Study of the developmental pattern of heme catabolism in liver and the effects of cobalt on cytochrome P-450 and the rate of heme oxidation during the neonatal period.

The comparative development patterns of heme oxidation andof cytochrome P-450 dependent drug oxidation in rat liver were examined. High levels of heme oxygenase activity were present in whole embryo preparations at day 13 of gestation. At birth this enzyme activity in liver was approximately equal to that of normal adult liver. In the immediate postnatal period the rate of hepatic heme oxidation increased sharply, reaching levels 3-5 times normal during the first week postpartum. Thereafter, this enzyme activity progressively decreased and returned to normal adult levels by the 28th postpartum day. The development of microsomal heme oxidation and of P-450 dependent drug oxidation exhibited reciprocal patterns, with the latter being at low levels of activity during the immediate postnatal period and reaching adult activity only 4 or more wk after birth. Cobalt injected into pregnant animals or in to nursing mothers did not induce heme oxygenase in the fetus or suckling neonate. However, when treated directly with the metal, 4-day old neonates exhibited a small induction response of this enzyme; and the inducibility of heme oxygenase increased gradually to fully adult levels by the end of the 4th postpartum week. Cobalt at all postnatal developmental stages was capable of diminishing hepatic contents of total microsomal heme and P-450; however this effect of the metal was small in the immediate period after birth and increased progressively with maturation. These findings demonstrate that the patterns of development of hepatic capacity for carrying out the oxidation of heme and the P-450 dependent oxidation of drugs are different and thus provide further evidence that these microsomal enzyme systems are distinct from each other and under separate regulatory mechanisms. The degree of induction response for hepatic heme oxygenase evoked by the trace metal, cobalt, was also shown to have developmental determinants as did the susceptibility of hepatic cytochrome P-450 to degradation by this metal. The very high levels of hepatic heme oxygenase activity which characterize neonates during the first week of life indicate that over-production of bilirubin contributes significantly to the mechanism of neonatal jaundice.

Characterization of an apically derived epithelial membrane glycoprotein from bovine milk, which is expressed in capillary endothelia in diverse tissues.

A glycoprotein (PAS IV) of apparent Mr 76,000 was purified from bovine milk-fat-globule membrane and partially characterized. PAS IV contained mannose, galactose, and sialic acid as principal sugars (approximately 5.3% total carbohydrate [wt/wt]) and existed in milk in at least four isoelectric variants. The glycoprotein appeared to be an integral membrane protein by several criteria. PAS IV was recovered in the detergent phase of Triton X-114 extracts of milk-fat-globule membrane at room temperature. When bound to membrane, PAS IV was resistant to digestion by a number of proteinases, although after solubilization with non-ionic detergents, the protein was readily degraded. Amino acid analysis of the purified protein revealed a high percentage of amino acids with nonpolar residues. The location of PAS IV was determined in bovine tissues by using immunofluorescence techniques. In mammary tissue, PAS IV was located on both the apical surfaces of secretory epithelial cells and endothelial cells of capillaries. This glycoprotein was also detected in endothelial cells of heart, liver, spleen, pancreas, salivary gland, and small intestine. In addition to mammary epithelial cells, PAS IV was also located in certain other epithelial cells, most notably the bronchiolar epithelial cells of lung. The potential usefulness of this protein as a specific marker of capillary endothelial cells in certain tissues is discussed.

Biochemical and morphological comparison of plasma membrane and milk fat globule membrane from bovine mammary gland.

Purified plasma membrane fractions from lactating bovine mammary glands and membranes of milk fat globules from the same source were similar in distribution and fatty acid composition of phospholipids. The sphingomyelin content of the phospholipid fraction of both membranes was higher than in these fractions from other cell components, beta-carotene, a constituent characteristic of milk fat, was present in the lipid fraction of the plasma membrane. Cholesterol esters of plasma membrane were similar in fatty acid composition to those of milk fat globule membranes. Disc electrophoresis of either membrane preparation on polyacrylamide gels revealed a single major protein component characteristic of plasma membrane from other sources. Distinct morphological differences between plasma membrane and milk fat globule membranes were observed in both thin sections and in negatively stained material. Plasma membrane was vesicular in appearance while milk fat globule membranes had a platelike aspect. These observations are consistent with derivation of fat globule membrane from plasma membrane accompanied by structural rearrangement of membrane constituents.

Mercury content of common foods determined by neutron activation analysis.

The mercury contents in samples of flour, sugar, nonfat dry milk, potatoes, hamburger, chicken breast, shrimp, liver, eggs, and whole milk were determined by neutron activation analysis. The mercury was separated by anion exchange chromatography and precipitated as the sulfide. The mercury concentrations for all these foods were below 50 parts per billion.

Dihydroxyphenylalanine in rat food containing wheat and oats.

Dopa has been identified in rat food by three different fluorimetric assays and paper chromatography. Incubation of the rat food with proteolytic enzymes dramatically increased the measurable free dopa. Analysis of samples of six individual protein-containing constituents of rat food revealed that both wheat and oats contain dopa.

Gonadotropin-releasing hormone in milk.

The hypothalamic hormone gonadotropin-releasing hormone (GnRH) has been found in milk of man, cow, and rat. Radioimmunoassays of acidified milk indicate concentrations of GnRH ranging between 0.1 and 3 nanograms per milliliter. Multistep extractions, followed by electrophoresis, reveal gonadotropin-releasing activity in the fraction that comigrates with the GnRH-marker. A second hypothalamic hormone, thyrotropin-releasing hormone, is present in milk at a much lower concentration. "Milk -GnRH" may influence the secretion of the gonadotropic hormones in neonates.

Serum complement-like opsonic activities in human, animal, vegetable, and proprietary milks.

Human, animal, proprietary, and soy milks are comparable to human serum C5 in opsonization of baker's yeast. Bovine milk and human serum opsonically reconstitute C5-deficient mouse serum. Such reconstitution is selectively inhibited by antiserum to human C5. Further characterization suggests that bovine milk contains material structurally and functionally similar, but not identical, to human C5.

Interferon administered orally: protection of neonatal mice from lethal virus challenge.

Interferon was identified in the milk of mice injected with an interferon inducer. The kinetics of interferon appearance in serum and in milk were similar, but maximum concentrations in milk were 10 to 20 percent of those in serum. Interferon administered orally to neonatal mice was detected in their serums. Significantly more newborns survived an oral challenge with vesicular stomatitis virus when interferon had been induced in the lactating mothers.

Morphine in cow and human milk: could dietary morphine constitute a ligand for specific morphine (mu) receptors?

Morphine has been found in cow and human milk at concentrations of 200 to 500 nanograms per liter. Multistep purification yields a material that has immunological, biological, pharmacological, and chemical properties identical to those of morphine. Similar morphine-like material, which has been tentatively identified in some common plant sources, may be a ubiquitous dietary constituent and a possible source for the material in milk. Since morphine (mu) receptors have a low affinity for enkephalins, and since morphine-like materials have been described in brain and intestine, it is possible that morphine in food may be the source of this material and a normal ligand specific for mu receptors.

United States population dose estimates for iodine-131 in the thyroid after the Chinese atmospheric nuclear weapons tests.

Analysis of samples collected within the United States after the Chinese atmospheric nuclear weapons tests of 26 September and 17 November 1976 indicates that the radiation dose to the thyroid from iodine-131 in milk was predominant. A U.S. population dose to the thyroid of 68,000 man-rads was calculated for the iodine-131 fallout. The four excess thyroid cancers that are estimated to occur as a result of the September test during the next 45 years will be masked by the 380,000 cases of thyroid cancer which are expected to occur in the United States from all causes during the same interval.

Milk-like fluid in a mammary adenocarcinoma: biochemical characterization.

The milk-like accumulation in the R3230AC mammary adenocarcinoma that follows treatment with estrogen contains lactose, fatty acids, and proteins with electrophoretic properties similar to those of casein and whey of rat milk. This mammary tumor retains the biochemical capacity of the mammary gland in its lactational response to administration of hormone.

Simultaneous detection of reverse transcriptase and high molecular weight RNA unique to oncogenic RNA viruses.

The simultaneous assay of the reverse transcriptase and 60S to 70S RNA of oncogenic RNA viruses is described. The virus can be detected at low concentrations in biological fluids containing enzymatically active cell fragments or other contaminants. The fact that two features diagnostic of the oncornaviruses are used in the tests increases the certainty with which a positive outcome can be interpreted.

Posttranslational processing of secretory component in the rat jejunum by a brush border metalloprotease.

Secretory component (SC) is a glycoprotein that mediates the transcellular transport of polymeric immunoglobulins into external secretions. SC is synthesized and inserted into the plasma membrane of epithelial cells and hepatocytes as a transmembrane protein, where it serves as a receptor for polymeric immunoglobulins. SC is posttranslationally cleaved to a soluble protein before secretion into external fluids. In the rat jejunum, we observed that the molecular weights of both the major membrane and soluble forms of SC were 10,000-20,000 smaller than the comparable hepatic forms of the glycoprotein. We therefore set out to determine the reason for the differences in size of SC between these two tissues. The smaller size of jejunal SC was not due to the action of pancreatic proteases or differential glycosylation but was due to proteolysis by a jejunal brush border protease. The protease was characterized as a metalloprotease, with a pH optimum of approximately 5. It is present in jejunal, ileal, and renal tubular brush borders as an integral membrane constituent. When the protease was inhibited in vivo, conversion of jejunal secretory component to the smaller size was partially prevented. Thus, in the rat jejunum, SC undergoes two posttranslational proteolytic events: conversion of membrane secretory component to the soluble form and conversion of soluble SC to a smaller size by a previously undescribed brush border protease.

Vitamin D supply to the rat fetus and neonate.

The prevention of neonatal rickets by oral supplementation with vitamin D2 (ergocalciferol) has tended to obscure our ignorance of the natural mechanism by which young mammals receive an adequate supply of vitamin D. To investigate the possibility of specific intrauterine transfer and storage of vitamin D in fetal tissues, vitamin D-deficient female rats were given depot injections of 3H- or 14C-labeled vitamin D3 (cholecalciferol) before mating and the 3H-labeled animals were killed at stages during the last third of gestation. Analysis of lipid extracts from whole fetuses revealed a linear increase in the concentration of 25-hydroxyvitamin D3, 24,25-dihydroxyvitamin D3, and D3 itself between days 14 and 19 of gestation. During this period the elimination half-time of 3H-labeled molecules in maternal plasma fell from 27.1 to 4.4 d, suggesting that a specific mechanism was transferring vitamin D molecules into the fetuses. The vitamin was stored predominantly as 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3, with the highest concentrations in fetal muscle. Immediately after birth, pups from 3H- and 14C-labeled mothers were exchanged and later killed after 1-3 wk of suckling. Analysis of total lipid extracts for 3H and 14C content determined the relative contributions of vitamin D supplied before birth via the placenta and after birth in the maternal milk. The vitamin D content of the rat milk was relatively high, between 1.0 and 3.5 micrograms/liter. Nevertheless, the supply of vitamin D in utero, rather than from milk, was the main determinant of vitamin D status in early neonatal life. This is the first indication in a mammal of a specific transfer mechanism that allows the fetus to accumulate vitamin D from the mother during the last third of gestation.

Trans unsaturated fatty acids in natural products and processed foods.

Various representatives of trans unsaturated fatty acids have been detected in plants. The main trans monoenoic acids in ruminants, elaidic acid and vaccenic acid are not found in plants. Most oil seeds used for production of edible fats do not contain any trans fatty acids. Trans fatty acids in non-ruminant animals are derived from food. In adult ruminants, however, trans fatty acids are produced by microbial hydrogenation of linoleic acid and linolenic acid in the rumen; in consequence, a variety of positional and stereoisomers of both cis and trans fatty acids appear in both meat and milk. The total trans content of 5-10% in beef fat is largely trans monoene, mainly 9-trans (elaidic acid), 10-trans and 11-trans (vaccenic acid). The trans fatty acid content of human fat is dependent on the uptake of trans fatty acid from food. The usual content is about 2%, though up to 15% has been recorded. The isomer distribution is similar to that of the trans isomers of butter and margarine. The trans fatty acid content in ruminant products such as milk, butter, cheese, curds and tallow is 5-10%. These products contain nearly all possible stereo- and positional isomers of oleic and linoleic acid. The trans fatty acid content of margarines fluctuates widely according to raw material and process conditions. According to one investigation, an average of 16% of all unsaturated fatty acids in margarines consists of geometrical and positional isomers of the "natural" acids, a similar percentage to that in butter. Diet and health margarines have much lower contents of trans fatty acids. The total trans content of margarines is largely due to trans-18:1 monoenes. The positional isomers of the trans-monoenes are more evenly distributed in margarine than in butter. Hardened oils do not contain trans fatty acid isomers other than those produced by the microflora of ruminants. Therefore, claims that trans fatty acid isomers are "synthetic", "nonphysiological" or "unnatural" are unjustified if these words are used to imply "not produced by the living organism".

Removal of inhibitors against RNA-directed DNA polymerase activity in human milk.

Milk from a number of species (e.g., man, mouse, rat, dog, and cow) contains inhibitors of the RNA-directed DNA polymerase. When attempts are made to isolate virions from the milk, part of the inhibitors follow the virions in the purification. The amount of inhibitors varies in different milk samples. These inhibitors can probably account for the large discrepancies reported in studies of the presence of oncornaviruses in human milk. Phosphatases bound to subcellular particles or fragments seem to be the most important inhibitors in the milk interfering with the RNA-directed DNA polymerase assay. It is shown that the inhibitory enzymes can be completely removed by sedimentation of the milk through a Metrizamide gradient.

The concentration of 67Ga and 45Ca in the lactating mammary gland and its relevance to the tumor uptake of 67Ga citrate.

67Ga is known to concentrate in the breasts of pregnant and postpartum women, and a case is now described in which 67Ga uptake was seen in the breasts of a woman who was neither pregnant nor postpartum, but was receiving chemotherapy for Hodgkin's disease. Comparative studies of the uptake of 67Ga and 45Ca in lactating dogs have shown that both nuclides are secreted in the milk in similar amounts and in protein-bound form. The concentration of 67Ga in mammary tissue is about one-half of that found in the milk at 5 hr postinjection but, by 48 hr, the concentrations are approximately equal. There were similarities in the subcellular distributions of 67Ga and 45Ca in the lactating mammary gland at 5 and 48 hr. Although there was a correlation between 67Ga and 45Ca in individual pieces of a lactating mammary gland at 5 hr after injection, no such correlation was seen between the two nuclides in multiple samples of a transmissible venereal tumor measured at various time intervals. The rate of dispersion of 67Ga and 45Ca from the lactating mammary gland was similar but, in the tumor, 67Ga was present in very much greater amounts than 45Ca and was retained longer. It is concluded that, although there may be similarities in the metabolic pathways of gallium and calcium in the lactating mammary gland, there is no similarity in the mechanism of uptake of these two elements into tumors.

Nature of micellar calcium phosphate in cows' milk as studied by high-resolution electron microscopy.

The nature of the inorganic calcium phosphate in the casein micelle of cows' milk has been studied by high-resolution electron microscopy. No periodic lattice spacings could be imaged, and diffraction patterns were of the diffuse amorphous type. Short-range order of less than 15 A may be present, but the results indicate that there is no long-range order in micellar calcium phosphate.

The secondary structure of peptides derived from caseins: a circular dichroism study.

Three peptides have been formed by proteolytic digestion of individual casein proteins and their secondary structures characterised by far-UV circular dichroism (CD). Peptide alpha s1(1-23), residues 1-23 of alpha s1-casein, was generated by treatment of the parent protein with chymosin. Peptides beta(1-28) and beta(1-52), residues 1-28 and 1-52 of beta-casein, were plasmin- and chymotrypsin-generated fragments, respectively. Analysis of the CD spectra revealed that in aqueous solution all three peptides have secondary structures composed exclusively of beta-sheet and random coil. A limited amount of alpha-helix was formed in two of the three peptides upon treatment with high concentrations (greater than 40% (v/v] of 2,2,2-trifluoroethanol. Partial dephosphorylation (60%) of beta(1-28) and beta(1-52) by treatment with alkaline phosphatase resulted in homogeneous preparations, as judged by polyacrylamide gel electrophoresis, which exhibited increased hydrophobicity. This reduction in the level of phosphorylation of serine residues 15, 17, 18 and 19 led to increased propensity for helix formation in the peptides in the presence of 2,2,2-trifluoroethanol, but no alpha-helical structures were detected in the dephosphorylated peptides in the absence of 2,2,2-trifluoroethanol.

Goat milk oligosaccharides: purification and characterization by HPLC and high-field 1H-NMR spectroscopy.

Three oligosaccharides were isolated from goat milk using Bio-Gel P-4 and reverse-phase C-18 HPLC and were characterized by high-field 1H-NMR spectroscopy as a trisaccharide, GlcNAc(beta 1-6)Gal(beta 1-4)Glc, a tetrasaccharide, Gal(beta 1-4)GlcNAc(beta 1-6)Gal(beta 1-4)Glc, and a pentasaccharide.