Butyrophilin,an apical plasma membrane-associated glycoprotein characteristic of lactating mammary glands of diverse species

Lipid globule membranes were isolated from human and bovine milk and from the milk of sheep, goat, pig, rat and guinea pig, and their polypeptide compositions were analyzed. The major polypeptides with molecular weights similar to that of bovine butyrophilin were separated by gel electrophoresis, isolated and characterized with respect to isoelectric point, molecular weight, immunological cross-reactivity and peptide composition after proteolytic cleavage. We show that in all species examined these proteins are similar to bovine butyrophilin in (i) their relative insolubility in buffers of low and high ionic strength and in non-denaturing detergents, (ii) the occurrence of several isoelectric variants, and (iii) patterns of peptides obtained by protease digestion. It is concluded that closely related proteins are major constituents of the cytoplasmic coat structures associated with milk lipid globule membranes of many species, and we propose the name butyrophilins for this group of proteins. Bovine and human butyrophilins are glycosylated with relatively large amounts of glucosamine, mannose, glucose and galactose but little fucose, sialic acids or galactosamine. Most if not all of the sugar residues are associated with an acetone-soluble peptide fragment of $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 12 000–16 000 focusing at about pH 4.0. We suggest that this fragment contains a membrane-spanning peptide sequence and is involved in the attachment of the cytoplasmic coat to the membrane of the milk lipid globule.     

Inhibition of Auxin-induced Deoxyribonucleic Acid Synthesis and Chromatin Activity by 5-Fluorodeoxyuridine in Soybean Hypocotyl

Rootless soybean (Glycine max) seedlings were used as a test system to examine the action of auxin on chromatin-directed RNA synthesis. Chromatin from the basal tissue of rootless seedlings (both control and auxin-treated) had RNA synthetic capacity similar to that of chromatin from comparably treated intact seedlings. When DNA synthesis normally induced in the basal tissue by auxin was blocked in the rootless seedlings by 5-fluorodeoxyuridine, the auxin enhancement of chromatin activity was inhibited 70%. This level was still three times the control level, indicating that auxin influenced the synthetic activity of existing DNA template. Experiments with Escherichia coli RNA polymerase revealed that chromatin from both auxin- and auxin plus 5-fluorodeoxyuridine-treated tissue saturated at higher levels than chromatin from control tissue.     

Glucose Metabolism in Neisseria gonorrhoeae

The metabolism of glucose was examined in several clinical isolates of Neisseria gonorrhoeae. Radiorespirometric studies revealed that growing cells metabolized glucose by a combination on the Entner-Doudoroff and pentose phosphate pathways. A portion of the glyceraldehyde-3-phosphate formed via the Entner-Doudoroff pathway was recycled by conversion to glucose-6-phosphate. Subsequent catabolism of this glucose-6-phosphate by either the Entner-Doudoroff or pentose phosphate pathways yielded CO(2) from the original C6 of glucose. Enzyme analyses confirmed the presence of all enzymes of the Entner-Doudoroff, pentose phosphate, and Embden-Meyerhof-Parnas pathways. There was always a high specific activity of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) relative to that of 6-phosphogluconate dehydrogenase (EC 1.1.1.44). The glucose-6-phosphate dehydrogenase utilized either nicotinamide adenine dinucleotide phosphate or nicotinamide adenine dinucleotide as electron acceptor. Acetate was the only detectable nongaseous end product of glucose metabolism. Following the disappearance of glucose, acetate was metabolized by the tricarboxylic acid cycle as evidenced by the preferential oxidation of [1-(14)C]acetate over that of [2-(14)C]acetate. When an aerobically grown log-phase culture was subjected to anaerobic conditions, lactate and acetate were formed from glucose. Radiorespirometric studies showed that under these conditions, glucose was dissimilated entirely by the Entner-Doudoroff pathway. Further studies determined that this anaerobic dissimilation of glucose was not growth dependent.     

The localization of a vitamin K-induced modification in an N-terminal fragment of human prothrombin

1. The N-terminal fragment (PF-I) split off from prothrombin during coagulation was purified to homogeneity from human serum. 2. The apparent molecular weight is 27000+/-2000 in sodium dodecyl sulphate-polyacrylamide-gel electrophoresis, whereas a value of about 19600 is obtained by calculation based on amino acid and carbohydrate analyses. The N-terminal sequence is an Ala-Asx bond. The fragment contains about 16% carbohydrate, binds phospholipids in the presence of Ca(2+) and is adsorbed to BaSO(4). The pK(a) of its BaSO(4)-binding group(s) is 3.1-3.5. 3. By CNBr cleavage of fragment PF-I two peptides (C-1 and C-2) were obtained with molecular weights of about 5900 (C-2) and 12400 (C-1) on the basis of amino acid and carbohydrate analyses. Only the smaller (N-terminal) peptide is adsorbed to BaSO(4) and, since the ability of the whole protein to bind to BaSO(4) is known to be absent in samples obtained from patients treated with vitamin K antagonists, this peptide probably contains the site of a modification to the structure of the protein which occurs during biosynthesis and depends on vitamin K. This peptide does not contain hexosamine or sialic acid.     

Differential cytotoxicity of activated lymphocytes on allogeneic and xenogeneic target cells. II. Activation by phytohemagglutinin

In the preceding paper it has been shown that human or mouse lymphocytes stimulated by a variety of agents, damaged allogeneic target cells while damage of xenogeneic target cells was weak or absent. In this study, the species specificity of the cytotoxicity of PHA activated lymphocytes has been studied in greater detail. Effector cells were purified lymphocytes either from human peripheral blood, or from spleen or lymph nodes of inbred mice. Target cells were ⁵¹Cr-labeled human Chang liver cells or mouse L cells.PHA stimulated human or mouse lymphocytes were significantly more cytotoxic to allogeneic than to xenogeneic target cells. At low PHA doses at which damage of allogeneic target cells was significant, damage of xenogeneic target cells was very weak or absent. At higher PHA doses, damage of xenogeneic target cells became also significant but always remained at a lower level than that of allogeneic target cells.Prestimulation of human lymphocytes with PHA for 3 days increased their cytotoxic efficiency. Furthermore, damage of human Chang cells by human lymphocytes had a dose-response relationship similar to that valid for stimulation of DNA synthesis. However, damage of mouse L cells by human lymphocytes increased at PHA-doses at which stimulation of DNA-synthesis declined. For mouse lymphocytes, these doseresponse relationships were less clear-cut, probably due to differences in origin and survival of the effector cells. This confirms previous observations that cytotoxicity and DNA-synthesis are different but probably interdependent expressions of lymphocyte activation.     

Hormonal Regulation of Cell Elongation in the Hypocotyl of Rootless Soybean: An Evaluation of the Role of DNA Synthesis

A method was developed where soybean seedlings were grown without roots to study the influence of hormones of root origin on shoot growth. Excision of the root resulted in inhibition of apical section growth and DNA synthesis and inhibited elongating section growth. A synthetic cytokinin restored DNA synthesis in the apical section, but did not influence growth in either the apical or elongating sections. Low concentrations of gibberellin with the cytokinin restored growth in the apical section. Gibberellin alone was sufficient to restore growth in the elongating section.An inhibitor of DNA synthesis, 5-fluorodeoxyuridine, inhibited the increase in apical section DNA without inhibiting control or gibberellin-induced growth in the elongating section. Experiments with (14)C-thymidine resulted in no DNA labeling differences in the elongating section under conditions where gibberellin-induced elongation varied from 50% to 73% above controls. It was concluded that gibberellin-induced elongation in soybean hypocotyl occurred in the absence of DNA synthesis. Gibberellin does stimulate DNA synthesis in the apical tissue apart from its effect on cell elongation.Excised soybean hypocotyl elongated maximally at 10(-6)m auxin. At higher auxin concentrations, fresh weight and ethylene production increased, but elongation was reduced. Addition of GA to the higher auxin concentrations resulted in a 50% inhibition in auxin-induced ethylene production and resumption in maximal elongation. Added ethylene inhibited elongation 30% at 2 mul/l. Addition of up to 100 mul/l ethylene did not inhibit elongation with GA present in the incubation medium. Thus GA may counteract ehtylene inhibition of cell elongation in addition to inhibiting ethylene production in auxin-treated tissues.     

The adenosine triphosphate inhibition of the pyruvate kinase reaction and its dependence on the total magnesium ion concentration

1. The effects of ATP, PP(i) and EDTA on the skeletal-muscle pyruvate kinase reaction at various concentrations of magnesium (where ;magnesium' refers to total Mg(2+), both free and in the form of complexes) were investigated. The reaction rate was determined as the amount of pyruvate formed in a recorded time of incubation. 2. At 44mm-magnesium the K(m) values for ADP and phosphoenolpyruvate were unaltered by the presence of ATP up to 6.8mm in systems buffered with either tris-hydrochloric acid or glycylglycine-sodium hydroxide, but the K(m) values were different in these systems. The K(m) for one substrate was independent of the concentration of the second substrate. 3. At 10mm-magnesium in the tris-hydrochloric acid system ATP inhibited the reaction competitively with respect to ADP and phosphoenolpyruvate. In the glycylglycine-sodium hydroxide system the inhibition appeared to be non-competitive. At 10mm-magnesium the K(m) values were lower than at 44mm-magnesium and dependent on the system used. 4. In the tris-hydrochloric acid system the reaction rate rose with increasing magnesium concentration up to a maximum at a concentration 10-20 times that of ADP. Further increase inhibited the reaction and at 44mm-magnesium the rate was 25-50% of its maximum. This inhibition paralleled that produced by increasing trimethylammonium chloride concentrations and was not due to a specific effect of the Mg(2+) ion. 5. In the presence of 6.8mm-ATP no reaction occurred below 4-6mm-magnesium, and further increase apparently abolished the inhibition as the reaction rate increased and became equal to those obtained in the absence of ATP at 10-25mm-magnesium. Further increase in magnesium concentration gave reaction rates that were slightly higher in the presence of ATP than in its absence. The maximal rate in the presence of ATP was distinctly lower than in its absence. When 6.8mm-PP(i) or 6.8mm-EDTA was present the variations in reaction rate with rising magnesium concentration were similar to that obtained in the presence of ATP below 6-8mm-magnesium but further increase in the magnesium concentration resulted in an increase in the rate up to a maximum comparable with that of the control. The effect of pure chelation was thus a displacement of the reaction maximum to higher magnesium concentrations without changing the maximal rate. When correction had been made for this effect, ATP gave inhibition at 44mm-magnesium that was competitive with respect to ADP (K(i) 2.1x10(-2)m). This degree of inhibition is far less than was reported earlier and its importance for the mechanism of the pyruvate kinase reaction is discussed.     

Cerebral O2 metabolism and cerebral blood flow in humans during deep and rapid-eye-movement sleep

It could be expected that the various stages of sleep were reflected in variation of the overall level of cerebral activity and thereby in the magnitude of cerebral metabolic rate of oxygen (CMRO2) and cerebral blood flow (CBF). The elusive nature of sleep imposes major methodological restrictions on examination of this question. We have now measured CBF and CMRO2 in young healthy volunteers using the Kety-Schmidt technique with 133Xe as the inert gas. Measurements were performed during wakefulness, deep sleep (stage 3/4), and rapid-eye-movement (REM) sleep as verified by standard polysomnography. Contrary to the only previous study in humans, which reported an insignificant 3% reduction in CMRO2 during sleep, we found a deep-sleep-associated statistically highly significant 25% decrease in CMRO2, a magnitude of depression according with studies of glucose uptake and reaching levels otherwise associated with light anesthesia. During REM sleep (dream sleep) CMRO2 was practically the same as in the awake state. Changes in CBF paralleled changes in CMRO2 during both deep and REM sleep.