bluuebunny/crossref_metadata_2025 huggingface dataset:
Psychological stress activates phosphorylase in the heart of the conscious pig without increasing heart rate and blood pressure.
Authors: Skinner J E , Beder S D , Entman M L
Published: May 2006

The present study uses a technique that enables the collection of multiple freeze-biopsy samples from the myocardium of the conscious pig (i.e., through a thoracic window). This technique enables sequential analysis of the metabolic state of the myocardium during different behavioral conditions. The results demonstrate that with daily adaptations to an unfamiliar environment (i.e., stress reduction), the phosphorylase activation ratio (phosphorylase a/total phosphorylase) in the quiescent pig declines steadily from approximately 80% to 30% (r = -0.91, P less than 0.01). This decline occurs with both the mean resting heart rate and left ventricular blood pressure remaining constant. The decline is seen within individual subjects during the whole adaptation sequence as well as between subjects whose samples were taken either early or late in the adaptation series. The dissociation of hemodynamic functional and metabolic activation in the unadapted, psychologically stressed pig may be associated with the occurrence of increased vulnerability of the ischemic heart to ventricular fibrillation, a phenomenon previously observed under the same behavioral conditions.
Biochemical and molecular analysis of naturally occurring Adh variants in Drosophila melanogaster.
Authors: Anderson S M , McDonald J F
Published: May 2006

The results of a detailed analysis of the biochemical and molecular basis of alcohol dehydrogenase (ADH) activity variation existing among six naturally occurring and one ethyl methanesulfonate-induced Adh variant strain of Drosophila melanogaster are presented. Significant specific activity differences exist among the strains but the majority of ADH activity variation can be accounted for by differences in levels of ADH protein. These protein level differences can, in turn, be accounted for by ADH synthesis rate variation, which positively correlates with in vivo levels of cytoplasmic Adh mRNA. The functional variability is correlated with known structural variation in and around the area of the Adh gene.
Recombination within and between the human insulin and beta-globin gene loci.
Authors: Lebo R V , Chakravarti A , Buetow K H , Cheung M C , Cann H , Cordell B , Goodman H
Published: May 2006

We detected a large number of polymorphic insulin restriction fragments in black Americans. These different size fragments were probably generated by unequal recombination on both sides of the human insulin gene. Population genetic analysis indicates that recombination occurred 33 times more frequently than expected to generate this large number of polymorphic fragments. Specific properties of the unique repeated 14- to 16-base-pair sequences 5' to the insulin gene suggest that this sequence would promote increased unequal recombination. Additional pedigree analysis showed that the recombination rate between the structural insulin and beta-globin gene loci was 14% with strong evidence for linkage. Since both insulin and beta-globin have been mapped to the short arm of human chromosome 11, this study establishes that the genetic map distance between these genes is 14.2 centimorgans.
Characterization of rat hypothalamic corticotropin-releasing factor.
Authors: Rivier J , Spiess J , Vale W
Published: May 2006

A polypeptide was purified from rat hypothalamic extracts on the basis of its high intrinsic activity to release corticotropin (ACTH) from cultured rat anterior pituitary cells and its immunoactivity in a radioimmunoassay directed against the NH2 terminus (residues 4-20) of ovine hypothalamic corticotropin-releasing factor (CRF). Based on Edman degradation, peptide mapping, and amino acid analysis, the primary structure of this rat CRF was established to be: H-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Met-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Met-Glu-Ile-Ile-NH2. The hypophysiotropic potency of synthetic rat CRF did not deviate significantly from the potencies of the isolated native peptide or of synthetic ovine CRF. The close structural relationship between rat and ovine hypothalamic CRF is indicated by an 83% sequence homology.
Receptor sites involved in posttranslational transport of apocytochrome c into mitochondria: specificity, affinity, and number of sites.
Authors: Hennig B , Koehler H , Neupert W
Published: May 2006

Assembly of cytochrome c involves a series of steps: synthesis of apocytochrome c on free ribosomes, specific binding of apocytochrome c to the mitochondrial surface, transfer across the outer membrane, covalent addition of protoheme, refolding of the polypeptide chain, and association of holocytochrome c with its functional sites at the inner membrane. The binding step of apocytochrome c to Neurospora crassa mitochondria was studied by inhibiting the subsequent transfer steps with the heme analogue deuterohemin. The binding sites are highly specific for mitochondrial apocytochromes c. Bound labeled Neurospora apocytochrome c was competitively displaced by unlabeled apocytochrome c from various species. These exhibited different abilities for displacement. Apocytochrome c from Paracoccus denitrificans, the amino-terminal (heme-binding) fragment of Neurospora apocytochrome c, and Neurospora holocytochrome c did not recognize the binding sites. Polylysine did not interfere with apocytochrome c binding. Apocytochrome c is reversibly bound. The binding sites are present in limited number. High-affinity binding sites were present at about 90 pmol/mg of mitochondrial protein. They displayed an association constant of 2.2 X 10(7) M-1. Apocytochrome c was imported into mitochondria and converted to holocytochrome c directly from the binding sites when inhibition by deuterohemin was relieved. We conclude that the apocytochrome c binding sites on mitochondria represent receptors that function in the recognition and import of this precursor by mitochondria.
Specific cell-cell contact serves as the developmental signal to deactivate discoidin I gene expression in
Dictyostelium discoideum
Authors: Berger Edward A. , Clark Judy M.
Published: May 2006

Specific cell-cell contact is a major regulatory signal controlling cell differentiation in
Dictyostelium discoideum
, causing dramatic changes in the developmental program of gene expression. In this report, we focus on the relationships between specific cell-cell contact and the activity of the genes for discoidin I, an endogenous lectin that has been implicated in the cell-cell cohesion process. By performing quantitative RNA dot-hybridization assays and RNA gel blot-hybridization analyses, using as a probe a recombinant plasmid containing a discoidin I cDNA insert, we have measured changes in discoiding I mRNA levels during normal development and in response to specific manipulations of the state of cellular aggregation. Our major findings are as follows. (
i
) During normal development on filters, there is a close temporal correspondence between the establishment of specific cell-cell contacts and the decline in discoidin I mRNA levels. By the tight-aggregate stage, discoidin I mRNA is barely detectable. (
ii
) When tight aggregates are disaggregated and the cells are maintained in the disaggregated state, there is a dramatic rise in discoidin I mRNA content. (
iii
) When cells are developed in suspension (conditions that interfere with the establishment of tight cell-cell contacts), discoidin I mRNA accumulates to abnormally high levels, and these persist well after the levels in filter-developed cells have declined. Taken together, these results strongly suggest that cell-cell contact is the normal developmental signal to deactivate discoidin I gene expression; thus, a contact-deactivated gene for which a recombinant DNA probe is available has now been identified. Furthermore, we demonstrate that exogenous cAMP almost completely blocks the disaggregation-induced reactivation of discoidin I gene expression. Possible mechanistic relationships between specific cell-cell contact, intracellular cAMP levels, and developmental gene expression are discussed.
Monoclonal IgM antibody exhibiting high-affinity binding and cryoglobulin properties.
Authors: Ballard D W , Kranz D M , Voss E W
Published: May 2006

A monoclonal IgM antibody (18-2-3) derived from cell fusion of (NZB X NZW) F1 splenocytes following secondary immunization with fluorescein-conjugated keyhole limpet hemocyanin was shown to exhibit high intrinsic binding affinity and cryoinsolubility. Affinity-purified preparations were determined to be IgM by immunochemical, electrophoretic, and chromatographic analyses. An intrinsic association constant (Ka) of 2.9 X 10(10) M-1 (at 2 degrees C) was measured by first-order dissociation-rate analysis. Antibody solubility at low concentration (approximately equal to 50 micrograms/ml) was shown, by absorption spectroscopy, to be temperature dependent between 4 degrees C and 32 degrees C. Insolubility at low temperature (4 degrees C) was reversible in the presence of homologous fluorescyl hapten, indicative of active site involvement in the mechanism of cryoglobulin-18-2-3 complex formation. Characteristics of clone 18-2-3 are discussed in terms of (i) its potential use as a model for examining the mechanism of cryoprecipitation and (ii) the proposed relationship between affinity maturation and the IgM to IgG class switch.
Human arterial wall cells secrete factors that are chemotactic for monocytes.
Authors: Mazzone T , Jensen M , Chait A
Published: May 2006

Macrophages and arterial smooth muscle cells comprise the cellular components of the atherosclerotic plaque. The vessel wall accumulation of macrophages occurs by a process of increased circulating monocyte migration into the vessel wall. In these studies it is demonstrated that human macrophages and arterial smooth muscle cells in culture secrete potent chemotactic factors for freshly isolated human monocytes. In contrast, human fibroblast-conditioned medium has no chemotactic activity. The effect of macrophage-conditioned medium is a function of macrophage differentiation and can be potentiated by macrophage activation. These results suggest that secretory products of human macrophages and arterial smooth muscle cells may be important stimuli for increased monocyte migration into the vessel wall in vivo.
Methylation involved in chemotaxis is regulated during Caulobacter differentiation.
Authors: Shaw P , Gomes S L , Sweeney K , Ely B , Shapiro L
Published: May 2006

Caulobacter crescentus carries a flagellum and is motile only during a limited time in its cell cycle. We have asked if the biochemical machinery that mediates chemotaxis exists coincident with the cell's structural ability to respond to a chemotactic signal. We first demonstrated that one function of the chemotaxis machinery, the ability to methylate the carboxyl side chains of a specific set of membrane proteins (methyl-accepting chemotaxis proteins, MCPs), is present in C. crescentus. This conclusion is based on the observations that (i) methionine auxotrophs starved of methionine can swim only in the forward direction (comparable to smooth swimming in the enteric bacteria), (ii) a specific set of membrane proteins was found to be methylated in vivo and the incorporated [3H]methyl groups were alkali sensitive, (iii) this same set of membrane proteins incorporated methyl groups from S-adenosylmethionine in vitro, and (iv) out of a total of eight generally nonchemotactic mutants, two were found to swim only in a forward direction and one of these lacked methyltransferase activity. Analysis of in vivo and in vitro methylation in synchronized cultures showed that the methylation reaction is lost when the flagellated swarmer cell differentiates into a stalked cell. In vivo methylation reappeared coincident with the biogenesis of the flagellum just prior to cell division. In vitro reconstitution experiments with heterologous cell fractions from different cell types showed that swarmer cells contain methyltransferase and their membranes can be methylated. However, newly differentiated stalked cells lack methyltransferase activity and membranes from these cells cannot accept methyl groups. These results demonstrate that MCP methylation is confined to that portion of the cell cycle when flagella are present.
Mitogenic hormones and tumor promoters greatly increase the incidence of colony-forming cells bearing amplified dihydrofolate reductase genes.
Authors: Barsoum J , Varshavsky A
Published: May 2006

Previous work has shown that the presence of a phorbol ester tumor promoter, phorbol 12-myristate 13-acetate (PMA), during a single-step selection for methotrexate (MTX)-resistant mouse 3T6 cells results in an up to 100-fold increase in the incidence of MTX-resistant, colony-forming cells. MTX resistance of most of these cells is due to amplification of the gene for dihydrofolate reductase (DHFR), the target enzyme for MTX. We show here that other active, noncytotoxic phorbol ester tumor promoters, such as phorbol 12, 13-didecanoate and 20-phorbol 12,13-butyrate, at their optimal concentrations (approximately equal to 0.1 microM) are approximately equal to PMA in increasing the incidence of MTX-resistant 3T6 colonies. Mezerein, a potent second-stage tumor promoter, but a weak complete promoter, increases the incidence of MTX resistance up to 350-fold, the strongest effect for any of the agents so far tested. PMA analogs that are inactive as tumor promoters, such as phorbol or phorbol 12,13,20-triacetate, have no effect on the incidence of MTX-resistant 3T6 colonies. Anthralin, a nonphorbol tumor promoter, is approximately equal to 40% as active as PMA in the MTX selection assay. Remarkably, the hormones insulin, arginine vasopressin, and epidermal growth factor, all of which are mitogenic for 3T6 cells, also exert a strong PMA-like effect on the incidence of MTX-resistant 3T6 colonies under conditions of MTX selection. The effect of insulin at its optimal concentration (approximately equal to 1 microgram/ml) is approximately equal to 70% that of PMA. Although the effect of PMA on the incidence of MTX-resistant 3T6 colonies does not significantly depend on the initial density of seeded cells or volume of the medium added, the analogous effect of insulin is strongly influenced by these parameters. Mevalonic acid, arachidonic acid, thymidine, caffeine, and nicotine, all of which are known to influence patterns of DNA synthesis in mammalian cells, were tested at their highest noncytotoxic concentrations and failed to produce any significant effect on the incidence of MTX-resistant 3T6 colonies. We discuss possible mechanisms of hormone- and tumor promoter-facilitated gene amplification in mammalian cells, relationship of mitogenic hormones to tumor promoters, and also implications of our findings for the problem of drug resistance in cancer chemotherapy.
Repeated sequences and open reading frames in the 3' flanking region of the gene for the RNA subunit of Escherichia coli ribonuclease P.
Authors: Reed R E , Altman S
Published: May 2006

We have determined the identity of about 700 nucleotides in the 3' flanking region of the gene for M1 RNA, the RNA component of RNase P (EC 3.1.26.5). This region begins with a 113-base-pair segment of DNA, which is repeated approximately 3.5 times. The first repeating unit originates within the gene sequence for the 3' end of mature M1 RNA. The repeating units are highly conserved but diverge considerably after the partial fourth repeat. Segments of sequence homologous to the repeats have been identified both upstream in the M1 RNA transcription unit and downstream from the repeating units. Several overlapping open reading frames, with the potential to encode small basic proteins, have been identified in the repeated sequences. The structure of the M1 RNA gene 3' flanking region is very similar to the corresponding region at the Tyr T locus. In vitro and in vivo, transcription of the M1 RNA gene appears to terminate about 40 nucleotides downstream from the 3' terminus of mature M1 RNA. Therefore, an RNA processing event is involved in the biosynthesis of M1 RNA.
Termination sites of the in vitro nick-translation reaction on DNA that had photoreacted with psoralen.
Authors: Piette J G , Hearst J E
Published: May 2006

A double-stranded circular DNA having a single nick at a specific site has been photochemically induced to react with 4'-hydroxymethyl-4,5', 8-trimethylpsoralen (HMT) and used as a substrate for nick-translation with Escherichia coli DNA polymerase I holoenzyme. By using the dideoxy chain-terminating sequencing procedure, it was possible to map the termination sites observed on the template that had photoreacted with HMT. These sites occur at nucleotides preceding potential psoralen crosslinking sites. Analysis of DNA products synthesized on templates that had photoreacted under conditions designed to maximize psoralen monoaddition revealed that DNA polymerase I is not stopped by this lesion. Psoralen monoadducts situated on the template strand act only as kinetic attenuators, whereas psoralen monoadducts localized on the nick-translated strand have no effect on the rate of synthesis. These data suggest that psoralen crosslinks are responsible for the lethal effects of psoralen photochemistry in E. coli. Mutagenesis may be associated, however, with the repair replication of psoralen monoadducts by E. coli DNA polymerase I.
Dephosphorylation of a major sperm membrane protein is induced by egg jelly during sea urchin fertilization
Authors: Ward Gary E. , Vacquier Victor D.
Published: May 2006

A 160-kilodalton (kDa) protein of
Arbacia punctulata
sperm is constitutively phosphorylated on serine residues, as shown by
in vivo
32
PO
4
3-
labeling. Exposure of sperm to solubilized egg jelly results in the immediate dephosphorylation (within 5 sec) of this protein and a simultaneous increase in its electrophoretic mobility (to an apparent molecular mass of 150 kDa). In its dephosphorylated form (150 kDa), the protein is a major component of the flagellar plasma membrane. In fact, silver-stained polyacrylamide gels show the 160/150-kDa protein to be the third most abundant protein in the whole flagellum. The spermatozoan must pass through the egg jelly layer on its way to the egg surface. The jelly-induced dephosphorylation of such an abundant sperm membrane protein may be an important event in the successful interaction of sperm and egg during fertilization.
Direct evidence for interaction between human erythroid progenitor cells and a hemoglobin switching activity present in fetal sheep serum.
Authors: Stamatoyannopoulos G , Nakamoto B , Kurachi S , Papayannopoulou T
Published: May 2006

An activity that induces Hb F to Hb A switching in human cells is present in fetal sheep serum. To test directly the role of cell-to-environment interactions in hemoglobin switching and to define the level of erythroid cell differentiation at which this activity operates, colony transfer experiments were done. Clones grown in the presence of switching activity-containing medium (fetal sheep serum) or control medium (fetal calf serum) were transferred, at the 16- to 30-cell stage, to either fetal sheep serum or fetal calf serum plates and Hb F synthesis was determined in the fully mature erythroid bursts. Fetal calf serum-to-fetal calf serum transfers produced colonies with the high Hb F levels characteristic of undisturbed fetal calf serum-grown clones. Fetal sheep serum-to-fetal calf serum transfers resulted in significant decrease in Hb F synthesis, revealing an interaction between hemoglobin switching activity and cells at an early stage of progenitor cell development. The reduction of Hb F synthesis in fetal calf serum-to-fetal sheep serum transfers indicated that hemoglobin switching activity interacts with cells at later stages of progenitor cell development. Maximal decrease in Hb F synthesis was observed in fetal sheep serum-to-fetal sheep serum transfers, indicating that optimal effects on Hb switching are obtained when the environment that induces Hb switching is present throughout the development of progenitor cells. By splitting single early clones into two parts and transferring them to either a fetal sheep serum or a fetal calf serum environment, these interactions were further demonstrated in the progeny of a single erythroid burst-forming unit. Since all clone transfers were done on cell-free plates, the results of fetal calf serum-to-fetal sheep serum and of fetal sheep serum-to-fetal sheep serum transfers indicated that the switching activity does not require helper cells for its action. These studies show directly that (i) Hb F synthesis is controlled at the level of progenitors and (ii) it involves interactions between progenitor cells and their environment.
Sensory distinction between H-2b and H-2bm1 mutant mice.
Authors: Yamazaki K , Beauchamp G K , Egorov I K , Bard J , Thomas L , Boyse E A
Published: May 2006

Genetic polymorphism in the H-2:Qa:Tla region of chromosome 17 is associated with constitutive variation of bodily odor phenotypes which permit individual olfactory recognition among mice. To determine whether known genes in the H-2:Qa:Tla complex are concerned in the constitution of odor phenotypes, mice were tested for their ability to sense a difference between the B6/By (H-2b) and congeneic B6.C-H-2bm1 strains, which differ genetically by mutation of the H-2K gene. As in previous studies of the sensory discrimination of H-2:Qa:Tla phenotypes, mice were trained by reward in a Y maze to distinguish the odors of urine samples, and the successful distinctions of B6/By from B6.C-H-2bm1 were confirmed by transfer of training, without reward, to coded samples of urine from genetically equivalent urine donor mice which the trained mice had not previously encountered. Cosegregation of odor phenotype with H-2b and H-2bm1 was demonstrated by transfer of training to typed H-2b and H-2bm1 homozygous segregants of F2 generations of appropriate crosses. Although it is not excluded that the differences in odor phenotype which distinguish H-2b and H-2bm1 mice are directly related to the structure of the H-2b and H-2bm1 products, it is equally possible that H-2-related odor phenotypes arise from effects of H-2 genetic variation on metabolic pathways either directly, or indirectly through developmental polymorphism.
Identification of the C3bi receptor of human monocytes and macrophages by using monoclonal antibodies.
Authors: Wright S D , Rao P E , Van Voorhis W C , Craigmyle L S , Iida K , Talle M A , Westberg E F , Goldstein G , Silverstein S C
Published: May 2006

We have obtained four monoclonal antibodies, IB4, OKM1, OKM9, and OKM10, all directed against the C3bi receptor of human monocytes and macrophages (M phi). Two criteria were used to determine the specificity of these antibodies. First, culture surfaces coated with the antireceptor antibodies caused specific down modulation of C3bi receptor activity on M phi adherent to these substrates. Second, receptor protein purified by using IB4 or OKM1 retained the ability to bind selectively to C3bi-coated erythrocytes. Each of the antibodies recognizes a distinct epitope on the C3bi receptor; they do not compete with one another for binding to monocytes. Further, when immobilized on a solid support, each of the antibodies binds a molecule from M phi lysates that can simultaneously bind one of the other monoclonal anti-C3bi receptor antibodies. OKM10 binds and masks the ligand-binding site of the C3bi receptor, while IB4, OKM1, and OKM9 bind to sites remote from the C3bi binding site. All four antibodies immunoprecipitated polypeptides of Mr 185,000 and 105,000 from 125I-surface-labeled M phi. IB4 also precipitates polypeptides of Mr 185,000, 153,000, and 105,000. We conclude that the C3bi receptor of human M phi is a complex composed of two polypeptides, Mr 185,000 and 105,000. We have identified monoclonal antibodies reacting with four distinct antigenic determinants of this complex. The determinant recognized by antibody OKM10 is at or near the ligand-binding site of the receptor. The determinant recognized by antibody IB4 is shared by at least two other leukocyte surface proteins.
Isolation of cDNA clones for the p33 invariant chain associated with HLA-DR antigens.
Authors: Long E O , Strubin M , Wake C T , Gross N , Carrel S , Goodfellow P , Accolla R S , Mach B
Published: May 2006

HLA-DR antigens are polymorphic cell surface glycoproteins involved in the control of the immune response in man. They consist of two subunits, the alpha and the beta chains. In addition, an invariant glycoprotein of Mr 33,000 (DRp33) is associated intracellularly with HLA-DR antigens. A cDNA clone for DRp33, called 33-10, was isolated. Because no amino acid sequence has yet been determined for DRp33 the identification of cDNA clone 33-10 was based on selection of mRNA by hybridization, subsequent translation in a rabbit reticulocyte lysate supplemented with microsomes, and translation in microinjected Xenopus oocytes followed by immunoprecipitation with an anti-DR antiserum. The translation products assembled with DR alpha and beta chains in oocytes coinjected with all three mRNAs. Assembly of DR alpha and beta chains was also observed in the absence of DRp33 mRNA. Furthermore, when compared with DRp33 immunoprecipitated from a human B-cell line, translation products of the hybrid-selected mRNA showed (i) identical migration in two-dimensional gel electrophoresis, (ii) identical apparent molecular weight in the absence of N-linked glycosylation, and (iii) a very similar two-dimensional peptide map. Transcription of the DRp33 gene into a mRNA 1,400 nucleotides long was observed in B cells but was undetectable in T-cell lines and was very low in liver. Thus, DRp33 appears to be coordinately expressed with DR alpha and beta chains. Hybridization to DNA of mouse-human somatic cell hybrids showed that DRp33 is encoded by a gene that is located outside the major histocompatibility complex.
Further characterization of HLA-DS molecules: implications for studies assessing the role of human Ia molecules in cell interactions and disease susceptibility.
Authors: Goyert S M , Silver J
Published: May 2006

HLA-DS molecules, the human homologs of murine I-A molecules, were analyzed and compared to DR molecules by two-dimensional gel analysis. These analyses allowed us to define six forms of DS molecules, suggesting that DS molecules were as polymorphic as DR molecules. The supertypic specificities, MT1 and MB3 were localized to DS molecules, whereas MT3 appeared to represent a crossreactive determinant present on products of two different loci encoding Ia-like molecules in man, a DS4 molecule and a DR7-like molecule. These studies also revealed that MT1 molecules isolated from DR1 and DR2 cell lines were different, as were MB3 molecules isolated from DR4 and DR5 cell lines. Extensive crossreactions between DR and DS molecules were observed by using several monoclonal antibodies. This sharing of epitopes between products of different loci for human Ia molecules has important functional implications.
Antigenic and structural differences in the catalytic subunits of the molecular forms of acetylcholinesterase.
Authors: Doctor B P , Camp S , Gentry M K , Taylor S S , Taylor P
Published: May 2006

A mixture of the 5.6S hydrophobic dimer and the asymmetric, tail-containing (17 + 13)S forms of acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) from Torpedo californica was used to immunize mice, and spleen cells from these mice were used to produce nine hybridoma lines secreting antibodies against acetylcholinesterase. Antibodies from one of the lines showed a 100-fold greater affinity for the 5.6S species when compared with the catalytic subunits of the (17 + 13)S species. This difference in specificity was retained after denaturation of the two acetylcholinesterase species. Another line produced antibody directed only to structural subunits of the (17 + 13)S species, whereas the remaining seven antibodies exhibited nearly equivalent crossreactivity for all of the forms of acetylcholinesterase. Tryptic peptides were generated from the catalytic subunits of the 5.6S and tail-containing acetylcholinesterase species, and high-pressure liquid chromatographic profiles show at least two distinct peptides in the catalytic subunits for each enzyme species. Some of these peptides exhibit retention times different from those of the identified glycopeptides. Thus, it is likely that the catalytic subunits of two molecular forms of acetylcholinesterase differ in primary structure and sites of antigenicity.
Protein p53 and inducer-mediated erythroleukemia cell commitment to terminal cell division.
Authors: Shen D W , Real F X , DeLeo A B , Old L J , Marks P A , Rifkind R A
Published: May 2006

Inducer-mediated murine erythroleukemia cell (MELC) differentiation provides a model for examining factors determining terminal cell differentiation. The nuclear protein, p53, has been implicated as a potential determinant of cell cycle progression and cell differentiation. In this study p53 content and synthesis, during inducer-mediated MELC differentiation, has been examined with monoclonal antibodies to p53. A decrease in p53 synthesis and content was demonstrated during induced differentiation. As determined by cell cycle fractionation, the decrease in p53 is manifest at all stages of the cell cycle. Hemin, which induces globin mRNA accumulation but not terminal cell division, fails to decrease p53 content. A MELC variant resistant to inducer-mediated commitment to terminal cell division also fails to decrease p53 levels in response to inducers. These experiments suggest that p53 is implicated in MELC cell proliferation and that an induced decrease in p53 may be responsible for G1 phase prolongation and terminal G1 arrest.
Molecular genetics of human serum albumin: restriction enzyme fragment length polymorphisms and analbuminemia.
Authors: Murray J C , Demopulos C M , Lawn R M , Motulsky A G
Published: May 2006

By using cDNA probes for the human albumin gene, four restriction enzyme fragment length polymorphisms (RFLPs) were discovered that were transmitted by codominant autosomal inheritance. Among Caucasians, the gene frequencies were 0.04/0.96 for Msp I/5', 0.43/0.57 for Hae III/3', 0.44/0.56 for Hae III/5', and 0.04/0.42/0.54 for Pst I/5'. These common variants provide a marker for chromosome 4 (q11-q13). A calculation of the extent of DNA variation at the albumin locus revealed that 1/95 nucleotide sites was affected by a RFLP, a figure similar to that found in the globin system. Restriction enzyme fragment study of the DNA of a human analbuminemic individual revealed no gross structural rearrangements of the albumin locus. The exact nature of abnormality will require more study.
Purification and partial sequence analysis of human T-cell growth factor.
Authors: Robb R J , Kutny R M , Chowdhry V
Published: May 2006

A murine monoclonal antibody directed against human T-cell growth factor (TCGF) from the JURKAT cell line was used for affinity column purification of the factor. Bound TCGF was eluted nearly quantitatively at low pH, and the recovered factor appeared homogeneous by two-dimensional gel electrophoresis. The molecule is markedly hydrophobic, with a high content of leucine. A single NH2-terminal sequence of 36 residues was obtained by automated Edman degradation, further supporting the homogeneity of the material. Thus, significant quantities of purified TCGF have been prepared in a single step, making possible detailed analysis of its molecular structure and biological role.
Adenovirus 2 early gene expression promotes susceptibility to effector cell lysis of hybrids formed between hamster cells transformed by adenovirus 2 and simian virus 40.
Authors: Cook J L , Hauser J , Patch C T , Lewis A M , Levine A S
Published: May 2006

Weakly oncogenic adenovirus 2 (Ad2)-transformed LSH hamster cells are sensitive to lysis by spontaneously cytolytic lymphoid cells and activated macrophages, whereas highly oncogenic simian virus 40 (SV40)-transformed LSH cells are relatively resistant to these nonspecific effector cells. Somatic cell hybrids formed between Ad2- and SV40-transformed hamster cells, which expressed Ad2 tumor (T) antigens, exhibited an increased cytolytic susceptibility compared to Ad2 T antigen-negative cell hybrids or nonhybrid SV40-transformed cells. No correlation was found between the expression of SV40 T antigen in hybrid cells and cytolytic susceptibility. The results suggest the existence of a novel function for early Ad2 genome-encoded polypeptides (T antigens) expressed in transformed hamster cells--the induction of susceptibility to destruction mediated by immunologically nonspecific effector cells.
Tangier disease: defective recombination of a specific Tangier apolipoprotein A-I isoform (pro-apo A-i) with high density lipoproteins.
Authors: Schmitz G , Assmann G , Rall S C , Mahley R W
Published: May 2006

Isoforms of apolipoprotein A-I (apo A-I) from subjects with Tangier disease were characterized, and their ability to recombine with normal high density lipoproteins (HDL) was studied. In contrast to normal serum, in which isoprotein 4 is the dominant species [79 +/- 1.8% (mean +/- SD)], the Tangier serum contained much less total apo A-I (approximately equal to 1% of that in normal serum), and isoproteins 2 and 4 were present in roughly equivalent amounts (35.3 +/- 2.5% and 42.7 +/- 3.6%, respectively). The Tangier isoprotein 2 was shown to correspond to pro-apo A-I, having a six-amino acid amino-terminal extension with the sequence: Arg-His-Phe-Trp-Gln-Gln-. The Tangier isoprotein 4 had the same amino-terminal sequence as normal circulating plasma apo A-I. Its association with normal HDL (70%) was similar to the association of normal apo A-I with HDL (80-90%) in recombination experiments. In marked contrast to this behavior, very little (less than 10%) of Tangier isoprotein 2 (pro-apo A-I) associated with HDL in recombination experiments. These results suggest that the underlying defect in Tangier disease may be a faulty conversion of pro-apo A-I to mature apo A-I, either due to a defect in the converting enzyme activity or to a further specific structural defect in Tangier apo A-I. The failure of Tangier pro-apo A-I to associate with HDL may be at least partially responsible for the HDL deficiency in Tangier subjects.
Secretion of lipoproteins from the liver of normal and Watanabe heritable hyperlipidemic rabbits.
Authors: Hornick C A , Kita T , Hamilton R L , Kane J P , Havel R J
Published: May 2006

We compared the rate of accumulation of lipoproteins in perfusates of isolated livers from normal New Zealand White rabbits and Watanabe heritable hyperlipidemic (WHHL) rabbits, in which a gene mutation has produced a virtually complete deficiency of low density lipoprotein (LDL) receptors. The rate of accumulation of apolipoprotein B-100 did not differ in perfusates of livers from normal and mutant animals and little or no apolipoprotein B-48 was detected. In both groups, virtually all apolipoprotein B accumulated in very low density lipoprotein (VLDL). Experiments in which [3H]lysine was added to the perfusates showed that the apolipoprotein B that accumulated in VLDL was newly synthesized by the liver whereas the small amount of apolipoprotein B found in lipoproteins of higher density appeared to be washed out of extravascular spaces during perfusion. Perfusate VLDL from both groups contained more triglycerides and less cholesteryl esters than their counterparts from blood plasma. As compared with perfusate VLDL from normal livers, those from livers of WHHL rabbits were enriched in cholesteryl esters. Experiments in which Triton WR-1339 was injected into the blood of intact rabbits confirmed the observations with perfused livers. Previous studies have shown that the extent to which VLDL is converted to LDL is increased several-fold in WHHL rabbits. Taken together with our present results, which fail to provide evidence for increased secretion of apolipoprotein B or de novo secretion of lipoproteins other than VLDL that contain apolipoprotein B, it can be concluded that overproduction of LDL in rabbits lacking LDL receptors is solely the result of altered metabolism of VLDL.
Molecular cloning of double-stranded RNA virus genomes.
Authors: Imai M , Richardson M A , Ikegami N , Shatkin A J , Furuichi Y
Published: May 2006

Genome double-stranded RNAs isolated from purified human reovirus (serotype 3) and rotavirus (Wa strain) were modified at the 3' termini by addition of oligo(C) approximately 15 with T4 RNA ligase. These RNAs were transcribed into cDNA by oligo(dG)-primed reverse transcriptase and cloned after insertion into pBR322 at the Pst I site. Hybridization of plasmid-transformed Escherichia coli RR1 colonies with 32P-labeled viral genome RNAs demonstrated the presence of DNA clones representative of each of the 10 reovirus RNAs and 10 of the 11 constituent segments of the rotavirus genome. Analyses of the size and terminal nucleotide sequences of insert DNAs indicated that some clones contained a full-length copy of the virus genome segment. The complete nucleotide sequence of rotavirus genome segment 11 double-stranded RNA was obtained by using this procedure. It provides a general method for cloning double-stranded RNAs and also nonpolyadenylylated single-stranded RNAs.
Isolation of plasma membrane domains from murine T lymphocytes.
Authors: Hoessli D C , Rungger-Brändle E
Published: May 2006

Murine T-lymphoma cells have been homogenized in dense sucrose solution and centrifuged under isopycnic conditions for membrane components. Floating fractions banding between 10% and 22.5% sucrose ("light" membranes) and between 22.5% and 35% sucrose ("heavy" membranes) were shown to consist of smooth membrane vesicles. The amounts of cholesterol and phospholipids recovered after chloroform/methanol extraction were similar in both fractions, but heavy membranes contained two to three times more protein than light membranes. The most striking difference between the two membrane fractions was revealed by their labeled surface glycoprotein patterns on polyacrylamide gels, suggesting that (i) the smooth membrane vesicles originated from the plasma membrane and (ii) two distinct segments of the plasma membrane can be recovered in fractions characterized by specific surface glycoproteins. Light membranes were enriched in Thy-1 antigen, whereas Ly-5 antigen and a 170,000-dalton surface glycoprotein were recovered almost exclusively from heavy membranes, as were metabolically labeled protein spots comigrating with the H-2k/d antigen in two-dimensional electrophoresis. The patterns of the unlabeled proteins in light and heavy membranes appeared similar, except for polypeptides of 180,000 and 85,000 daltons that were found preferentially in heavy membranes. These results support the concept of plasma membrane domains by showing that two distinct populations of plasma membrane vesicles can be isolated and that these populations contain different sets of cell surface glycoproteins.
Induction of leukemia by both fractionated x-irradiation and radiation leukemia virus involves loci in the chromosome 2 segment H-30-A.
Authors: Meruelo D , Offer M , Rossomando A
Published: May 2006

A common link between the induction of leukemia by (i) fractionated doses of x-irradiation and (ii) radiation leukemia virus in mice may be established by the observation that the segment of chromosome 2 between the loci for the minor histocompatibility antigen H-30 and color coat agouti (H-30-A) includes distinct loci involved in susceptibility to leukemogenesis induced by factors i and ii.
Multiple DNA fragment polymorphisms associated with immunoglobulin mu chain switch-like regions in man.
Authors: Migone N , Feder J , Cann H , van West B , Hwang J , Takahashi N , Honjo T , Piazza A , Cavalli-Sforza L L
Published: May 2006

DNA probes containing the switch region (S) associated with the human immunoglobulin heavy chain mu gene were used to investigate polymorphisms in the germ-line human DNA. Six polymorphisms, detected by a single restriction enzyme (Sst I) are described. Linkage studies in 29 families show that five of the six polymorphisms, although relatively unassociated in random individuals, segregate in complete linkage one to the other and to Gm allotypes (markers on the heavy chain of IgG), while the sixth segregates independently. Altogether, when one considers the DNA markers at the five closely linked loci and the IgG1 and IgG3 heavy chain allotypes, 33 different haplotypes have been described; of these, 28 are detected by the DNA polymorphism alone. Study of 158-187 random haplotypes showed strong linkage disequilibrium only between one DNA polymorphism (Sst A) and Gm. Of the polymorphic Sst I loci, one, Sst E [associated with 2.2- to 2.7-kilobase (kb) fragments], is included in the mu chain S region (S mu); another, Sst A (6.8-7.4 kb), must be very close to the gamma 1-gamma 3 chain gene cluster. Based on studies of an IgE human myeloma, a third polymorphism, Sst C (4.8-5.5 kb), should map 3' of the active epsilon chain gene. An Sst I restriction enzyme map of phage clones carrying the two alpha chain genes indicates that Sst A and Sst C loci probably overlap with the alpha 1 and alpha 2 S regions, respectively. Both deletion/duplications and point mutations were detected.
Evolution of altruistic behavior by kin selection: an alternative approach.
Authors: Engels W R
Published: May 2006

The evolution of altruistic and selfish behavior by kin selection has been analyzed previously by asking which types of behavior are favored by natural selection. A type of behavior is defined as a given cost/benefit ratio, c/b, in terms of Darwinian fitness. An alternative approach is to consider c/b itself as a quantitative character subject to natural selection and evolving toward an equilibrium. This approach allows consideration of a continuum of behavioral options as opposed to just two alternatives as in the previous work. Environmental restrictions on the availability of opportunities for fitness transactions are imposed so that the total benefit an individual can provide for or take from his relatives increases with his c/b ratio. General conditions for stable equilibria are derived. These depend only on the coefficient of relationship between donor and recipient (r) and the function describing the availability of benefit. They are independent of the heritability and variance in the population. Under weak selection, the equilibrium c/b ratio will be r/2 for altruistic behavior and 1/(2r) for selfish behavior. By contrast, standard theory predicts that all altruistic acts with c/b ratios less than r will be favored, and no equilibrium can be predicted except under certain special conditions where only two behavioral options are available. In general, these results show that evolution of the donor's behavior (altruism) tends to maximize the quantity rb--c, and evolution of the recipient (selfish behavior) maximizes b--rc.
Association arrays in assessing forms of dependencies between bivariate random variables
Authors: Karlin Samuel
Published: May 2006

The bivariate distribution of pairs of random variables (
X,Y
) is said to be associated with respect to the classes of functions [unk] and [unk] if the product-moment correlation
r
[Φ(
X
),Ψ(
Y
)] ≥ 0 for all Φ € [unk] and Ψ € [unk]. In the case in which both [unk] = [unk] = [unk]
*
consist of all increasing functions, then the bivariate distribution of (
X,Y
) is said to be positive quadrant dependent. To apply the concept to data, I examine the correlations for classes of extremal functions that span by positive combinations the totality of functions Φ € [unk] and Ψ € [unk] to investigate whether the pair of random variables (
X,Y
) are associated with respect to [unk] and [unk] and to assess the relative degree (or strength) of association when comparing two sets of random variables (
X,Y
) and (
Z,W
).
Purification of photochemically active halorhodopsin.
Authors: Taylor M E , Bogomolni R A , Weber H J
Published: May 2006

We have developed a procedure for the purification of halorhodopsin in a photochemically active state. Solubilization of membranes from a bacteriorhodopsin-negative Halobacterium strain with octyl glucoside was followed by chromatography on hydroxylapatite and octyl-Sepharose gels. All steps were carried out in high-ionic-strength solutions. The procedure resulted in 270-fold enrichment with a 35% yield. The eluted pigment had an absorption maximum at 575 nm and an A280/A575 ratio of 2. On removal of the detergent by dialysis, the purified halorhodopsin was chemically bleached, regenerated with [3H]retinal, and reduced with cyanoborohydride. Such samples showed one main and one satellite band after staining or fluorography of NaDodSO4/polyacrylamide gels. The apparent molecular weight of the main band was 25,000. Purified halorhodopsin underwent a photocycle after excitation with pulsed laser light and showed a 9-nm blue shift (at neutral pH) on removal of chloride ion. The pigment also underwent a photoreversible shift at alkaline pH to a form absorbing maximally at 410 nm. All three reactions closely resembled those of membrane-bound halorhodopsin.
Perturbation of lac operator DNA modification by tryptic core protein from lac repressor.
Authors: Manly S P , Bennett G N , Matthews K S
Published: May 2006

Operator DNA fragments were modified in the presence of lac repressor protein or its trypsin-resistant core. Operator DNA was alkylated or cleaved enzymatically with these related proteins present to compare the influences of their binding on the reactivities or enzymatic susceptibilities of individual bases in the sequence. These two protein species have pronounced and distinguishable effects on the reactivity of the bases of the operator fragment toward methylation by dimethyl sulfate. Perturbation of base alkylation by the trypsin-resistant core repressor is most pronounced in the inner, asymmetric region of the operator DNA, while repressor effects extend further on either end of the operator sequence. Digestion of the two protein-operator complexes by DNase I yields fragment patterns that differ primarily in extent of protection. These data extend the experimental base supporting the involvement of the core region of the lac repressor in addition to its NH2 termini in the operator-specific binding activity of this protein.
Structural analysis of antigen-specific Ia-bearing regulatory T-cell factors: gel electrophoretic analysis of the antigen-specific augmenting T -cell factor.
Authors: Miyatani S , Hiramatsu K , Nakajima P B , Owen F L , Tada T
Published: May 2006

An antigen-specific T-cell factor (TaF) that specifically augments the antibody response was purified and biochemically analyzed by NaDodSO4/polyacrylamide gel electrophoresis and isoelectric focusing. Biosynthetically labeled TaF was separated from the Nonidet P-40 extract of T-cell hybridoma FL10, which produces a keyhole limpet hemocyanin-specific TaF, by affinity chromatography either with antigen or with monoclonal anti-I-A antibodies. The material thus obtained was composed of two different types of molecules of molecular weights of 67,000 and 33,000 under nonreducing conditions. After reduction with dithiothreitol, all the molecules migrated to the position of molecular weight 33,000. The absorption studies with immunoadsorbents of antigen and antibodies revealed that the intact TaF is a heterodimer of two discrete polypeptide chains, one carrying a determinant detectable by a monoclonal anti-Tindd directed to an Igh-I -linked allotypic structure of T cells and being associated with the antigen-binding site and the other expressing a unique determinant controlled by the I-A subregion of murine H-2 major histocompatibility complex but being different from known class II polypeptide chains. The antigen-binding polypeptide has an isoelectric point of pH 5.6, and the I-A polypeptide has an isoelectric point of pH 6.3.