme by published methods were not successful.     

The molecular composition of square arrays

Square arrays are prominent structures in plasma membranes of brain, muscle, and kidneys with an unknown function. So far, the analysis of these arrays has been restricted to freeze fracture preparations, which have shown square arrays to contain the water channel Aquaporin-4 (AQP4). Using Blue-Native PAGE immunoblots, we provide evidence that higher-order AQP4 complexes correspond to square arrays, with the AQP4 isoform M23 playing a dominant role. Our data are consistent with the idea that square arrays consist of aggregates of AQP4 tetramers complexed with multiples of dimers. By comparison, Aquaporin-1 and Aquaporin-9 form tetramers, but not higher-order complexes. AQP4 square arrays are stable under several biochemical purification steps. Analyzing the internal composition of the higher-order complexes by 2D gels, we demonstrate that the square arrays in addition to M23 also invariably contain AQP4, M1, and a novel AQP4 isoform that we call Mz. The visualization AQP4 square arrays by a rapid, biochemical assay provides new insight in the molecular organization of square arrays and gives further proof of the heterogeneity of AQP4 square arrays in vivo.     

The chemical composition of interstellar molecular clouds

Relative abundances for molecules observed by radio astronomical techniques have been determined for several interstellar regions with differing physical properties.     

The composition of cheetah (Acinonyx jubatus) milk

Milk was obtained from two captive bred cheetahs. The nutrient content was 99.6 g protein; 64.8 g fat; and 40.21 g lactose per kg milk. Small amounts of oligosaccharides, glucose, galactose and fucose were noted. The protein fraction respectively consisted of 34.2 g caseins per kg milk and of 65.3 g whey proteins per kg milk. Very little variation in milk composition among the individual cheetahs was noted. Electrophoresis and identification of protein bands showed a similar migrating sequence of proteins as seen in lion's and cat's milk, with small differences in the β-caseins. The lipid fraction contains 290.4 g saturated and 337.3 g mono-unsaturated fatty acids per kg milk fat respectively. The high content of 279.5 g kg^− 1 milk fat of polyunsaturated fatty acids is due to a high content in α-linolenic acid. No short chain fatty acids, but substantial levels of uneven carbon chain fatty acids were observed.     

The chemical composition of interstellar molecular clouds

Relative abundances for molecules observed by radio astronomical techniques have been determined for several interstellar regions with differing physical properties.     

产溶剂梭菌分子遗传操作技术研究进展

产溶剂梭菌是一类重要的工业微生物.通过遗传改造以优化产溶剂梭菌的发酵性能一直是溶剂制造技术研究的重要课题,但长期受限于该类菌并不完善的遗传操作工具,未见明显突破.近年来,随着TargeTron基因中断、大片段基因整合等新技术和新方法的出现,其分子遗传改造已取得较大进展.文中对产溶剂梭菌的分子遗传操作工具研究进展进行了总结,并指出了现有技术在效率及全面性方面的不足.基于此,今后应进一步优化现有的梭菌基因失活技术,如建立基于同源重组的基因删除和替换;同时也应发展新的分子操作技术,如基因组多位点共编辑、多拷贝定点和随机整合等.     

The composition of milk from captive aardvark (Orycteropus afer)

Evidence indicates that hand-rearing of exotic species may require specialized milk formulae. Milk samples collected from a captive aardvark (Orycteropus afer) between days 3 and 32 postpartum have been analysed for fat, protein, lactose, and minerals. The level of nutrients determined may be at variance with those of a normal lactating aardvark because of the low volumes of milk removed. The analysis indicates that the commercial preparations may be inadequate in protein and provide high levels of energy in the form of lactose. The energy level from fat appears to be similar to that of other species, but it is unlikely that the commercial formulae would provide the level of the long chain polyunsaturated fatty acids found in aardvark milk. It is clear that the energy available from the milk protein is significant.     

The composition of serval (Felis serval) milk during mid-lactation

Milk was obtained from three captive servals. The average nutrient content was 158.3+/-44.4 g protein; 152.6+/-62.3 g fat; and 68.7+/-31.4 g lactose per kg milk. The protein fraction respectively consisted of 117.7+/-44.8 g caseins per kg milk and of 40.6+/-6.7 g whey proteins per kg milk. Electrophoresis and identification of protein bands showed a similar migrating sequence of proteins as seen in cheetah and cat milk, with small differences in the beta-caseins. The lipid fraction contains 313.3+/-18.8 g saturated and 338.6+/-11.9 g mono unsaturated fatty acids per kg milk fat respectively. The high content of 292.4+/-24.9 g kg(-1) milk fat of polyunsaturated fatty acids is due to a high content in linolenic acid. No short chain fatty acids, but substantial levels of uneven carbon chain fatty acids were observed. In general, serval milk has a higher protein and fat content than that of the domestic cat and cheetah, and a lower content of unsaturated fatty acids than that of the domestic cat.     

The molecular composition of the volutin granule of yeast.

The effects of insulin on glucose uptake and lactate release in the perfused working rat heart have been investigated in three types of preparation: (i) a control low-workload preparation; (ii) an increased-pressure-workload preparation, simulating conditions of aortic pressure encountered in vivo; (iii) an increased-volume-workload preparation, where pumping work done is approximately the same as (ii) but coronary flow is restricted because of the decreased aortic pressure. Insulin stimulated glucose uptake and lactate release in preparations (i) and (ii), but failed to do so in preparation (iii). It was considered possible that preparation (iii) was hypoxic, thus necessitating a maximal stimulation of glucose uptake. This was confirmed by improving cardiac oxygenation by addition of stroma-free haemoglobin to the perfusate in preparation (iii). Under these conditions in the absence of insulin, glucose uptake and lactate release were decreased compared with perfusions in the absence of haemoglobin. Insulin stimulation of both processes was restored. We conclude that the failure of other workers to observe insulin effects on glucose uptake and lactate release under physiological workloads [preparation (ii)] may be a consequence of intracellular hypoxia in their preparations.     

The fatty acid composition of milk fat as influenced by feeding oilseeds

The fatty acid composition of bovine milk fat can be substantially altered by feeding lipid sources which alter the fatty acid profile of lipid entering the intestine from the rumen. As long-chain fatty acids of dietary origin can be incorporated directly into milk fat the opportunity exists to alter the ratio of short and long-chain fatty acids as well as the degree of saturation of milk fat. In practice our ability to alter the fatty acid profile of milk fat is limited not by the synthetic capacity of the mammary gland, but rather by the challenge of achieving effective protection of unsaturated dietary fatty acids from biohydrogenation in the rumen, as well as keeping the level of polyunsaturated fatty acids within the range where the organoleptic quality and shelf-life of milk and dairy products are not compromised. The fatty acid composition of oilseeds such as canola are considered desirable from a human health perspective and thus their inclusion in the diet of dairy cattle as a means of achieving a more desirable fatty acid profile in milk fat may enhance the nutritive quality of milk.     

The calculation of molecular similarity: alternative formulas,data manipulation and graphical display

The use of electrostatic potential comparisons between molecules for the elucidation of structure activity relationships is now a well-established modeling technique. The Carbo and Hodgkin similarity indices are used extensively to make quantitative comparisons of this nature; yet their roots are found in the overlap of electron density distribution, with both formulas utilizing a product-based numerator.Two new similarity indices are suggested that calculate the electrostatic potential similarity using a difference-based numerator. The form of the new indices allows the creation of additional software functions that enhance the flexibility of similarity calculations and permit the creation of similarity maps.The general properties of these software functions and all indices are discussed and applied to a series of dopamine D2 receptor agonists.     

The manipulation of fatty acid composition in L-M cell monolayers supplemented with cyclopentenyl fatty acids

Mouse L-M fibroblasts, grown in a serum-free medium, were supplemented with fatty acids of 16 and 18 carbon chain lengths that contain a cyclopentene ring in the ω position. These fatty acids, unnatural to mammalian systems, were incorporated into the major lipid classes of L-M fibroblasts. Supplementation with the cyclopentenyl fatty acids caused an accumulation of neutral glycerolipids and marked inhibition of cell growth. Following the addition of supplement, the cells became more rounded. Of particular interest was the fact that the phospholipid fraction isolated from treated cells contained cyclic fatty acids that accounted for as much as 24% of the total phospholipid acyl groups. Unlike the pattern of distribution displayed by endogenous natural monoenes, the majority of the cyclic acid present was esterified in the sn-1 position of both phosphatidylcholine and phosphatidylethanolamine. The 18-carbon cyclic fatty acid [chaulmoogric acid, 13-(2-cyclopenten-1-yl)tridecanoic acid] was incorporated at the expense of the endogenous C-16:0, C-18:0, and C-18:1 fatty acids of the glycerophospholipids. The esterification altered the ratio of saturated to unsaturated acyl groups in the cellular phospholipids. No biochemical modification of chaulmoogric acid was detected.Our results imply that incorporation of unnatural fatty acid analogs, such as chaulmoogric acid, into cellular membranes would alter the functional properties of biological membranes that are dependent on membrane fluidity and structural organization.     

Genome-scale genetic manipulation methods for exploring bacterial molecular biology

Bacteria are diverse and abundant, playing key roles in human health and disease, the environment, and biotechnology. Despite progress in genome sequencing and bioengineering, much remains unknown about the functional organization of prokaryotes. For instance, roughly a third of the protein-coding genes of the best-studied model bacterium, Escherichia coli, currently lack experimental annotations. Systems-level experimental approaches for investigating the functional associations of bacterial genes and genetic structures are essential for defining the fundamental molecular biology of microbes, preventing the spread of antibacterial resistance in the clinic, and driving the development of future biotechnological applications. This review highlights recently introduced large-scale genetic manipulation and screening procedures for the systematic exploration of bacterial gene functions, molecular relationships, and the global organization of bacteria at the gene, pathway, and genome levels.     

Alteration of the phospholipid composition of Escherichia coli through genetic manipulation

In order to study the function of individual phospholipids, we have constructed a strain of Escherichia coli in which the ratio of phosphatidylethanolamine to phosphatidylglycerol plus cardiolipin can be regulated. In this strain (HDL1001) the normal expression of the phosphatidylglycerophosphate synthase does not occur due to the presence of the pgsA30 allele (Heacock, P. N., and Dowhan, W. (1987) J. Biol. Chem. 262, 13044-13049). A second chromosomal copy of the pgsA gene is fused to the lacOP region in single copy within the lac operon. Strain HDL1001 is absolutely dependent for growth on an inducer of the lac operon. In addition, the level of the pgsA gene product, the content of the two major acidic phospholipids, and the growth rate are dependent on the level of inducer in the growth medium. Cells remain viable in the absence of inducer as evidenced by a rapid return to normal growth after the readdition of inducer. The growth rate and phospholipid composition are affected only after the level of phosphatidylglycerophosphate synthase drops below about 15% of normal levels; both phosphatidic acid and (d)CDP-diacylglycerol also begin to increase to significant levels. At the point of cell arrest the level of the major acidic phospholipids is reduced by about 90% of wild type levels.