Comparison of the whey acidic protein genes of the rat and mouse.

Whey acidic protein (WAP), a hormonally-regulated 14,000 dalton cysteine-rich protein, is the principal whey protein found in rodent milk. Genomic clones encompassing both the 2.8 Kb rat and 3.3 Kb mouse WAP genes have been characterized. The genes consist of four exons and three introns. The middle two exons encode the two cysteine-rich regions which probably form separate protein domains. Homology in the 5' flanking DNA of the mouse and rat extends at least 325 bp upstream of the putative CAP site, including a precisely conserved stretch of 50 bp around the unusual TATA and CAAT sites. The homology previously observed between the 3' noncoding sequences of the rat and mouse mRNAs extends at least 20 bp into the 3' flanking region. Several potential glucocorticoid receptor binding sites have been found in the 5' flanking region of the WAP gene. The conservation of the 5' flanking region of the WAP genes may be related to regulation of expression of WAP by peptide and/or steroid hormones.     

Metastasis of cancer of the thyroid gland as a cause of goitre.

In three cases of cancer (arising in the pancreas, the breast and the lung) that had metastasized to the thyroid an unusual primary thyroid lesion was the presenting problem. While reputedly rare, metastases in the thyroid gland are not infrequently encountered at necropsy. These cases illustrate the varied causes of goitre, the value of needle aspiration and cytologic study in thyroid disease and the need for histologic vigilance. Worthwhile palliation by simple means can be obtained in such cases.     

Functional mosaicism of membrane proteins in vesicles of Escherichia coli.

Membrane vesicles of Escherichia coli prepared by osmotic lysis of lysozyme ethylenediaminetetracetate (EDTA) spheroplasts have approximately 60% of the total membrane-bound reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase (ED 1.6.99.3) and Mg2+-adenosine triphosphatase (ATPase) (EC 3.6.1.3) activities exposed on the outer surface of the inner membrane. Absorption of these vesicles with antiserum prepared against the purified soluble Mg2+-ATPase resulted in agglutination of approximately 95% of the inner membrane vesicles, as determined by dehydrogenase activity, and about 50% of the total membrane protein. The unagglutinated vesicles lacked all dehydrogenase activity and may consist of outer membrane. Lysozyme-EDTA vesicles actively transported calcium ion, using either NADH or adenosine 5'-triphosphate (ATP) as energy source. However, neither D-lactate nor reduced phenazine methosulfate energized calcium uptake, suggesting that the observed calcium uptake was not due to a small population of everted vesicles. Transport of calcium driven by either NADH or ATP was inhibited by simultaneous addition of D-lactate or reduced phenazine methosulfate. Proline transport driven by D-lactate oxidation was inhibited by either NADH oxidation or ATP hydrolysis. These results suggest that the portion of the total population of vesicles capable of active transport, i.e., the inner membrane vesicles, are functionally a homogeneous population but cannot be categorized as either right-side-out or everted, since activities normally associated with only one side of the inner membrane can be found on both sides of the membrane of these vesicles. Moreover, the data indicate that oxidation of NADH or hydrolysis of ATP by externally localized NADH dehydrogenase or Mg2+-ATPase establishes a protonmotive force of the opposite polarity from that established through D-lactate oxidation.     

Effects of neuraminidase on lectin binding by wild-type and ricin-resistant strains of hamster fibroblasts.

The nature of cell surface receptors for ricin on wild-type and ricin-resistant variants of baby hamster kidney fibroblasts has been studied. Neuraminidase stimulated ricin binding threefold by wild-type cells, and increased their susceptibility to ricin toxicity as measured by inhibition of [3H]leucine uptake (LD30 fell from 5.0 to 0.5 microgram/mL). Basal ricin binding by ricin-resistant variants (10-300% that of wild type) was also stimulated (2- to 17-fold) by neuraminidase in all seven clonal strains examined; susceptibility to ricin was greatly increased by neuraminidase in these variants. Neuraminidase did not affect the binding of concanavalin A by wild type or a ricin-resistant variant, but decreased the binding of wheat-germ agglutinin by 90% in both cell types. The trivial binding of peanut agglutinin by wild type and a ricin-resistant variant was markedly enhanced (14- to 22-fold) by neuraminidase. Neither collagenase (50 U/mL) nor Pronase (0.0001%) affected ricin binding by wild type or a ricin-resistant variant. These data suggest the existence of "exposed" and "cryptic" oligosaccharide receptors for ricin on the cell membrane glycoproteins of baby hamster kidney fibroblasts. The cryptic ricin receptors probably include at least the sequence D-galactosyl-beta-(1 replaced by 3)-N-acetylhexosamine substituted by sialic acid residues. Exposed and cryptic ricin receptors appear to be different and under separate genetic control.     

Translational control by messenger RNA competition for eukaryotic initiation factor 2

Translation of globin mRNA in a micrococcal nuclease-treated reticulocyte lysate was studied in the presence of increasing amounts of Mengovirus RNA, under conditions in which the number of translation initiation events remains constant as judged by the transfer of label from N-formyl[35S]methionyl-tRNAf into protein. The translation of globin mRNA is progressively inhibited by low concentrations of Mengovirus RNA, free of detectable traces of double-stranded RNA, concomitant with the increasing synthesis of Mengovirus RNA-directed products. On a molar basis, Mengovirus RNA apparently competes about 35 times more effectively than globin mRNA for a critical component in translation. The competition is relieved by the addition of highly purified eukaryotic initiation factor 2 (eIF-2). Addition of eIF-2 does not stimulate overall protein synthesis, but shifts it in favor of globin synthesis. No stimulation of globin mRNA translation by eIF-2 is seen when Mengovirus RNA is absent. These experiments show that Mengovirus RNA competes, directly or indirectly, with globin mRNA for eIF-2. In direct binding experiments using isolated mRNA and eIF-2, Mengovirus RNA is shown to compete with globin mRNA for eIF-2 and to exhibit a 30-fold higher affinity for this factor. The binding of Mengovirus RNA to eIF-2 is much more resistant to increasing salt concentrations than is the binding of globin mRNA, again reflecting its high affinity. These results reveal a direct correlation between the ability of these mRNA species to compete in translation and their ability to bind to initiation factor eIF-2. They suggest that the affinity of a given mRNA species for eIF-2 is essential in determining its translation, relative to that of other mRNA species. Messenger RNA competition for eIF-2 may contribute significantly to the selective translation of viral RNA in infected cells.     

Tunicamycin-mediated depletion of insulin receptors in 3T3-L1 adipocytes.

Tunicamycin, an antibiotic that inhibits protein glycosylation, elicited a rapid depletion of insulin binding activity at the surface of 3T3-L1 adipocytes. Disappearance of insulin receptors occurred more rapidly in the presence of tunicamycin than when protein synthesis was inhibited by cycloheximide and was accompanied by a diminution in sensitivity of the adipocytes to the acute effects of insulin and anti-insulin receptor antibody on hexose uptake and metabolism.     

Inhibition of adenosine 3':5'-monophosphate-dependent protein kinase: comparison of a protein inhibitor with polyanions and substrate analogs.

The mechanism of inhibition of adenosine 3':5'-monophosphate (cyclic AMP)-dependent protein kinase was studied using a protein inhibitor isolated by a non-denaturing procedure from bovine heart. This protein inhibitor interacts with the catalytic subunit of protein kinase and binds to some substrates of the kinase. Protein kinase activity can also be inhibited by polyanions which, like the protein inhibitor, bind to basic substrates but do not bind to the catalytic subunit of protein kinase. Peptides such as L-lysyl-L-tyrosyl-L-threonine that resemble the phosphate accepting site of protein kinase substrates competitively inhibit phosphorylation of histone. Protein kinase activity can thus be inhibited in vitro by interaction of the protein inhibitor with substrates, and/or the catalytic subunit of the kinase, by competition of substrate analogs with "natural" substrates and by direct interaction of polyanions with basic protein substrates for the phosphotransferase reaction.     

Orientation of the protonmotive force in membrane vesicles of escherichia coli

Membrane vesicles of Escherichia coli can be produced by 2 different methods: lysis of intact cells by passage through a French pressure cell or by osmotic rupturing of spheroplasts. The membrane of vesicles produced by the former method is everted relative to the orientation of the inner membrane in vivo. Using NADH, D-lactate, reduced phenazine methosulfate, or ATP these vesicles produce protonmotive forces, acid and positive inside, as determined using flow dialysis to measured the distribution of the weak base methylamine and the lipophilic anion thiocyanate. The vesicles accumulate calcium using the same energy sources, most likely by a calcium/proton antiport. Calcium accumulation, therefore, is presumably indicative of a proton gradient, acid inside. The latter type of vesicle, on the other hand, exhibits D-lactate-dependent proline transport but does not accumulate calcium with D-lactate as an energy source. NADH oxidation or ATP hydrolysis, however, will drive the transport of calcium but not proline in these vesicles. Oxidation of NADH or hydrolysis of ATP simultaneous with oxidation of D-lactate does not result in either calcium or proline transport. These results suggest that the vesicles are a patchwork or mosiac, in which certain enzyme complexes have an orientation opposite to that found in vivo, resulting in the formation of electrochemical proton gradients with an orientation opposite to that found in the intact cell. Other complexes retain their original orientation, making it possible to set up simultaneous proton fluxes in both directions, causing an apparent uncoupling of energy-linked processes. That the vesicles are capable of generating protonmotive forces of the opposite polarity was demonstrated by measurements of the distribution of acetate and methylamine (to measure the ΔpH) and thiocyanate (to measure the Δψ).     

Detection of Mouse Mammary Tumor Virus RNA in BALB/c Tumor Cell Lines of Nonviral Etiologies

A complementary DNA (cDNA) probe to mouse mammary tumor virus (MMTV) RNA was synthesized using calf thymus DNA oligonucleotides as a random primer. This probe was then used to study the expression of MMTV RNA in cell lines from BALB/c tumors induced in vivo either spontaneously or in response to viral, chemical, or hormonal stimuli. The cDNA had a length of approximately 400 to 500 nucleotides and specifically hybridized to MMTV RNA and BALB/c lactating mammary gland RNA, but not to Moloney leukemia virus RNA. Calf thymus DNA-primed cDNA could protect 50% of iodinated MMTV RNA from S1 nuclease digestion at cDNA-RNA ratios of 1:1 and 90% of labeled viral RNA at ratios of 10:1. Thermal denaturation of MMTV RNA-cDNA hybrids yielded a T(m) of 88.5 degrees C, indicative of a well-base-paired duplex. Screening of mouse mammary tumor cells for MMTV sequences revealed that three out of five lines of BALB/c origin had undetectable levels of viral RNA (相似文献   

Plasma norepinephrine concentrations: No differences among normal volunteers and low,high or normal renin hypertensive patients

Plasma norepinephrine concentrations were measured by a sensitive radioenzymatic method in 51 patients with essential hypertension and 26 age-matched normal volunteers under conditions of ad libitum sodium intake, after volume expansion by infusion of saline intravenously, and after volume contraction by administration of furosemide orally. The hypertensive patients were classified into low, normal and high renin groups both by renin-sodium indexing and by their renin response to furosemide and saline administration. Plasma norepinephrine concentrations were similar among normal volunteers and patients with low, normal or high renin hypertension while the people were either recumbent or after they stood for 5 min. These and other results do not support the hypothesis that abnormal activity of the sympathetic nervous system accounts for the low or high renin values seen in many hypertensive patients.