bluuebunny/crossref_metadata_2025 huggingface dataset:
Imaging of hepatic low density lipoprotein receptors by radionuclide scintiscanning in vivo.
Authors: Huettinger M , Corbett J R , Schneider W J , Willerson J T , Brown M S , Goldstein J L
Published: May 2006

The low density lipoprotein (LDL) receptor mediates the cellular uptake of plasma lipoproteins that are derived from very low density lipoproteins (VLDL). Most of the functional LDL receptors in the body are located in the liver. Here, we describe a radionuclide scintiscanning technique that permits the measurement of LDL receptors in the livers of intact rabbits. 123I-labeled VLDL were administered intravenously, and scintigraphic images of the liver and heart were obtained at intervals thereafter. In seven normal rabbits, radioactivity in the liver increased progressively between 1 and 20 min after injection, while radioactivity in the heart (reflecting that in plasma) decreased concomitantly. In Watanabe-heritable hyperlipidemic rabbits, which lack LDL receptors on a genetic basis, there was little uptake of 123I-labeled VLDL into the liver and little decrease in cardiac radioactivity during this interval. These findings demonstrate that the LDL receptor is necessary for the hepatic uptake of VLDL-derived lipoproteins in the rabbit. Two conditions that diminish hepatic LDL receptor activity, cholesterol-feeding and prolonged fasting, also reduced the uptake of 123I-labeled VLDL in the liver as measured by scintiscanning. The data suggest that radionuclide scintiscanning can be used as a noninvasive method to quantify the number of LDL receptors expressed in the liver in vivo.
Isolation of the gene encoding the human T-lymphocyte differentiation antigen Leu-2 (T8) by gene transfer and cDNA subtraction.
Authors: Kavathas P , Sukhatme V P , Herzenberg L A , Parnes J R
Published: May 2006

We report the isolation of genomic and cDNA clones encoding the human T-lymphocyte cell-surface differentiation antigen, Leu-2 (T8), by use of a combination of transfection, fluorescence-activated cell-sorting, and subtractive cDNA hybridization. We constructed a cDNA library with mRNA from a mouse L-cell transfectant in which the human Leu-2 gene is expressed and amplified. We identified Leu-2 cDNA clones by screening with a selected cDNA probe from a second amplified Leu-2 transfectant. This probe contained cDNA species not removed by hybridization with L-cell mRNA. A Leu-2 cDNA clone was used to isolate a genomic clone. Transfection with DNA from this clone resulted in a high number of Leu-2 transfectants. This approach can be used to clone genes coding for other cell-surface molecules.
Dissociation between testicular organogenesis and endocrine cytodifferentiation of Sertoli cells.
Authors: Magre S , Jost A
Published: May 2006

Differentiation of the rat testis from the undifferentiated primordium begins with the appearance of a new cell type characterized by a large and clear cytoplasm. These cells aggregate, enclose germ cells, and progressively form seminiferous cords. Therefore, they were considered primordial Sertoli cells. A similar process was obtained in vitro in explants cultured in a synthetic medium. On the contrary, when fetal calf serum was added to the medium, the organization of seminiferous cords was impaired; large clear cells appeared, but they did not aggregate. Instead, they remained scattered throughout the abnormal gonad. The present experiments were undertaken to verify whether these cells are in fact Sertoli cells. The production of Müllerian inhibitor is a marker of fetal Sertoli cells. Therefore, undifferentiated gonadal primordia from 12-day 16-hr old male rat fetuses were cultured for 2 days in vitro with serum and then associated for 3 days with 14.5-day-old sex ducts from female fetuses. Müllerian ducts were inhibited as well by the abnormal cordless gonads as by those with differentiated sex cords. These experiments confirm previous views on testicular development and demonstrate that differentiation of Sertoli cells may take place quite independently of the testicular cord formation.
Structure linkage, and sequence of the two genes encoding the delta and gamma subunits of the nicotinic acetylcholine receptor.
Authors: Nef P , Mauron A , Stalder R , Alliod C , Ballivet M
Published: May 2006

We have cloned and sequenced a fragment of the chicken genome approximately 9 kilobases in length that comprises the genes encoding the delta and gamma subunits of the nicotinic acetylcholine receptor. The two genes are homologous and have identical structures: both consist of 12 exons, some of which precisely correspond to predicted structural domains of the receptor subunits. The delta and gamma subunit genes are encoded by the same DNA strand and are very closely linked, there being only 740 base pairs between the last codon of delta and the initiator codon of gamma. Blot analysis demonstrates that the genes we describe are unique in the genome. Comparison of the predicted protein sequence for the corresponding subunits of chicken and of the elasmobranch Torpedo reveals a high degree of conservation in some but not all of the protein domains.
Major coat protein and single-stranded DNA-binding protein of filamentous virus Pf3.
Authors: Putterman D G , Casadevall A , Boyle P D , Yang H L , Frangione B , Day L A
Published: May 2006

The region of the Pf3 virus genome encoding its major coat protein and its single-stranded DNA-binding protein is organized somewhat like the corresponding region of the fd (M13, f1) genome. Nevertheless, the major coat protein is unique among the major coat proteins of fd and the other filamentous phages studied in that it lacks a signal sequence and appears to be a direct translation product and in that it has fewer basic amino acid residues than its equivalent of DNA phosphates in the virion. These features are relevant to considerations of both protein insertion into membranes and DNA structure in filamentous viruses. The single-stranded DNA-binding protein also has a sequence that is different from the sequences of single-stranded DNA-binding proteins from other filamentous viruses.
Spider sedation induced by defensive chemicals of milliped prey
Authors: Carrel James E. , Eisner Thomas
Published: May 2006

Wolf spiders (
Lycosa
spp.) show delayed induced sedation (total immobilization) of prolonged duration (in the order of days) after attacks upon millipeds (
Glomeris marginata
). The sedation is specifically attributable to glomerin and homoglomerin, two previously characterized quinazolinones present in the defensive secretion of
Glomeris
. Median sedative doses for the quinazolinones are in the range of 1-7 μg per spider, a fraction of the total (60-90 μg) present in the secretion of medium to full-grown millipeds. A sedative effect upon an invertebrate predator has not previously been demonstrated for an animal defense. Quinazolinones include the synthetic drug methaqualone (Quaalude), a potent human sedative.
Temperature dependence of the resonance Raman spectra of plastocyanin and azurin between cryogenic and ambient conditions.
Authors: Woodruff W H , Norton K A , Swanson B I , Fry H A
Published: May 2006

Resonance Raman spectra of spinach plastocyanin and Pseudomonas aeruginosa azurin were studied as a function of temperature between 10 K and 300 K. The spectra are markedly improved both in signal/noise ratio and in resolution at low temperatures. The assignments of the resonance Raman-active vibrations are reinterpreted in view of the number and intensities of peaks observed in the low-temperature spectra. Features appear in the low-temperature spectra of azurin that may be due to copper-bound methionine. The plastocyanin spectra undergo a transition between 220 K and the melting point of water that results in dramatically narrowed peaks at lower temperature and a shift in the carbon-sulfur stretching frequency of the copper-bound cysteine, suggesting a structural change in the active site and an accompanying effect on vibrational dephasing. Considering that the structures and nonvibrational spectroscopies of these two proteins are similar, the substantial differences in the resonance Raman spectra are striking and significant.
Increased intracellular concentration of an initiator protein markedly reduces the minimal sequence required for initiation of DNA synthesis.
Authors: Dotto G P , Zinder N D
Published: May 2006

One of the most common sites used for cloning in the filamentous phages f1, fd, and M13 lies within the phage "functional origin," a sequence of 140 nucleotides that is required for phage replication. Even small insertions (four nucleotides) at this location severely reduce origin function. Secondary trans-acting mutations in the phage genome are necessary to restore efficient replication. One of these mutations, present in one of our cloning vectors, R218, has been fully characterized. It consists of a regulatory mutation within gene V that leads to a marked increase in the intracellular level of the phage gene II protein, the "initiator" of viral replication. Increased gene II protein production is sufficient to reduce the minimal sequence required for a functional origin to only 40 nucleotides, while the remaining 100 (containing the cloning site) become entirely dispensable. The general implications of these findings are discussed.
Analysis of the pleiotropic regulation of flagellar and chemotaxis gene expression in Caulobacter crescentus by using plasmid complementation.
Authors: Bryan R , Purucker M , Gomes S L , Alexander W , Shapiro L
Published: May 2006

The biosynthesis of the single polar flagellum and the proteins that comprise the chemotaxis methylation machinery are both temporally and spacially regulated during the Caulobacter crescentus cell-division cycle. The genes involved in these processes are widely separated on the chromosome. The region of the chromosome defined by flaE mutations contains at least one flagellin structural gene and appears to regulate flagellin synthesis and flagellar assembly. The protein product of the adjacent flaY gene was found to be required to regulate the expression of several flagellin proteins and the assembly of a functional flagellum. We demonstrate here that each of these genes is also required for the expression of chemotaxis methylation genes known to map elsewhere on the chromosome. In order to study the regulation of these genes, plasmids were constructed that contain either an intact flaYE region or deletions in the region of flaY. These plasmids were mated into a wild-type strain and into strains containing various Tn5 insertion and deletion mutations and a temperature-sensitive mutation in the flaYE region. The presence of a plasmid containing the flaYE region allowed the mutant strains to swim and to exhibit chemotaxis, to synthesize increased amounts of the flagellins, to methylate their "methyl-accepting chemotaxis proteins" (MCPs), and to regain wild-type levels of methyltransferase activity. Chromosomal deletions that extend beyond the cloned region were not complemented by this plasmid. Plasmids containing small deletions in the flaY region failed to restore to any flaY or flaE mutants the ability to swim or to assemble a flagellar filament. When mated into a wild-type strain, plasmids bearing deletions in the flaY region were found to be recessive. The pleiotropic regulation of flagellin synthesis, assembly, and chemotaxis methylation functions exhibited by both the flaY and flaE genes suggest that their gene products function in a regulatory hierarchy that controls both flagellar and chemotaxis gene expression.
beta-endorphin-(1-27) is an antagonist of beta-endorphin analgesia.
Authors: Hammonds R G , Nicolas P , Li C H
Published: May 2006

beta h-Endorphin-(1-27), a naturally occurring fragment of human beta-endorphin (beta h-endorphin), diminishes the analgesic effect of beta h-endorphin when coinjected intra-cerebroventricularly into mice. A parallel shift in the dose-response curve of beta h-endorphin in the presence of beta h-endorphin-(1-27) suggests competition at the same site. The potency of beta h-endorphin-(1-27) in antagonizing analgesia is greater than 4 times greater than that of the opiate antagonist naloxone.
The effects of water-soluble solutes on the phase transitions of phospholipids.
Authors: Sturtevant J M
Published: May 2006

A previous treatment [Sturtevant, J.M. (1982) Proc. Natl. Acad. Sci. USA 79, 3963-3967], based on ideal solution theory, of the influence of small molecules on the main phase transitions of phospholipid bilayers is extended to include the effect of water solubility of the added solute. This effect is manifested primarily in a sharpening of the phase transition, resulting from a buffering by the aqueous phase of the concentration of the solute in the lipid phases.
Structural studies of the primary donor cation radical P
870
+·
in reaction centers of
Rhodospirillum rubrum
by electron-nuclear double resonance in solution
Authors: Lubitz W. , Lendzian F. , Scheer H. , Gottstein J. , Plato M. , Möbius K.
Published: May 2006

The light-induced cation radical of the primary electron donor, P
870
+·
, in photosynthetic reaction centers from
Rhodospirillum rubrum
G-9, has been investigated by electron-nuclear double resonance (ENDOR) in liquid aqueous solution. The measured hyperfine coupling constants are assigned to specific molecular positions by partial deuteration. Comparison with the bacteriochlorophyll a cation radical shows different reduction factors of the individual coupling constants deviating from the value 2.0 reported in earlier investigations in frozen solutions. The average of the coupling constants is, however, reduced by a factor very close to 2.0. EPR simulations using the ENDOR coupling constants support a dimer model for P
870
+·
with C
2
symmetry, where the two macrocycles are close enough to form a supermolecular orbital resulting in a different distribution of the unpaired electron, compared with the monomeric bacteriochlorophyll a cation radical. Molecular orbital calculations were used to obtain structural information about this dimer.
kappa gene diversity among the clonal progeny of pre-B lymphocytes.
Authors: Ziegler S F , Treiman L J , Witte O N
Published: May 2006

Some clonal pre-B cell lines, when transformed by Abelson murine leukemia virus, are able to rearrange and express kappa light chain genes. We have analyzed the light chains expressed in sets of early B-cell subclones derived from two pre-B cell clones. Each subclone makes an indistinguishable mu heavy chain, while the kappa gene rearrangements and proteins synthesized were distinct. All members of one set of subclones expressed a V kappa 21 kappa light chain. Only one of the members of the other two sets of subclones expressed V kappa 21. Thus, a single pre-B-cell clone can select a kappa variable region from more than one family. In each subclone of the set that expressed V kappa 21 light chain the same member appears to be used. The differences detected in the expressed proteins can best be explained by primary sequence alterations in the rearranged V kappa 21 segment. These sequence alterations have resulted in a restriction site polymorphism in the expressed V kappa 21 gene and charge and size differences in the expressed proteins. These data suggest that diversification of kappa light chains can occur at the pre-B- to early B-cell stage of development.
Segregation of on- and off-center afferents in mink visual cortex.
Authors: McConnell S K , LeVay S
Published: May 2006

In the lateral geniculate nucleus of the mink, on-center and off-center neurons occupy separate layers [LeVay, S. & McConnell, S.K. (1982) Nature (London) 300, 350-351]. To study the mode of termination of geniculate afferents in area 17, we recorded from their terminal arborizations in layer IV after the destruction of cortical neurons by injection of kainic acid. At the majority of recording sites, multifiber responses were entirely or predominantly of one type: on-center or off-center. Responses obtained during perpendicular penetrations showed the same preferred sign of contrast throughout the thickness of layer IV. During tangential penetrations through the layer, we encountered sequences of on- and off-center activity separated by stretches of mixed responses. We conclude that on- and off-center afferents terminate in separate, alternating patches that occupy the full thickness of layer IV. These coexist with another set of patches in which the same afferents are segregated by eye of origin.
Purified reconstituted lac carrier protein from Escherichia coli is fully functional.
Authors: Viitanen P , Garcia M L , Kaback H R
Published: May 2006

Proteoliposomes reconstituted with lac carrier protein purified from the plasma membrane of Escherichia coli catalyze each of the translocation reactions typical of the beta-galactoside transport system (i.e., active transport, counterflow, facilitated influx and efflux) with turnover numbers and apparent Km values comparable to those observed in right-side-out membrane vesicles. Furthermore, detailed kinetic studies show that the reconstituted system exhibits properties analogous to those observed in membrane vesicles. Imposition of a membrane potential (delta psi, interior negative) causes a marked decrease in apparent Km (by a factor of 7 to 10) with a smaller increase in Vmax (approximately equal to 3-fold). At submaximal values of delta psi, the reconstituted carrier exhibits biphasic kinetics, with one component manifesting the kinetic parameters of active transport and the other exhibiting the characteristics of facilitated diffusion. Finally, at low lactose concentrations, the initial velocity of influx varies linearly with the square of the proton electro-chemical gradient. The results provide quantitative support for the contention that a single polypeptide species, the product of the lac y gene, is responsible for each of the transport reactions typical of the beta-galactoside transport system.
Chloroplast DNA transcripts are encapsidated by tobacco mosaic virus coat protein
Authors: Rochon D'Ann , Siegel Albert
Published: May 2006

Preparations of tobacco mosaic virus contain pseudovirions, particles resembling virions but containing host rather than viral RNA. The encapsidated host RNA was found to be composed of discrete-sized species derived from a large portion of the chloroplast genome except that very little, if any, ribosomal RNA is present. Pseudovirions contain the same chloroplast DNA transcripts as those detected in extracts from uninfected leaves, although not always in the same relative amounts. Several strains of tobacco mosaic virus were tested and all were found to contain pseudovirions, with the U2 strain containing more than the others.
Evidence for the multicentric origin of the sickle cell hemoglobin gene in Africa.
Authors: Pagnier J , Mears J G , Dunda-Belkhodja O , Schaefer-Rego K E , Beldjord C , Nagel R L , Labie D
Published: May 2006

Previous studies of the Hpa I cleavage site-sickle cell hemoglobin gene linkage in various African populations suggested that the sickle gene arose independently more than once. In the present study we have performed restriction endonuclease haplotype analysis for the beta-globin-like gene cluster from four separate geographic areas in Africa, all of which possess the sickle gene. In Benin (Central West Africa) and Algeria (Arab North Africa) all chromosomes carrying the sickle gene possess an identical haplotype as defined by 11 different polymorphic restriction endonuclease sites within the 60-kilobase region of the beta-globin-like gene cluster. In the Central African Republic (Bantu-speaking Africa) and in Senegal (Atlantic West Africa) a very large proportion of the sickle gene chromosomes were associated with a haplotype specific for each country. Thus, three different haplotypes are shown to be associated with the sickle gene in Africa, and each is present at a very high frequency in geographically separate regions. Since the three haplotypes differ from each other by at least three sites residing both 5' and 3' to a putative hot spot for recombination, it is most likely that the sickle gene arose at least three times on separate preexisting chromosomal haplotypes. This may have implications for a better understanding of the variable nature of the expression of sickle cell anemia, because clinically relevant sequences (for example, gamma-globin gene regulatory sequences responsive to anemia) might be linked polymorphically to these haplotypes.
Class II HLA-DC beta-chain DNA restriction fragments differentiate among HLA-DR2 individuals in insulin-dependent diabetes and multiple sclerosis.
Authors: Cohen D , Cohen O , Marcadet A , Massart C , Lathrop M , Deschamps I , Hors J , Schuller E , Dausset J
Published: May 2006

HLA-DR2 allele is negatively associated with insulin-dependent diabetes and positively associated with multiple sclerosis (MS). A 2.2-kilobase-pair EcoRI DNA restriction fragment detected with a beta-chain HLA-DC cDNA probe was found to be strongly correlated with HLA-DR2 in the normal population, but was absent in HLA-DR2 insulin-dependent diabetic patients. This fragment was found in HLA-DR2 multiple sclerosis patients with the same frequency as in controls. A beta-chain HLA-DC 12-kilobase-pair BamHI fragment might differentiate multiple sclerosis patients from healthy individuals.
Kappa-chain allotypes and isotypes in the rabbit: cDNA sequences of clones encoding b9 suggest an evolutionary pathway and possible role of the interdomain disulfide bond in quantitative allotype expression.
Authors: McCartney-Francis N , Skurla R M , Mage R G , Bernstein K E
Published: May 2006

The constant regions of rabbit kappa light chains are unusual because the sequences of the allotypic forms can differ more from each other than do some variable regions with which they associate. We report the nucleic acid sequence of a full-length cDNA clone of b9 allotype and show comparisons to available sequences of the rabbit kappa allotypes b4, b5, and bas-N4. Our analyses suggest that the primordial rabbit kappa gene encoded a bas-like sequence. They also reveal a surprising difference in the position of the variable region cysteine that forms the interdomain disulfide bond that is unique to most rabbit kappa chains. One b9 cDNA sequence lacks the usual cysteine-80 and instead encodes cysteine-108, which in three-dimensional models appears capable of forming the interdomain disulfide bond with cysteine-171 in the constant region. A partial sequence of a second b9 clone encodes both cysteine-80 and cysteine-108; the translation product of this clone could have a free reactive sulfhydryl group that might lead to an unstable nonfunctional Ig molecule. The fact that pre-B cells with b9 kappa chains do not differentiate and expand into productive Ig-producing cells with frequencies comparable to the other allotypes may be explained if a substantial proportion of the gene products have a free sulfhydryl group. Our sequence results suggest that in cells differentiating to produce kappa light chains of b9 allotype the number and location of the cysteines influence immunoglobulin expression.
Time-resolved photolabeling by the noncompetitive blocker chlorpromazine of the acetylcholine receptor in its transiently open and closed ion channel conformations.
Authors: Heidmann T , Changeux J P
Published: May 2006

A rapid-mixing photolabeling apparatus is developed to resolve the kinetics of association of the noncompetitive channel blocker [3H]chlorpromazine (CPZ) with the membrane-bound acetylcholine (AcCho) receptor from Torpedo marmorata and to photolabel its subunits in the 100-milli-seconds to seconds time range. Rapid mixing of AcCho and [3H]CPZ with the receptor followed by brief (less than 20 msec) UV irradiation results in the selective labeling of the four chains of the AcCho receptor, according to a rapid bimolecular association process close to diffusion-controlled. Rapid association is not observed with the competitive antagonists d-tubocurarine or flaxedil or the snake venom alpha-toxins. Its initial rate increases with agonist concentration, with maxima of 0.6 for carbamoylcholine and 0.2 for phenyltrimethylammonium taking 1 for AcCho, with apparent dissociation constants of 30 microM, 400 microM, and 300 microM for AcCho, carbamoylcholine, and phenyltrimethylammonium, respectively, and with sigmoid shape (Hill coefficients of 1.1-1.3). Under conditions in which the receptor "desensitizes" and the ionic channel closes (preincubation with AcCho), rapid [3H]CPZ association decreases in parallel. It is concluded that the agonist-dependent rapid association of [3H]CPZ takes place at the level of a site common to all five subunits, which lies within the ion channel and becomes accessible when the channel opens.
Spectroscopic studies of ferricyanide oxidation of Azotobacter vinelandii ferredoxin I.
Authors: Morgan T V , Stephens P J , Devlin F , Stout C D , Melis K A , Burgess B K
Published: May 2006

The Fe(CN)3-(6) oxidation of the crystallographically characterized [[3Fe-3S], [4Fe-4S]] ferredoxin I of Azotobacter vinelandii has been studied using absorption, circular dichroism, magnetic circular dichroism, and EPR spectroscopies. A paramagnetic intermediate is observed en route to Fe-S cluster-free apoprotein, possessing an anisotropic g approximately equal to 2 EPR signal, surviving to temperatures greater than 77 K. This species is shown to result from 3-electron oxidation of the [4Fe-4S] cluster, without modification of the [3Fe-3S] cluster. However, it does not give rise to observable paramagnetic magnetic circular dichroism in the visible-near UV spectral region and is therefore neither an oxidized HIPIP [4Fe-4S] cluster nor an oxidized [3Fe-3S] cluster. We identify the paramagnetic species as a cysteinyldisulfide radical formed on dissociation of an oxidized cysteinate and an oxidized sulfide ion from the [4Fe-4S] cluster. This conclusion is consistent with the observed reaction stoichiometry, the spectroscopic results obtained, known EPR spectra of disulfide radicals, and the reconstitution of the native [4Fe-4S] cluster by dithiothreitol alone. This reaction, earlier interpreted as a HIPIP-type oxidation, is a previously uncharacterized oxidation reaction of [4Fe-4S] clusters.
Calmodulin inhibitor in senescing apples and its physiological and pharmacological significance
Authors: Paliyath Gopinadhan , Poovaiah B. W.
Published: May 2006

While assaying calmodulin activity in senesced apple extracts by using its property of promoting the activity of activator-deficient 3′,5′-cyclic AMP 5′-nucleotidohydrolase (phosphodiesterase, EC 3.1.4.17) from bovine heart, we detected a heat-stable, dialyzable, low molecular weight component that inhibited calmodulin activity. Specific activity of calmodulin as calculated from the linearly increasing portion of the activity curve was in the range of 150 to 160 units/mg of protein in crude extracts from apples stored at 2°C for a period of 6 months with or without calcium treatment. Apple extract that was passed through a phenothiazine-Sepharose affinity column did not promote phosphodiesterase activity, whereas the EGTA eluate of the column promoted phosphodiesterase activity similar to the original extract. The inhibition of calmodulin activity appeared to be lower in extracts from apples stored at 2°C after calcium treatment. The inhibition was found to increase after storage of apples at room temperature for 30 days. Activity of purified bovine brain calmodulin was also inhibited by the inhibitor present in apple extracts, which indicated that the inhibition was not specific to plant calmodulin alone and could have wide applications. The importance of the inhibitor in relation to senescence/aging and its possible pharmacological applications are discussed.
Expression and rearrangement of homologous immunoglobulin VH genes in two mouse strains.
Authors: Near R I , Juszczak E C , Huang S Y , Sicari S A , Margolies M N , Gefter M L
Published: May 2006

A family of murine anti-p-azophenylarsonate (Ars) antibodies share a variable (V) region serologically defined marker, the 36-60 idiotype (Id36-60). Most mouse strains possess five genes highly homologous to the gene encoding the heavy (H) chain V region of antibodies bearing Id36-60 (VH36-60); however, only one of these genes is ever utilized by hybridomas whose antibodies bind Ars and bear Id36-60. The relevant VH genes were cloned from A/J and BALB/c mouse DNA libraries. Their DNA sequences were found to differ at only two positions. Southern blot analysis, protein sequence determination, and nucleic acid sequence determination indicate that the above hybridomas utilize the same joining (JH3), diversity (D), and VH gene segments regardless of BALB/c or A/J strain origin. Despite this virtual identity, BALB/c and A/J mouse strains express quite different serum levels of Id36-60-bearing antibodies when immunized with Ars. The basis of this regulatory process is discussed.
Inhibition of the mitogenic effects of plasma by a monoclonal antibody to somatomedin C.
Authors: Russell W E , Van Wyk J J , Pledger W J
Published: May 2006

Immunoneutralization studies with a monoclonal antibody to somatomedin C (Sm-C) were undertaken to further determine the role of this peptide in cellular proliferation. For our model we used density-arrested cultures of BALB/c 3T3 cells. Transient exposure of these cells to platelet-derived growth factor enables them to respond to platelet-poor plasma by progressing through the G1 stage and undergoing renewed DNA synthesis. In this system, the combination of nanogram concentrations of Sm-C and epidermal growth factor can fully substitute for plasma, and microgram concentrations of insulin can substitute for Sm-C by crossreacting with the Sm-C receptor. We now show that a monoclonal antibody to Sm-C, which in defined medium blocks the mitogenic effect of Sm-C but not insulin, also blocks the stimulation of DNA synthesis by human plasma or calf serum. Furthermore, by adding the antibody at progressively later times during G1, we show that these cells escape from their dependence on Sm-C for DNA synthesis after traversing G1 to a point at or near the G1/S boundary.
Increased adhesiveness of Down syndrome fetal fibroblasts in vitro.
Authors: Wright T C , Orkin R W , Destrempes M , Kurnit D M
Published: May 2006

We compared the in vitro rate of divalent cation-independent aggregation of fibroblasts derived from abortuses with normal karyotypes and with trisomy 21 (Down syndrome). Fibroblasts from five lung and two of three cardiac cultures from subjects with Down syndrome aggregated more rapidly than matched fibroblasts from normal controls or lung fibroblasts from an abortus with trisomy 13. In contrast, skin fibroblasts derived from the trisomy 21 subjects had low rates of aggregation. The high rates of aggregation of trisomy 21 lung fibroblasts were not affected by hyaluronidase treatment. Lung fibroblasts from both normal and Down syndrome subjects had similar membrane polarization values in an assay using the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene. Thus, the increased aggregation rate we observed for trisomy 21 fibroblasts was restricted to specific fibroblast cultures, was not mediated by hyaluronic acid or gross membrane lipid alterations, and was specific for trisomy of chromosome 21. As illustrated in computer simulations presented elsewhere, increased intercellular adhesiveness during organogenesis could explain the frequent occurrence of malformations, including pulmonary hypoplasia and congenital heart defects, in Down syndrome.
Inhibition of 3-hydroxy-3-methylglutaryl-CoA reductase by mevinolin in familial hypercholesterolemia heterozygotes: effects on cholesterol balance.
Authors: Grundy S M , Bilheimer D W
Published: May 2006

Patients with heterozygous familial hypercholesterolemia (FH) have a deficiency of receptors for plasma low-density lipoprotein (LDL) that impairs removal of LDL from plasma. In these patients, mevinolin, an inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase [mevalonate:NAD+ oxidoreductase (CoA-acylating), EC 1.1.1.88], increases receptors for LDL and decreases LDL concentrations. To determine whether mevinolin also causes severe decreases in total body synthesis of cholesterol, fecal excretions of neutral steroids and acidic steroids were determined in five FH heterozygotes before and during treatment with mevinolin. The drug produced an average decrease in plasma total cholesterol of 23% and in LDL cholesterol of 24%. Mevinolin caused a significant decrease in the output of neutral and acidic steroids in three patients, but it caused no alterations in two others. Changes in fecal output of steroids did not correlate with the degree of lowering of the patients' LDL-cholesterol level. In none of the patients did the output of fecal steroids fall below the values seen in normal subjects studied under similar conditions. One patient had a previous ileal exclusion operation and had a massive output of acidic steroids in the control period; mevinolin therapy caused a slight decrease in excretion of acidic steroids, but the output was still markedly above normal. We conclude that the LDL lowering action of mevinolin does not appear to require a severe decrease in cholesterol synthesis that might lead to depletion of vital body stores of cholesterol.
Carbonyl oxygen exchange evidence of imine formation in the glutamate dehydrogenase reaction and identification of the "occult role" of NADPH.
Authors: Fisher H F , Viswanathan T S
Published: May 2006

Although an imine intermediate has long been postulated as participating in the reaction catalyzed by glutamate dehydrogenase (EC 1.4.1.4), direct evidence for a kinetically competent intermediate of this kind has not heretofore been found. We have sought such evidence by studying the exchange of the carbonyl oxygen atom of alpha-ketoglutarate in a variety of binary, ternary, and quaternary enzyme complexes. We have found that the time course of this exchange is biphasic when the enzyme, alpha-ketoglutarate, NADPH, and ammonia are all present initially and that the rapid initial phase ends when ammonia is depleted. We present evidence that this rapid exchange is due to an imine form of the enzyme-reduced-coenzyme-substrate-ammonia complex. Formed very rapidly but in very small amounts, this imine can undergo one of two competing fates: (i) hydrolytic reversal to form carbonyl-exchanged alpha-ketoglutarate with regeneration of ammonia, and (ii) an internal hydride transfer converting the iminoglutarate to glutamate, whereby ammonia is consumed. The agreement of the amplitudes of rapid 18O exchange with predictions based on direct transient-state spectroscopic kinetic studies supports the identity of an enzyme-NADPH-alpha-iminoglutarate complex as an obligatory intermediate on the enzyme-catalyzed reaction path. The corresponding enzyme-alpha-iminoglutarate binary complex (previously suggested as an intermediate) is formed at a rate that is less than 1/1000th of that of the NADPH-containing complex shown here, and it therefore lacks kinetic competence. This finding points up an important catalytic role for NADPH that does not involve its obvious function as a hydride donor and is distinctly separate from this role. In the case of the glutamate dehydrogenase-catalyzed reaction, this "occult role" clearly involves the induction of ketimine formation on the enzyme surface.
beta-Thalassemia in Chinese: use of in vivo RNA analysis and oligonucleotide hybridization in systematic characterization of molecular defects.
Authors: Cheng T C , Orkin S H , Antonarakis S E , Potter M J , Sexton J P , Markham A F , Giardina P J , Li A , Kazazian H H
Published: May 2006

To perform a systematic analysis of beta-thalassemia genes among Chinese, we have determined the DNA haplotype in the beta-globin gene region of 37 Chinese beta-thalassemia chromosomes. Only four haplotypes were found. Blot hybridization analysis of erythroid RNA from patients homozygous for haplotypes 1, 2, and 3 demonstrated different patterns, suggesting that a different mutation was associated with each haplotype. The mutation associated with haplotype 1 was a C----T substitution at IVS-2, position 654. This mutation produces a new donor splice site and leads to formation of a beta-globin RNA with an insertion of 73 nucleotides. The mutation associated with haplotype 2 was a nucleotide insertion of A between codons 71 and 72, which results in a frameshift and premature termination of beta-globin synthesis. Haplotype analysis suggests that these two mutations may account for up to 85% of beta-thalassemia genes in this ethnic group. The haplotype 3 gene contained a transcriptional "TATA" box mutation that has been previously reported. Oligonucleotide hybridization demonstrated that the mutation associated with haplotype 4 was the same IVS-1 position 5 substitution commonly observed among beta-thalassemia genes in Asian Indians. Since haplotype 4 of Chinese differs at polymorphic sites on either side of the IVS-1 position 5 mutation from the haplotype associated with this mutation in Indians, the mutation presumably arose independently in these two populations.
Genetic analysis of beta-adrenergic receptor internalization and down-regulation.
Authors: Mahan L C , Koachman A M , Insel P A
Published: May 2006

We have used wild-type and variants of the T-lymphoma cell line S49 to explore internalization and down-regulation of adenylate cyclase-linked beta-adrenergic receptors. Internalization was defined by the loss in "surface receptors" detected at 4 degrees C on intact cells by the antagonists [3H]CGP-12177 or [125I]iodocyanopindolol, whereas down-regulation was defined as the loss in total cellular content of receptors [( 125I]iodocyanopindolol binding assayed at 37 degrees C). In wild-type cells, the beta-adrenergic agonist isoproterenol induced a rapid (t 1/2, approximately equal to 1 min) and reversible loss in surface receptors. The surface sites were lost at a rate similar to the rate of desensitization of beta-adrenergic receptor-mediated cyclic AMP generation of S49 cells. A series of S49 variants (cyc-, UNC, H21a) having lesions in NS (the guanine nucleotide binding protein that couples beta-receptors to adenylate cyclase) or with absent cAMP-dependent protein kinase activity (kin-), had a loss in surface sites that was equivalent to that of wild-type cells. By contrast, S49 variant cells having lesions in NS showed variable rates and extents of down-regulation of beta-adrenergic receptors. In wild-type and kin- S49 cells, beta-receptors down-regulated with a t 1/2 of approximately equal to 4 hr. Down-regulation was blunted in the cyc- and UNC variants that have altered coupling of receptors to NS, but it was faster in the H21a variant that retains receptor-NS interaction. Recovery of receptors after down-regulation occurred at a similar rate (t 1/2, approximately equal to 6 hr) in wild-type, UNC, and H21a cells. These results demonstrate that internalization of beta-adrenergic receptors may be necessary, but is not sufficient, to explain agonist-induced receptor down-regulation in S49 cells. The variable expression in the development of down-regulation in S49 variants implies that receptor-NS interaction regulates the fate of receptors linked to the stimulation of adenylate cyclase.
Functional reconstitution of the purified brain sodium channel in planar lipid bilayers.
Authors: Hartshorne R P , Keller B U , Talvenheimo J A , Catterall W A , Montal M
Published: May 2006

The ion conduction and voltage dependence of sodium channels purified from rat brain were investigated in planar lipid bilayers in the presence of batrachotoxin. Single channel currents are clearly resolved. Channel opening is voltage dependent and favored by depolarization. The voltage at which the channel is open 50% of the time is -91 +/- 17 mV (SD, n = 22) and the apparent gating charge is approximately 4. Tetrodotoxin reversibly blocks the ionic current through the sodium channels. The Ki for the tetrodotoxin block is 8.3 nM at -50 mV and is voltage dependent with the Ki increasing e-fold for depolarizations of 43 mV. The single channel conductance, gamma, is ohmic. At 0.5 M salt concentrations gamma = 25 pS for Na+, 3.5 pS for K+, and 1.2 pS for Rb+. This study demonstrates that the purified brain sodium channel--which consists of three polypeptide subunits: alpha (Mr approximately 260,000), beta 1 (Mr approximately 39,000), and beta 2 (Mr approximately 37,000)--exhibits the same voltage dependence, neurotoxin sensitivity, and ionic selectivity associated with native sodium channels.
Use of a cDNA expression vector for isolation of mouse interleukin 2 cDNA clones: expression of T-cell growth-factor activity after transfection of monkey cells.
Authors: Yokota T , Arai N , Lee F , Rennick D , Mosmann T , Arai K
Published: May 2006

A cDNA sequence coding for mouse interleukin 2 (IL-2) has been cloned from a cDNA library prepared from mRNA derived from a concanavalin A-activated mouse T-cell clone. The library was constructed by using the pcD vector system, which permits the expression of cDNA inserts in mammalian cells. Screening of the library was performed by transfecting COS-7 monkey cells with pools of cDNA clones in order to express the products encoded by full-length cDNA inserts. By assaying the supernatant fluid, IL-2 cDNA clones that express T-cell growth-factor (TCGF) activity were identified. The DNA sequence codes for a polypeptide of 169 amino acid residues including a putative signal peptide. The mouse IL-2 amino acid sequence deduced from the nucleotide sequence of its cDNA shares extensive homology with the human IL-2 amino acid sequence reported previously. These results demonstrate that identification of full-length cDNA clones for many lymphokines may be achieved entirely on the basis of detection of the functional polypeptides in mammalian cells.
Common evolutionary origin of alpha 2-macroglobulin and complement components C3 and C4.
Authors: Sottrup-Jensen L , Stepanik T M , Kristensen T , Lønblad P B , Jones C M , Wierzbicki D M , Magnusson S , Domdey H , Wetsel R A , Lundwall A
Published: May 2006

A comparison of the sequence of the subunit of human alpha 2-macroglobulin (alpha 2M; 1451 amino acid residues) with that of murine complement component pro-C3 (1639 amino acid residues) reveals eight extended regions of sequence similarity. These regions contain between 19% and 31% identically placed residues and account for 75% and 67%, respectively, of the polypeptide chains of alpha 2M and pro-C3. Published sequence data for complement component C4 show that segments of this protein match well with corresponding stretches in alpha 2M and pro-C3. It is proposed that alpha 2M, C3 and C4, which all contain a unique activatable beta-cysteinyl-gamma-glutamyl thiol ester, have a common evolutionary origin and are homologous proteins. Several larger regions of low sequence similarity indicate the presence of structural domains in each of these proteins that specifically modify an underlying common gross structure. The quartets of basic residues in pro-C3 and pro-C4, at which cleavage takes place to produce the mature subunits of these proteins, and most of the residues forming the anaphylatoxin peptides of C3 and C4 (C3a and C4a) are absent in alpha 2M. In addition, C3 and C4 contain large portions, which extend beyond the COOH terminus of alpha 2M.
Electrostatic influence on energetics of electron transfer reactions.
Authors: Rees D C
Published: May 2006

Electron transfer chains in biological systems must operate efficiently to satisfy metabolic energetic requirements. The component proteins in these chains are expected to exhibit characteristic structural features that facilitate electron transfer to the appropriate donor and acceptor proteins. A survey of soluble one-electron carrier proteins indicates a significant tendency for lower potential proteins to be more negatively charged than higher potential proteins. Consideration of the electrostatic consequences of this pattern of charge asymmetry suggests that the reduction potential difference between the two proteins will be minimized in the precursor complex associated with electron transfer. An equivalent statement is that the change in free energy accompanying electron transfer in the complex will approach zero. This behavior is consistent with theoretical arguments advanced by Albery and Knowles [Albery, W. J. & Knowles, J. R. (1976) Biochemistry 15, 5631-5640], which suggest that for the most efficient enzymes, the free energy difference between enzyme-bound species should approach zero. A more general derivation of this prediction is provided. The observed charge asymmetry in electron transfer proteins provides a structural mechanism for satisfying this requirement, thus accelerating the overall rate of electron transfer.
Developmentally regulated expression of a chicken muscle-specific gene in stably transfected rat myogenic cells.
Authors: Nudel U , Greenberg D , Ordahl C P , Saxel O , Neuman S , Yaffe D
Published: May 2006

To test the evolutionary conservation of DNA sequences specifying the developmentally regulated expression of the skeletal muscle actin gene, a recombinant plasmid containing the chicken skeletal muscle actin gene was introduced into rat myogenic cells. In a significant number of isolated clones, the accumulation of chicken actin mRNA increased greatly during differentiation. To test the expression in myogenic cells of a gene that is normally expressed during terminal differentiation of another tissue, rat myogenic cells were transfected with a mouse/human beta-globin chimeric gene. A decrease by a factor of 2-3 in the amount of globin mRNA during differentiation was observed in most clones in which the gene was expressed. The results indicate the conservation of the muscle-specific regulatory DNA sequences for more than 300 Myr.
Amino acid sequence of a variant pro-form of insulin-like growth factor II.
Authors: Zumstein P P , Lüthi C , Humbel R E
Published: May 2006

Human serum contains, in addition to the "classical" 7.5-kDa insulin-like growth factors (IGFs) I and II, small amounts of larger IGF-II. A 10-kDa IGF-II was isolated by gel filtration, immunoaffinity chromatography, and reversed-phase HPLC. Upon amino acid sequence determination, a substitution of Cys-Gly-Asp for Ser-33 was found as well as a COOH-terminal extension of 21 residues (E peptide). These sequence differences suggest that 10-kDa IGF-II is a precursor of a variant IGF-II. Since the substitution is not located at a known intron/exon hinge region, the finding of this variant IGF-II is evidence for the presence of more than one gene for IGF-II.
The rate-determining step in cAMP-mediated exocytosis in the rat parotid and submandibular glands appears to involve analogous 26-kDa integral membrane phosphoproteins.
Authors: Quissell D O , Deisher L M , Barzen K A
Published: May 2006

The possible direct involvement of protein phosphorylation in the regulation of exocytosis during beta-adrenergic receptor stimulation in rat parotid and submandibular salivary glands was investigated in vitro using dispersed cells. The dispersed cells were labeled with [32P]orthophosphate for 40 min prior to experimental manipulation. Subcellular fractions were isolated, the proteins were separated using sodium dodecylsulfate/polyacrylamide gel electrophoresis (NaDodSO4/PAGE), and the phosphoproteins were detected by autoradiography. Changes in the extent of phosphorylation for each phosphoprotein were determined indirectly by densitometric analyses. The analogous parotid and submandibular 26-kDa membrane phosphoproteins had a rapid phosphate turnover rate (t 1/2 = 5-6 min) whereas the analogous 21-kDa membrane phosphoproteins had a much slower phosphate turnover rate (t 1/2 greater than 20 min). The results of Triton X-114 extraction indicated that the 26- and 21-kDa phosphoproteins were integral membrane proteins. The rate of phosphate turnover for the analogous 26-kDa phosphoproteins is compatible with a regulatory role in exocytosis, whereas the slower phosphate turnover rate for the analogous 21-kDa phosphoproteins suggests that these proteins may play a more subordinate role in secretion or they may coordinate secretion with other cellular metabolic events.