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Many biological fluids, including blood, milk, extracellular fluid, saliva, urine, synovial fluid and cerebrospinal fluid, are usually supersaturated with respect to hydroxyapatite (HA) [1][2][3][4][5], but generally remain stable. Nevertheless, dystrophic calcification does occur, and vascular calcification or stone-forming biofluids, for example, have serious consequences for human health. Genetic ablation and other experiments on individual serum proteins have demonstrated the importance of serum fetuin A (FETUA), osteopontin (OPN) and matrix Gla protein (MGP) for inhibiting the precipitation of calcium phosphate (CaP) in serum and preventing ectopic calcification of soft tissues [6][7][8]. A metastable, colloidal, complex of CaP with FETUA, MGP and secretory phosphoprotein 24 (SPP-24) forms when the serum is destabilized [9,10], but the physiological mechanism is still unclear.Milk provides an example of a biofluid that seldom forms CaP precipitates or causes dystrophic calcification of the mammary gland, even though it may contain very much higher concentrations of calcium (Ca) and inorganic phosphorus (P i ) than does serum [11]. In milk, casein micelles sequester CaP through phosphate centre (PC) sequences, typically pSpSpSEE, in Calcium phosphate nanoclusters are equilibrium particles of defined chemical composition in which a core of amorphous calcium phosphate is sequestered within a shell of casein phosphopeptides. Sequence analyses and a structure prediction method were applied to secreted phosphoproteins of known importance in controlling calcification, and eight noncasein phosphoproteins were identified as containing one or more subsequences capable of forming nanoclusters. Small-angle X-ray scattering was used to confirm that a plasmin phosphopeptide of one of the identified proteins, osteopontin, formed a novel type of calcium phosphate nanocluster in which the radius of the amorphous calcium phosphate core was four times larger than is typical of casein nanoclusters. A thermodynamic treatment of nanocluster formation identified the factors of importance in determining the equilibrium size of the core, and showed how a nanocluster solution could be thermodynamically stable yet supersaturated with respect to the mineral phase of bones and teeth. It is suggested that the ability of some secreted phosphoproteins to form nanoclusters is physiologically important for the control or inhibition of calcification in soft and mineralized tissues, the extracellular matrix and a wide range of biofluids, including milk and blood.Abbreviations ACP, amorphous calcium phosphate; CaP, calcium phosphate; CPN, calcium phosphate nanocluster; DCPD, di-calcium phosphate di-hydrate; DMP1, dentin matrix acidic phosphoprotein 1; FETUA, fetuin A; HA, hydroxyapatite; MGP, matrix Gla protein; OCP, octacalcium phosphate; OPN, osteopontin; PC, phosphate centre; pS, phosphoseryl residue; RBP, riboflavin-binding protein; SAXS, small-angle X-ray scattering; SCPP, secretory calcium-binding phosphoprotein; SP, secreted phosphoprot...

1. Changes in the mean volume, the rate of fatty acid and acylglycerol glycerol synthesis, the activity of lipoprotein lipase and the numbers and affinities of insulin receptors of subcutaneous adipocytes are reported for sheep at different stages of pregnancy and lactation. In addition, the serum concentrations of insulin, progesterone, prolactin, choriomammotropin, somatotropin, glucose, acetate, L-lactate, glycerol and unesterified fatty acids are reported for these sheep. 2. A switch from lipid accumulation to net lipid mobilization accompanied by a decline in the capacity for lipid synthesis, occurred at the onset of the last third of pregnancy. Net lipid mobilization continued during lactation. 3. The changes that occurred in the serum concentrations of the various hormones listed above are discussed in relation to their possible roles in the modulation of adipose tissue metabolism in sheep during pregnancy and lactation. The observations are compared with those from previous studies on the hormonal control of adipose tissue metabolism in the rat during pregnancy and lactation.

Changes in the volume, the rates of fatty acid synthesis and synthesis of the glycerol moiety of acylglycerols, the activity of lipoprotein lipase, and the number and affinity of insulin receptors of adipocytes, and concentrations of serum insulin, prolactin and progesterone were determined in virgin rats and in rats at various stages of pregnancy and lactation. Changes in the metabolic activities of adipose tissue appeared to be synchronized and primarily comprised a marked decrease in anabolic activity around parturition. In contrast, the number of insulin receptors (Kd 1.5 nM) per adipocyte doubled during pregnancy before returning to normal values around parturition. It is postulated that the increase in the number of insulin receptors is an adaptation to counteract the effects of insulin-antagonistic hormones during pregnancy and that the decrease in the number of receptors is primarily responsible for the loss of anabolic activity around parturition.

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1. Nitrate reductase was purified 134-fold from Escherichia coli K12. The purification procedure involves the release by Triton X-100 of the enzyme from the cell envelope. i. The purified enzyme exists in aqueous solution either as a monomer (mol. wt. about 220 000) or as an associated form (probably a tetramer; mol.wt. about 880 000). 3. The purified enzyme has three subunits with apparent mol.wts. of 150 000, 67000 and 65000. An additional subunit of apparent mol.wt. 20000 is present in a haem-containing fraction that is also produced by the preparative procedure described. 4. None of the enzyme subunits is present in the cell envelope of cells grown in the absence of nitrate. 5. Reversible changes in the activity of nitrate reductase in vitro with FMNH2 as reductant can be induced under circumstances which are without effect on the reduced Benzyl Viologen-NO3-activity.