bluuebunny/crossref_metadata_2025 huggingface dataset:
Direct demonstration of immunoglobulin κ chain RNA in thymus T cells by
in situ
hybridization
Authors: Storb Ursula
Published: May 2006

Mouse thymuses with more than 99% T cells have been reported to contain immunoglobulin κ mRNA-like molecules (κ RNA) in relatively large quantities. The present study was undertaken to rule out the possibility that the κ RNA was mainly a product of a few contaminating B cells of the thymus and to determine whether all T-cell subpopulations contained κ RNA. By
in situ
hybridization with DNA complementary to κ mRNA (κ cDNA) the following observations were made: 98.5% of thymus cell preparations hybridized with κ cDNA; the 1.5% unlabeled cells were generally larger and paler staining than the majority of thymus cells. Only 0.015% of thymus cells were intensely labeled and appeared to be plasma cells. Also, 87% of spleen cells hybridized with κ cDNA; most of these showed similar labeling intensity to the majority of thymus cells. The number of unlabeled cells corresponded to the percentage of hemopoietic cells and macrophages in the spleen. Spleen cells in the range of 0.37-0.85% were intensely labeled and appeared to be plasma cells. The following controls supported the conclusion that the results with thymus and spleen were due to specific hybridization: most of the κ mRNA-deficient tissue culture cells of the plasmocytoid tumor ABPL-4 did not hybridize with κ cDNA. The κ mRNA-producing cells from myeloma PC 3741 hybridized
in situ
with κ cDNA. Furthermore, all cells from this tumor and all spleen cells hybridized uniformly with a cDNA probe complementary to most of the total cellular poly(A)-containing RNA species of these cells. These results indicate that T cells of all types in the thymus as well as in the periphery contain substantial quantities of κ RNA.
Large T1 oligonucleotides of Moloney leukemia virus missing in an env gene recombinant, HIX, are present on an intracellular 21S Moloney viral RNA species.
Authors: Faller D V , Rommelaere J , Hopkins N
Published: May 2006

HIX, a recombinant derived from Moloney leukemia virus, has an envelope glycoprotein different from that of the Moloney virus. HIX and Moloney viruses share the majority of the large T1 oligonucleotides derived from their genomes but each possesses a set of distinctive oligonucleotides that lie clustered in corresponding regions in the 3' halves of their oligonucleotide maps. These regions presumably contain envelope glycoprotein coding sequences. The type C viral envelope glycoprotein is believed to be translated from a 21S RNA. Thus, at least part of the region of the Moloney virus genome that is altered relative to HIX was expected to be present on such a species. To test this prediction, we purified an intracellular 21S Moloney viral RNA species and analyzed its large T1 oligonucleotides by two-dimensional polyacrylamide gel electrophoresis. This RNA contains one T1 oligonucleotide that is probably derived from the 5' end of the Moloney virus genome, the Moloney virus T1 oligonucleotides that are missing in HIX, and those that lie to their 3' side on the Moloney virus T1 oligonucleotide map.
Ontogenetic appearance and disappearance of tyrosine hydroxylase and catecholamines in the rat embryo.
Authors: Cochard P , Goldstein M , Black I B
Published: May 2006

The ontogenetic pattern of noradrenergic differentiation in rat embryonic autonomic neuroblasts was defined in vivo. Noradrenergic specialization was examined by documenting the immunohistochemical appearance of tyrosine hydroxylase [Tyr-OH; tyrosine 3-monooxygenase; L-tyrosine,-tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2] and the development of histofluorescence due to catecholamine (CA). Tyr-OH and CA were undetectable in the dorsal neural crest or the ventrally migrating crest cells and first appeared at 12.5 days of gestation (36--37 somite stage) in sympathoblasts that had formed sympathetic ganglion primordia. Fluorescence intensity and the number of fluorescent cells increased progressively thereafter. In addition, Tyr-OH and CA transiently appeared in scattered presumptive neuroblasts in the gut. The enzyme and transmitter were first detectable at 11.5 days of gestation and thereafter decreased progressively so that, by 14.5 days, only rare cells were encountered. There was remarkable synchrony in the appearance and disappearance of Tyr-OH and CA. These observations suggest that a number of noradrenergic transmitter mechanisms develop simultaneously in the differentiating neuroblast. The relevance of these results to the elucidation of developmental regulatory mechanisms is discussed.
Oxygen-mediated heterogeneity of apo-low-density lipoprotein.
Authors: Schuh J , Fairclough G F , Haschemeyer R H
Published: May 2006

Mild oxidation of human serum low-density lipoprotein (LDL) converts the apoprotein from a nearly homogeneous component of high apparent molecular weight to a mixture of apparently lower molecular weight polypeptide components, as characterized by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. This protein alteration, which correlates temporally with increases in the formation of lipid oxidation products and in the fluorescence of the apoprotein, is markedly reduced when oxygen is excluded or when EDTA or the free-radical-scavenging antioxidants, butylated hydroxytoluene or propyl gallate, are added. The conversion thus appears to be due to a reaction between the protein moiety and auto-oxidizing lipid. The presence of the antibacterial agent sodium azide markedly accelerates the oxidation, suggesting that it should only be used with caution in lipid-containing solutions.
Interaction of methotrexate, folates, and pyridine nucleotides with dihydrofolate reductase: calorimetric and spectroscopic binding studies.
Authors: Subramanian S , Kaufman B T
Published: May 2006

The thermodynamic parameters, deltaG, deltaH, and deltaS characterizing the tight binding of methotrexate, folates, and pyridine nucleotides to chicken liver dihydrofolate reductase (5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) have been determined from calorimetric and fluorescence measurements. At 25 degrees the binding of NADPH and NADP+ is characterized by small negative enthalpies and large positive entropies whereas the binding of the folates and methotrexate is accompanied by large negative enthalpies and small negative entropies. In addition, the enthalpy of methotrexate-enzyme interaction demonstrates a proton transfer associated with binding; this is not the case with folate and dihydrofolate, thus confirming the conclusions drawn from the observed difference spectra characteristic of the interaction of methotrexate and substrates with the enzyme. The implications of these results are discussed in terms of the nature of the binding process, conformational changes in the enzyme, and the nature of the active site region.
Identification, localization, and role of fibronectin in cultured bovine endothelial cells.
Authors: Birdwell C R , Gospodarowicz D , Nicolson G L
Published: May 2006

We have examined bovine aortic endothelial cell cultures for the presence of fibronectin, a high molecular weight cell-surface glycoprotein. Sparse cultures contain fibronectin only on dorsal cell surfaces at regions of cell-cell contact, as detected by immunofluorescence. In contrast, when the endothelial cells reached confluence as a highly contact-inhibited monolayer, fibronectin was detected in an extracellular matrix underneath the cell monolayer but not on top of the monolayer. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of isolated extracellular matrix revealed that a predominant component of the matrix is a protein of approximately 2.3 X 10(5) molecular weight, which has been identified as fibronectin.
Separation of younger red cells with improved survival
in vivo
: An approach to chronic transfusion therapy
Authors: Piomelli Sergio , Seaman Carol , Reibman Joan , Tytun Alex , Graziano Joseph , Tabachnik Nina , Corash Laurence
Published: May 2006

Transfusion of donor blood containing predominantly younger red cells with prolonged survival
in vivo
could significantly reduce the iron overload of patients requiring chronic transfusion. Age-dependent separation of red cells can be obtained by buoyant density centrifugation on isotonic solutions of arabino-galactane. By this technique, rabbit red cells were separated on a single layer of arabino-galactane and the appropriate fraction, after being labeled with
51
Cr, was reinfused into the donor. The survival
in vivo
was calculated by a mathematical model which corrects for both
51
Cr elution and random loss. There was a significant difference in survival
in vivo
between the light young red cells and the heavy old red cells. The potential survival
in vivo
of the 50% lightest red cells was 56 days, compared to 28 days for the heaviest red cells. Arabino-galactane appeared to be devoid of acute toxicity and of strong antigenicity and it did not appear to adhere to the red cell stroma. These data extrapolated to humans indicate that it may be feasible and advantageous to use red cells fractionated by this technique for transfusion. The 50% lightest human red cells can be expected to have a mean survival of 88 days, compared with 60 days for unfractionated blood. Transfusion of young red cells could significantly reduce the iron overload for patients requiring chronic transfusion, by avoiding infusion of the oldest red cells, which contribute equally to iron overload yet offer only transient survival
in vivo.
X-ray sensitivity of diploid fibroblasts from patients with hereditary or sporadic retinoblastoma.
Authors: Weichselbaum R R , Nove J , Little J B
Published: May 2006

Fibroblasts derived from patients with hereditary retinoblastoma appear to be more sensitive to the lethal effects of x-rays than do fibroblasts from patients with sporadic retinoblastoma or normal controls. A defect in DNA repair is postulated to account for the high incidence of second tumors in these patients. Retinoblastoma appears to be an interesting model for the study of genetic susceptibility to the development of spontaneous or radiation-induced tumors.
Generalizing Fisher's "reproductive value": overlapping and nonoverlapping generations with competing genotypes.
Authors: Samuelson P A
Published: May 2006

How to go beyond Fisher's 1930 linear eigenvector definition of reproductive value has been established for dilute systems whose dynamic relations are first-degree-homogeneous functions so that intensive ratios are scale-free. Here such an extension is applied to standard mendelian models. It is shown that, aside from singular cases like that of the Hardy-Weinberg razor's-edge labile equilibrium, such general systems are irreducibly nonlinear and admit of reproductive value functions that are calculable only in an infinite number of steps.
Phospholipid synthesis: Oxazaphospholanes and dioxaphospholanes as intermediates
Authors: Eibl Hansjörg
Published: May 2006

The synthesis of phospholipids described herein allows an almost quantitative conversion of 1,2-diacylglycerols to phospholipids via cyclic intermediates such as oxazaphospholanes or dioxaphospholanes, which are the only products observed when ethanolamine or ethylene glycol is allowed to react with phosphatidic acid dichlorides. The acidic hydrolysis of the cyclic intermediates occurs rapidly without the formation of by-products and results in phospholipids differing in the structure of the polar part depending on which vicinal aminoalcohols or diols are used. For instance, phosphatidylethanolamines, -(
N
-methyl)ethanolamines, and -serine benzyl esters are directly formed from the corresponding oxazaphospholanes. Phosphatidyl(
N,N
-dimethyl)ethanolamines and -cholines are prepared by amination of the bromoethyl esters of phosphatidic acids, which can be obtained by treatment of dioxaphospholanes with sodium bromide. A similar sequence of reactions starting from 1-acyl-2-benzylglycerols or monoacylpropane-1,3-diols will result in the respective monoacyl analogues such as lysophospholipids or the so-called desoxylysophospholipids. The mild reaction conditions, good yields (>90% based on diacylglycerol), and high purity of the products make this route an attractive procedure for phospholipid synthesis; in addition, it avoids the need for chromatography and thus can be utilized on a large scale.
RNA metabolism, manganese, and RNA polymerases of zinc-sufficient and zinc-deficient Euglena gracilis.
Authors: Falchuk K H , Hardy C , Ulpino L , Vallee B L
Published: May 2006

The three major RNA classes from zinc-sufficient [(+Zn)] and zinc-deficient [(=Zn)] Euglena gracilis have been separated by affinity chromatography on oligo(dT)- and N-[N'-[m-(dihydroxyboryl)phenyl]succinamoyl]aminoethyl (DBAE)-celluloses. The total RNA content and the ribosomal and transfer RNA fractions are the same in (+Zn) and (=Zn) cells. IN (-Zn) cells, the messenger RNA fraction increases, and its altered base composition reveals additional bases and a 2-fold increase in the (G+C)/(A+U) ratio. Since the intracellular content of manganese increases in (-Zn) cells, we have examined its role in determining these changes in RNA composition. An increase in the Mn2+ content from 1 to 10 mM in assays with RNA polymerases I and II from (+Zn) cells and those with the single RNA polymerase from (-Zn) cells decreases the ratio of UMP to CMP incorporated from 1.7 to 1.0, 2.1 to 0.8 and 3.5 to 0.4, respectively. Thus, Mn2+ concentration can significantly alter the products of the enzymatic action of RNA polymerases from both (+Zn) and (-Zn) E. gracilis cells.
Inhibition of mammalian protein kinase and phosphodiesterase activities by a cyclic AMP-like compound isolated from higher plants.
Authors: Wood H N , Pomerantz A H , Binns A N , Allfrey V G , Braun A C
Published: May 2006

A cyclic AMP-like substance has been isolated from higher plant tissues which can be quantitated with the use of a radioimmunoassay similar to that described by A. L. Steiner, D. M. Kipnis, R. Utiger, and C. Parker [(1969) Proc. Natl. Acad. Sci. USA 64, 367-373]. This compound has been extensively purified and is chromatographically distinct from authentic cyclic AMP. This cyclic AMP-like compound inhibited beef heart 3':5'-cyclic-nucleotide phosphodietsterase (3':5'-cyclic-nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17), with half-maximal inhibition occurring at a concentration of 7.6 X 10(-10) M cyclic AMP equivalents. The compound also inhibited cyclic AMP-dependent protein kinase (ATP:protein phosphotransferase; EC 2.7.1.37) from bovine heart, with half-maximal inhibition of mixed histone phosphorylation occurring at 8.0 X 10(-11) M cyclic AMP equivalents. Equipotent inhibition of phosphorylation and associated trace ATPase activity were observed with the purified catalytic subunit of cyclic AMP-dependent protein kinase from calf thymus with a synthetic heptapeptide as substrate. Moreover, steady-state kinetic analysis of this inhibition in the latter system showed it to be nonlinear and noncompetitive versus MgATP.
Temperature dependence of calcium-induced fusion of sonicated phosphatidylserine vesicles.
Authors: Sun S T , Day E P , Ho J T
Published: May 2006

We have measured the temperature dependence calcium-induced fusion of sonicated phosphatidylserine vesicles. The vesicles were incubated in the presence of calcium at a specified temperature until the resulting aggregation or fusion process had gone to completion. EDTA was then added and the resulting final size of the vesicle population was measured by using dynamic light scattering. This final size was plotted against incubation temperature to show the temperature dependence of calcium-induced fusion. This curve has a peak near 11 degrees C which may be associated with the phase transition of the sonicated phosphatidylserine vesicles in the presence of calcium prior to the aggregation or fusion process.
Origin of the genetic code: a testable hypothesis based on tRNA structure, sequence, and kinetic proofreading.
Authors: Hopfield J J
Published: May 2006

We hypothesize that the origin of the genetic code is associated with the structure of the tRNA that existed in primal cells. The sequences of modern tRNA contain correlations which can be understood as "fossil" evidence of the secondary structure of primal tRNA. Kinetic proofreading through diffusion can amplify a low level of intrinsic selectivity of tRNA for its amino acid. Experimental tests of the theory are suggested.
Coated vesicles: characterization, selective dissociation, and reassembly.
Authors: Woodward M P , Roth T F
Published: May 2006

Sodium dodecyl sulfate/polyacrylamide gels of coated vesicles from porcine brain (mean 76% coated vesicles) show three major proteins (180,000, 125,000, and 55,000 daltons) that account for 73% of the total protein. Preparations consisting predominantly of coats (65%) have less of the 55,000-dalton protein. Clathrin (180,000 daltons) comprises 40% of the protein of a coated vesicle. Conditions of 2 M urea, 0.25 M MgCl2, or pH 7.5 disrupt the coat and solubilize clathrin. Solubilized clathrin reforms coat structures after dilution of urea or MgCl2. High-pH-solubilized clathrin reassembles after dialysis against buffer at pH 6.5 containing dithiothreitol (5 mM). Reassembled coats are predominantly clathrin.
Genetic regulation of tissue-specific expression of amylase structural genes in Drosophila melanogaster.
Authors: Abraham I , Doane W W
Published: May 2006

Laboratory strains of Drosophila melanogaster were screened for spatial variations in adult midgut alpha-amylase (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1) expression. No strain-specific differences were found anteriorly, but three patterns of activity were discerned in the posterior midgut: A, activity throughout most of the region; B, activity in the anterior part of the region; and C, little or no activity. Alleles of a control gene, map, are responsible for this tissue-specific regulation of activity; e.g., mapA homozygotes produce the A pattern and mapC homozygotes the C pattern. The map locus was placed at 2--80 +/- on the genetic map of chromosome 2R, about two crossover units distal to the Amy structural gene region for alpha-amylase. Electrophoretic studies showed that mapA is trans acting in mapA/mapC flies, allowing expression of amylase isozymes coded for by genes on the opposite chromosome. The map gene behaves as a temporal gene that is clearly separable from the tightly linked, duplicated Amy structural genes.
Amplification of DNA sequences during chicken cartilage and neural retina differentiation.
Authors: Strom C M , Moscona M , Dorfman A
Published: May 2006

[3H]BrdUrd-substituted DNA probes were prepared from organ cultures of differentiating chicken neural retina and cell cultures of stage 24 chicken limb buds. Reassociation reactions using the neural retina probe demonstrated amplification of DNA sequences during differentiation of neural retina. This probe also contained sequences present in greater numbers in heart DNA than in DNA from undifferentiated neural retina. Reassociation reactions of both differentiated cartilage and differentiated neural retina DNA with both the neural retina probe and the cartilage probe demonstrated that at least part of the amplified sequences are tissue specific.
Relative sensitivities of forward and reverse mutation assays in Salmonella typhimurium.
Authors: Skopek T R , Liber H L , Kaden D A , Thilly W G
Published: May 2006

Forward mutation to 8-azaguanine resistance and reverse mutation to histidine prototrophy were measured in Salmonella typhimurium after treatment with 16 mutagens of both base-substitution and frameshift classes. The two approaches were found to be equisensitive for all 16 mutagens--i.e., induction of significant mutation occurred at similar concentrations in the forward mutation assay and in the most sensitive of the five Ames tester strains.
Assignment of genes for immunoglobulin kappa and heavy chains to chromosomes 6 and 12 in mouse.
Authors: Hengartner H , Meo T , Müller E
Published: May 2006

Using somatic cell hybrids from fusions of lymphocytes of two different mouse stocks with the myeloma cell line X63-Ag8, we have assigned genes for the immunoglobulin heavy and kappa-type light chains to chromosomes 12 and 6, respectively. The two mouse stocks exhibit karyotypes consisting of nine pairs of metacentric chromosomes as a result of centric fusions of acrocentric chromosomes in different combinations. In the hybrid cells these metacentric chromosomes can be distinguished from the acrocentric chromosomes of myeloma origin, permitting correlation of Ig chain expression with mitotic loss of individual metacentric chromosomes.
Caulobacter crescentus nucleoid: analysis of sedimentation behavior and protein composition during the cell cycle.
Authors: Evinger M , Agabian N
Published: May 2006

The envelope-associated nucleoid (EAN) of Caulobacter crescentus has been isolated during major developmental stages in the cell cycle. The sedimentation coefficient of the Caulobacter EAN changes as a function of development and is closely correlated with the periodicity of DNA synthesis in this bacterium. The contribution of proteins to the structure of the Caulobacter nucleoid has been analyzed by using polyacrylamide gel electrophoresis of proteins labeled both for short intervals and continuously throughout the cell cycle. The EAN proteins are derived primarily from membranes and are sequentially associated with the EAN during the cell cycle. Several distinct proteins are isolated with the EAN at specific stages of differentiation. A 26,000 Mr protein, which appears uniquely associated with the swarmer cell EANs, was identified and shown to segregate preferentially with the swarmer cell EAN at cell division.
Isolation of a 7S particle from Xenopus laevis oocytes: a 5S RNA-protein complex.
Authors: Picard B , Wegnez M
Published: May 2006

Previtellogenic oocytes of Xenopus laevis contain a free 5S RNA-protein complex sedimentating at 7 S. This particle consists of one molecule of 5S RNA and one 45,000-dalton protein. The protein of the 7S particle and the protein that is released in association with 5S RNA when the ribosome is treated with EDTA are unrelated. Because the 5S RNA accumulated by small oocytes in storage particles is incorporated into the ribosome later in oogenesis, we conclude that 5S RNA is succesively associated with two proteins during the life span of the oocyte.
Evidence for tetradecanal as the natural aldehyde in bacterial bioluminescence.
Authors: Ulitzur S , Hastings J W
Published: May 2006

Dim aldehyde mutants of the luminous bacterium Beneckea harveyi emit light with exogenously added long-chain aliphatic aldehyde. In one class of these mutants, luminescence is also stimulated by myristic (tetradecanoic) acid. In such mutants the amount of light obtained by the addition of a small (limiting) amount of either tetradecanal or myristic acid may be increased 60-fold by cyanide and other agents that block respiration. This indicates that the fatty acid product of the luminescent reaction is recycled. The effect, like the stimulation by exogenous fatty acid, exhibits specificity for the 14-carbon compound, suggesting that tetradecanal is the natural aldehyde. In those aldehyde mutants that are not stimulated to emit light by fatty acids, and thus presumably lack the recycling system, the chain-length-specific stimulation by cyanide does not occur.
Translocation of intracellular glutathione to membrane-bound γ-glutamyl transpeptidase as a discrete step in the γ-glutamyl cycle: Glutathionuria after inhibition of transpeptidase
Authors: Griffith Owen W. , Meister Alton
Published: May 2006

Several inhibitors of γ-glutamyl transpeptidase
in vitro
[L-serine plus borate, 6-diazo-5-oxo-L-norleucine, and L- and D-γ-glutamyl-(
o
-carboxy)phenylhydrazide] are active
in vivo
, as indicated by their effect in decreasing the conversion of administered D-γ-glutamyl-L-α-amino[
14
C]butyrate to respiratory
14
CO
2
in mice. The hydrazides (both L and D isomers) are the most potent inhibitors
in vitro
and
in vivo
. Inhibition of γ-glutamyl transpeptidase
in vivo
by the hydrazides is accompanied by extensive glutahionuria. The evidence suggests that a substantial fraction of the urinary glutathione arises from the kidney. The findings support the view that renal intracellular glutathione is normally translocated to the membrane-bound γ-glutamyl transpeptidase as a separate step in the γ-glutamyl cycle. Studies on
in vivo
inhibition of glutathione synthesis and of γ-glutamyl transpeptidase provide direct evidence that glutathione is normally translocated from tissues to the blood plasma and that the turnover of plasma glutathione is relatively high. The data suggest that the low but significant steady-state level of glutathione in the plasma reflects synthesis of glutathione (predominantly in the liver) and its utilization by γ-glutamyl transpeptidase (predominantly in the kidney). Thus, glutathione synthesized in cells that have transpeptidase may be translocated to and used by the membrane-bound enzyme, whereas glutathione synthesized in cells that lack the transpeptidase may be transported via the plasma to transpeptidase located on the membranes of other cells.
Single-step selection of clones of a mouse hepatoma line deficient in aryl hydrocarbon hydroxylase.
Authors: Hankinson O
Published: May 2006

The mouse hepatoma line Hepa-1 has high and inducible aryl hydrocarbon (benzo[a]pyrene) hydroxylase[benzo[a]pyrene, reduced-flavoprotein:oxygen oxidoreductase (3-hydroxylating), EC 1.14.14.2] activity. Fourteen subclones of a clonal derivative of Hepa-1 were isolated and shown not to display heterogeneity in aryl hydrocarbon hydroxylase activity beyond what could be ascribed to experimental error. Hepa-1 was found to be very sensitive to benzo[a]pyrene (BaP) toxicity and a single-step selection procedure for isolating clones resistant to BaP at 4 microgram/ml was designed. Those BaP-resistant variants tested had much reduced aryl hydrocarbon hydroxylase activities under both inducing and noninducing conditions and they retained their resistance to BaP and low aryl hydrocarbon hydroxylase activities over considerable periods of time of culture in the absence of BaP. The spontaneous rate of origin of the BaP-resistant clones was estimated, by Luria-Delbrück fluctuation analysis, to be 2 X 10(-7) events per cell per generation. It is concluded that the properties of the variants are consistent with the notion that they are mutational in origin and that this system is a promising one for carrying out a somatic cell genetic analysis of aryl hydrocarbon hydroxylase.
Enkephalin inhibits release of substance P from sensory neurons in culture and decreases action potential duration.
Authors: Mudge A W , Leeman S E , Fischbach G D
Published: May 2006

Sensory neurons grown in dispersed cell culture in the absence of non-neuronal cell types contain immunoreactive substance P that is chemically similar to synthetic substance P. When depolarized in high-K+ media (30-120 mM), the neurons release this peptide by a Ca2+-dependent mechanism. An enkephalin analogue, [D-Ala2]enkephalin amide, at 10 micron inhibits the K+-evoked release of substance P. At the same or lower concentrations, [D-Ala2]enkephalin amide and enkephalin decrease the duration of the Ca2+ action potential evoked and recorded in dorsal root ganglion cell bodies without affecting the resting membrane potential or resting membrane conductance. This modulation of voltage-sensitive channels may account for the inhibition of substance P release.
Insulin receptor: covalent labeling and identification of subunits.
Authors: Jacobs S , Hazum E , Shechter Y , Cuatrecasas P
Published: May 2006

Two methods were used to label insulin receptors covalently with 125I. In the first, an aryl azide derivative of insulin, 125I-labeled 4-azido-2-nitrophenyl-insulin, was synthesized and used to photolabel the binding region of the insulin receptor in rat liver membranes and human placenta membranes. In the second, insulin receptors were purified from rat liver membranes and labeled with 125I by use of chloramine-T; this method presumably has no specificity for the binding region of the receptor. The proteins labeled by both methods were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis after or without reduction by dithiothreitol. The photoaffinity label specifically labeled a single band in both liver and placenta that had an apparent molecular weight of 135,000 after reduction. A band with similar mobility was present in the chloramine-T-labeled preparation, which also contained a second major band with an apparent molecular weight of 45,000. Without reduction, both methods resulted in a single labeled band with an apparent molecular weight of about 310,000. These results indicate that the insulin receptor of both liver and placenta has a subunit of molecular weight 135,000 that binds insulin and that the receptor may be composed of at least two different subunits that are linked together or greatly stabilized by disulfide bonds.
Active site trapping of nucleotides by crosslinking two sulfhydryls in myosin subfragment 1.
Authors: Wells J A , Yount R G
Published: May 2006

Studies with reagents that crosslink two thiol groups have shown that it is possible to trap nucleotides at the active site of myosin chymotryptic subfragment 1. Subfragment 1 incorporates nearly stoichiometric quantities of [14C]ATP or [14C]ADP in a manner that depends linearly on the extent of inactivation by either N,N'-p-phenylenedimaleimide or Co(II)phenanthroline/[Co(III)(phenanthroline)2CO3]+ complexes. The incorporated radioactive nucleotide is retained after gel filtration, even when the enzyme derivatives are stored in the presence of EDTA or nonradioactive nucleotides (t 1/2 approximately 5 days). The nucleotide incorporated is not covalently bound because HClO4 denaturation allows immediate release of bound nucleotide. The nucleotide retained is ADP because the gamma-phosphate of [gamma-32P]ATP is lost after trapping. Subfragment 1 inactivated as above does not bind the competitive inhibitor adenosine 5'-[beta, gamma-imido]triphosphate, indicating that the active site is blocked. It is proposed that a jawlike nucleotide cleft closes on MgADP or MgATP, which can be locked shut by crosslinking two thiol groups by reaction with N,N'-p-phenylenedimaleimide or cobalt phenanthroline complexes.
Evidence for a tyrosine residue at the active site of phosphoglucomutase and its interaction with vanadate.
Authors: Layne P P , Najjar V A
Published: May 2006

The rate of transfer of [32P]phosphate from [32P]-labeled phosphoglucomutase (alpha-D-glucose-1,6-bisphosphate:alpha-D-glucose-1-phosphate phosphotransferase, EC 2.7.5.1) to glucose increases dramatically between pH 8.5 and 10.5 with a half maximal rate at pH 9.8. This suggests the participation of a residue containing an ionizable group with a pK close to 10. The inhibition of enzyme activity obtained with tyrosine-derivatizing reactions--iodination, nitration, acetylation, and diazo coupling--is strongly indicative of tyrosine participation. Thiol reagents, p-hydroxymercuribenzoate and ethyleneimine, were without effect. Vanadate and arsenate augmented the transfer reaction 200- and 2.5-fold, respectively, and lowered the pH optimum of the reaction.
Regulation of normal differentiation in mouse and human myeloid leukemic cells by phorbol esters and the mechanism of tumor promotion
Authors: Lotem Joseph , Sachs Leo
Published: May 2006

The control of cell multiplication and differentiation by tumor-promoting phorbol esters including 12-
O
-tetradecanoylphorbol-13-acetate (TPA) has been studied with different clones of mouse myeloid leukemic cells, a line of human myeloid leukemic cells, and normal mouse bone marrow myeloblasts. TPA induced normal cell differentiation in one of the mouse leukemic clones and this was mediated by induction of the protein inducer of differentiation to macrophages or granulocytes (MGI) in the cells that then differentiated. Other mouse clones were not induced to differentiate by TPA. In one of these clones, TPA induced cell susceptibility to externally added MGI. This effect was not due to a general induction of susceptibility to all compounds because TPA did not induce susceptibility to lypopolysaccharide or dexamethasone in this clone. In the human leukemic cell line, TPA also induced differentiation with the induction of MGI activity and enhanced susceptibility to added MGI. It is suggested that the clonal differences in induction of MGI activity and increased susceptibility to MGI may be associated with differences in receptors for TPA and the ability of TPA to modify receptors for MGI. Studies with normal bone marrow cells have indicated that TPA stimulated MGI activity and also increased susceptibility of normal myeloblasts to induction of multiplication by MGI. The ability of different phorbol esters to produce these effects on normal myeloblasts and myeloid leukemic cells paralleled their ability to act as tumor promoters. The results indicate that a tumor promoter such as TPA can induce the production of and increase cell susceptibility to a normal regulator of cell multiplication and differentiation. TPA has pleiotropic effects. It is suggested that, by these mechanisms, TPA may thus act as a tumor promoter by increasing cell multiplication in initiated cells, induce differentiation in some cells, or inhibit differentiation in other cells, depending on which molecules are being regulated in the TPA-treated cells.
Contractile protein isozymes in muscle development: identification of an embryonic form of myosin heavy chain.
Authors: Whalen R G , Schwartz K , Bouveret P , Sell S M , Gros F
Published: May 2006

The nature of the myosin heavy chain in embryonic muscle tissue, cultured muscle cells, and several adult muscles was investigated. After denaturation with sodium dodecyl sulfate, purified rat myosins were subjected to partial proteolytic cleavage or immunological analysis using microcomplement fixation. Three types of myosin heavy chains could be demonstrated by both approaches. Whereas adult muscles contain fast- or slow-type myosin heavy chains, embryonic tissue and cultured muscle cells harbor a distinct embryonic form. The existence of this distinct form further characterizes the isozymic transitions of contractile proteins during muscle development.
Transformation by Rous sarcoma virus: effects of src gene expression on the synthesis and phosphorylation of cellular polypeptides.
Authors: Radke K , Martin G S
Published: May 2006

Infection of chicken embryo fibroblasts by Rous sarcoma virus induces a variety of alterations in cellular growth and morphology. We have used two-dimensional polyacrylamide gel electrophoresis to examine the effects of viral transformation on the pattern of synthesis and phosphorylation of cellular polypeptides. Infection by Rous sarcoma virus does not appear to induce the de novo synthesis, or the complete suppression, of any of the [35S]methionine-labeled cellular polypeptides that can be resolved with this technique; however, there are quantitative changes in a minor fraction (approximately 4%) of the [35S]methionine-labeled polypeptides. When cells labeled with [32P]orthophosphate were examined, a phosphorylated polypeptide, Mr 36,000, was detected in transformed cells; this polypeptide appears within 20 min when cells infected by a temperature-sensitive mutant of Rous sarcoma virus are shifted from the nonpermissive to the permissive temperature. Phosphorylation of the 36,000 Mr polypeptide thus represents an early event in the process of transformation, and it is possible that this polypeptide is a target for the kinase activity associated with pp60src.
Mathematical model for studying genetic variation in terms of restriction endonucleases.
Authors: Nei M , Li W H
Published: May 2006

A mathematical model for the evolutionary change of restriction sites in mitochondrial DNA is developed. Formulas based on this model are presented for estimating the number of nucleotide substitutions between two populations or species. To express the degree of polymorphism in a population at the nucleotide level, a measure called "nucleotide diversity" is proposed.
Platelet receptors for human Factor VIII/von Willebrand protein: functional correlation of receptor occupancy and ristocetin-induced platelet aggregation.
Authors: Kao K J , Pizzo S V , McKee P A
Published: May 2006

Previous studies of von Willebrand disease indicate that a deficiency of blood clotting Factor VIII/von Willebrand factor (FVIII/vWF) activity is responsible for the failure of platelets to participate fully in the initial stages of hemostasis. We have recently identified specific FVIII/vWF binding sites on platelets, suggesting that the interaction of these sites with FVIII/vWF may be functionally important in the development of platelet clumps. We have now studied how different ristocetin concentrations, various known platelet aggregation inhibitors, and the exposure of platelets to proteases affect the ability of platelets to bind FVIII/vWF and to form aggregates. Our results demonstrate a highly significant linear correlation between the degree of FVIII/vWF receptor binding and the extent of ristocetin-induced platelet aggregation. Because neither FVIII/vWF binding nor platelet aggregation occurs after platelets are exposed to low concentrations of proteases, the FVIII/vWF receptors must be in the platelet membrane. We conclude that the interaction between FVIII/vWF protein and its receptors on the platelet membrane is an important mechanism by which platelet aggregation occurs during primary phase hemostasis.
Theory of the dependence of population levels on environmental history for semelparous species with short reproductive seasons
Authors: Coleman Bernard D. , Hsieh Ying-Hen
Published: May 2006

A population that is strongly self-regulating through density-dependent effects is expected to be such that, if many generations have elapsed since its establishment, its present size should not be sensitive to its initial size but should instead be determined by the history of the variables that describe the influence of the environment on fecundity, mortality, and dispersal. Here we discuss the dependence of abundance on environmental history for a semelparous population in which reproduction is synchronous. It is assumed that at each instant
t:
(
i
) the rate of loss of members of age
a
by mortality and dispersal is given by a function ρ of
t, a
, and the present number
x = x
(
a,t
) of such members; and (
ii
) the number
x
(0,
t
) of members born in the population is given by a function
F
of
t
and the number of
x
(

a
f
,t

) at a specified age

a
f

of fecundity. It is further assumed that the functions ρ and
F
have the forms ρ(
x,a,t
) = π
1
(
a,t
)
x
+ π
2
(
a,t
)
x
2
and
F
(
x
(

a
f
,t

),
t
) = ν

t
x

(

a
f
,t

). For such a population, a change in the environment is significant only if it results in a change in ν

t

π
1
(
a,t
), or π
2
(
a,t
), and, hence, the history of the environment up to time
t
is described by giving ν
τ
, π
1
(
a
,τ), and π
2
(
a
,τ) for each τ ≤
t
and all
a
in [0,

a
f

]. We show that the dependence of
x
on the history of the environment can be calculated explicitly and has certain properties of “fading memory”; i.e., environmental events that occurred in the remote past have less effect upon the present abundance than comparable events in the recent past.
Initiation of Escherichia coli ribosomal RNA synthesis in vivo.
Authors: Lund E , Dahlberg J E
Published: May 2006

The 5'-terminal sequences of Escherichia coli ribosomal RNA precursors (pre-rRNAs) synthesized in vivo were characterized by RNA oligonucleotide sequence analysis. The 60- to 170-nucleotide-long 5'-end-specific fragments were produced by RNase III treatment of 30S and 18S pre-rRNAs. Comparison of the RNA oligonucleotides of these fragments with known DNA sequences of the promoter regions of several ribosomal RNA operons allows us to determine the start points of transcription of each operon. We conclude that transcription of most (and perhaps all) rRNA operons is initiated in vivo at two tandem promoters, called P1 and P2, which have recently been identified by in vitro transcription studies of several groups. Depending on the transcription unit, the initiating nucleotide at P1 promoters is either ATP or GTP, whereas at P2 promoters it is either CTP or GTP.
Vitamin D metabolism during pregnancy and lactation in the rat.
Authors: Halloran B P , Barthell E N , DeLuca H F
Published: May 2006

The plasma concentration of the major vitamin D metabolites; 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D, and 24,25-dihydroxyvitamin D were measured during pregnancy and lactation in the adult female rat. The concentrations of these metabolites were also measured in rat pups during lactation and after weaning. The plasma concentration of 1,25-dihydroxyvitamin D in the adult female increases from a control value of 26 pg/ml to 86 pg/ml during the latter stages of pregnancy, reaches a peak of 158 pg/ml during lactation, and then returns to control levels by 3 weeks postweaning. Plasma concentrations of 24,25-dihydroxyvitamin D fall dramatically during pregnancy from a control level of 3.9 ng/ml to 1.6 ng/ml remain low during lactation, and return to control levels by 3 weeks postweaning. In the neonatal rat pup at 14 days postpartum, 1,25-dihydroxyvitamin D plasma concentration is 25 pg/ml and 24,25-dihydroxyvitamin D concentration is 2.8 ng/ml. By day 25 postpartum, 1,25-dihydroxyvitamin D concentrations reached levels of 101 pg/ml, whereas 24,25-dihydroxyvitamin D concentrations fell to 1.9 ng/ml.
Calcium-dependent increase in adenosine 3′,5′-monophosphate and induction of the acrosome reaction in guinea pig spermatozoa
Authors: Hyne Ross V. , Garbers David L.
Published: May 2006

Experiments were designed to determine the interrelationship between cyclic AMP and Ca
2+
during the processes of sperm capacitation and the acrosome reaction. In minimal culture media containing pyruvate and lactate as substrates, guinea pig spermatozoa required a minimum of 1.0-1.5 hr to capacitate in the presence of 1.7 mM Ca
2+
and a minimum of 0.5-1.0 hr to capacitate in the absence of added Ca
2+
. Sperm cyclic AMP concentrations were increased by as much as 30-fold within 0.5 min after addition of cells to various media containing Ca
2+
, and the concentrations then remained increased for up to 4 hr. When the cells were added to several Ca
2+
-deficient media, however, cyclic AMP concentrations increased only about 3-fold within 0.5 min and then returned to basal concentrations within 2 min. D-600, a calcium transport antagonist, completely blocked the Ca
2+
-induced increase in sperm cyclic AMP concentrations. In contrast to capacitation, the acrosome reaction failed to occur in the absence of extracellular Ca
2+
. After capacitation of spermatozoa in a Ca
2+
-free medium, addition of Ca
2+
caused an increase in sperm cyclic AMP concentrations within 1 min and a maximal number of spermatozoa showing an acrosome reaction within 10 min. The addition of 1-methyl-3-isobutylxanthine along with Ca
2+
had a synergistic effect on the increase in cyclic AMP. Neither 1-methyl-3-isobutylxanthine nor 8-Br cyclic AMP induced an acrosome reaction in capacitated spermatozoa in the absence of Ca
2+
, but both significantly decreased the time required for maximal expression of the acrosome reaction in the presence of Ca
2+
. These results suggest that the sperm acrosome reaction is associated with both a primary transport of Ca
2+
and a Ca
2+
-dependent increase in sperm cyclic AMP concentrations. Because a cyclic AMP analogue did not induce an acrosome reaction in the absence of added Ca
2+
, the increase in sperm cyclic AMP concentrations induced by Ca
2+
probably reflects one of a number of Ca
2+
-dependent events associated with the acrosome reaction.
Passage of phenotypes of chemically transformed cells via transfection of DNA and chromatin.
Authors: Shih C , Shilo B Z , Goldfarb M P , Dannenberg A , Weinberg R A
Published: May 2006

DNA was prepared from 15 different mouse and rat cell lines transformed by chemical carcinogens in vitro and in vivo. These DNAs were applied to NIH3T3 mouse fibroblast cultures by using the calcium phosphate transfection technique. DNAs of five donor lines were able to induce foci on the recipient monolayers. Ten other donor DNAs yielded few or no foci. DNAs from control, nontransformed parental cell lines induced few or no foci. Chromosomes were transfected from one donor whose naked DNA was unable to induce foci, and morphologic transformation of recipients was observed. These experiments prove that in five of these cell lines the chemically induced phenotype is encoded in DNA, and the sequences specifying the transformed phenotype behave as a dominant allele in the NIH3T3 recipient cells. The sequences encoding the transformation are likely found on a single fragment of DNA.
Antibodies to Epstein-Barr virus-determined antigens in normal subjects and in patients with seropositive rheumatoid arthritis.
Authors: Catalano M A , Carson D A , Slovin S F , Richman D D , Vaughan J H
Published: May 2006

Prior studies have shown that patients with seropositive rheumatoid arthritis (RA) have an increased frequency of precipitating antibody against a nuclear antigen, the RA nuclear antigen, detected in human B lymphoblastoid cell lines infected by Epstein-Barr virus. The present investigations demonstrate that patients with seropositive RA also have specifically elevated titers of antibodies to another, better-characterized Epstein-Barr virus-associated B cell antigen, the Epstein-Barr nuclear antigen, which is detected by anti-complement immunofluorescence. Titers of these two antibodies were not affected by absorption of rheumatoid factor from serum. Furthermore, patients with RA did not have elevated titers of antibodies against the Epstein-Barr virus capsid antigen or to three other species of human herpesviruses: herpes simplex type 1, varicella-zoster virus, and cytomegalovirus. In both normal individuals and RA patients there was a significant association between the presence of antibodies to RA nuclear antigen and the titers of antibody to Epstein-Barr nuclear antigen. Thus, normal subjects with antibody to RA nuclear antigen had titers of antibody to Epstein-Barr nuclear antigen equivalent to those of patients with RA and significantly higher than normal subjects lacking antibody to RA nuclear antigen. One interpretation of these results is that patients with seropositive RA derive from a larger population with enhanced immune responsiveness to B lymphocyte nuclear antigens determined by the Epstein-Barr virus.
Amino acid sequence of an immunoglobulin-like HLA antigen heavy chain domain.
Authors: Trägärdh L , Rask L , Wiman K , Fohlman J , Peterson P A
Published: May 2006

The classical human transplantation antigens, derived from the HLA-A, -B, and -C loci, are cell-surface-expressed glycoproteins. On the exterior of the cell the transplantation antigen heavy chain exposes two disulfide-containing domains and a glycosylated NH2-terminal extension. The disulfide-containing domain closest to the membrane has been isolated and its amino acid sequence has been determined. The HLA antigens used for the sequence analysis were derived from two and possibly three loci and comprised several allelic forms. The primary structure was remarkably invariant, and amino acid variations were observed only at three positions. Whether this suggests that the allelic variation of the HLA antigens is preferentially confined to other regions of the molecule or is a result of fortuitous selection of peptides remains to be established. The sequenced portion of the HLA antigen heavy chain is as homologous to beta 2-microglobulin and immunoglobulin light and heavy chains as are the latter to one another. This observation strengthens the notion that the transplantation antigens and the immunoglobulins are evolutionarily related.