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Biomedical subjects

W Gilbert

Publications and source records attributed to W Gilbert.

At least 91 records · Page 5Linked to original sources
PubMed1989

The monoclonal antibody B30 recognizes a specific neuronal cell surface antigen in the developing mesencephalic trigeminal nucleus of the mouse.

A monoclonal antibody, B30, obtained with whole cells from embryonic brain as an immunogen, recognizes a neuronal cell surface antigen that appears only in 2 distinct systems in the developing mouse brain: the trigeminal system and the cerebellum. In the trigeminal system, B30 labels the surface of neurons, including their axons and their transient dendrites, in 2 groups of cells: the centrally located mesencephalic trigeminal nucleus and the peripheral trigeminal ganglion. Immunoreactivity is detectable during axon outgrowth, peaks around the seventh postnatal day, and disappears around 2 weeks after birth. In the cerebellum, B30 labels 2 layers of cells during development. Perinatally, and for about a week after birth, the layer of premigratory granule cells stains. After their maturation, Purkinje cells start to stain and by 12 d postnatally all the Purkinje cell bodies, their axons, and their dendritic trees show strong immunoreactivity. Subsequently, and in the adult, this staining is lost from some cells to reveal bands of antigen positive and negative Purkinje cells. Initial biochemical characterization of the epitope shows that it is carried on 2 minor gangliosides.

Animals↗
PubMed1988

Formation of parallel four-stranded complexes by guanine-rich motifs in DNA and its implications for meiosis.

We have discovered that single-stranded DNA containing short guanine-rich motifs will self-associate at physiological salt concentrations to make four-stranded structures in which the strands run in parallel fashion. We believe these complexes are held together by guanines bonded to each other by Hoogsteen pairing. Such guanine-rich sequences occur in immunoglobulin switch regions, in gene promoters, and in chromosomal telomeres. We speculate that this self-recognition of guanine-rich motifs of DNA serves to bring together, and to zipper up in register, the four homologous chromatids during meiosis.

Base Sequence↗
PubMed1987

Basic protein enhances the incorporation of DNA into lipid vesicles: model for the formation of primordial cells.

DNA can be encapsulated into lipid vesicles formed by sonication. The presence of a basic protein, lysozyme, enhances the incorporation 100-fold above the level expected by random trapping. This is demonstrated by the ability of the lipid vesicles to protect DNA from digestion with DNase. Such an enhancement of nuclei acid incorporation into vesicles by basic polypeptides and the sharply increased concentration of these macromolecules in the internal volume may have been advantageous in prebiotic evolution.

Biological Evolution↗
PubMed1986

Reaction energetics of a mutant triosephosphate isomerase in which the active-site glutamate has been changed to aspartate.

The essential catalytic base at the active site of the glycolytic enzyme triosephosphate isomerase is the carboxylate group of Glu-165, which directly abstracts either the 1-pro-R proton of dihydroxyacetone phosphate or the 2-proton of (R)-glyceraldehyde 3-phosphate to yield the cis-enediol intermediate. Using the methods of site-directed mutagenesis, we have replaced Glu-165 by Asp. The three enzymes chicken isomerase from chicken muscle, wild-type chicken isomerase expressed in Escherichia coli, and mutant (Glu-165 to Asp) chicken isomerase expressed in E. coli have each been purified to homogeneity. The specific catalytic activities of the two wild-type isomerases are identical, while the specific activity of the mutant enzyme is reduced by a factor of about 1000. The observed kinetic differences do not derive from a change in mechanism in which the aspartate of the mutant enzyme acts as a general base through an intervening water molecule, because the D2O solvent isotope effects and the stoichiometries of inactivation with bromohydroxyacetone phosphate are identical for the wild-type and mutant enzymes. Using the range of isotopic experiments that were used to delineate the free-energy profile of the wild-type chicken enzyme, we here derive the complete energetics of the reaction catalyzed by the mutant protein. Comparison of the reaction energetics for the wild-type and mutant isomerases shows that only the free energies of the transition states for the two enolization steps have been seriously affected. Each of the proton abstraction steps is about 1000-fold slower in the mutant enzyme. Evidently, the excision of a methylene group from the side chain of the essential glutamate has little effect on the free energies of the intermediate states but dramatically reduces the stabilities of the transition states for the chemical steps in the catalyzed reaction.

Aspartic Acid↗
PubMed1986

The triosephosphate isomerase gene from maize: introns antedate the plant-animal divergence.

We have cloned and characterized a cDNA and genomic DNA for the triosephosphate isomerase expressed in maize roots. The gene is interrupted by eight introns. If we compare this gene with that for the protein in chicken, which has six introns, we see that five of the introns are at identical places, one has shifted by three codons, and two are totally new. This great matching leads us to conclude that the introns were in place before the plant-animal divergence, and that the parental gene had at least eight introns, two of which were lost in the line that leads to animals.

Animals↗
PubMed1985

B lineage--specific interactions of an immunoglobulin enhancer with cellular factors in vivo.

The mouse heavy chain immunoglobulin gene contains a tissue-specific enhancer. The enhancer and flanking sequences were studied in vivo by carrying out dimethyl sulfate protection experiments on living cells, in combination with genomic sequencing. Relative to reactions on naked DNA, there are changes (protections and enhancements) in the reactivity of guanine residues to dimethyl sulfate within the enhancer sequence in myeloma, B, and early B cells, whereas virtually no alterations appear in cells of non-B lineage. Most of the affected residues are in four clusters, in sequences homologous to the octamer 5'CAGGTGGC 3' (C, cytosine; A, adenine; G. guanine; T, thymine). The alterations in the pattern of G reactivity are consistent with the tissue-specific binding of molecules to the mouse immunoglobulin heavy chain enhancer.

Animals↗
PubMed1985

d-Fenfluramine selectively suppresses carbohydrate snacking by obese subjects.

Twenty obese inpatients who claimed to crave carbohydrate-rich foods were given d-fenfluramine (15 mg p.o., twice daily) or its placebo, double-blind, for two consecutive eight-day periods. Food choices were measured on treatment days 1, 7, and 8 by giving the subjects access to unlimited portions of six isocaloric meal foods (three high in carbohydrate and three high in protein) and of 10 isocaloric snack foods (five high in protein and five high in carbohydrate) available 24 hours a day in a computerized vending machine. d-fenfluramine reduced mealtime calorie intake by only 16% (from 1940 +/- 94 to 1630 +/- 92; p < .001), mealtime carbohydrate by 22%, and had no significant effect on mealtime protein consumption; in contrast, snack calorie intake was reduced by 41% (from 707 +/- 97 to 414 +/- 46; p < .001), and snack carbohydrate intake by the same proportion. The mean number of carbohydrate-rich snacks consumed per day decreased from 5.8 +/- 0.8 to 3.4 +/- 0.4 (p < .01), while that of protein-rich snacks failed to change signficantly (i.e., from 0.7 +/- 0.2 to 0.5 +/- 0.2).

Adult↗
PubMed1985

Rous sarcoma virus encodes a transcriptional activator.

Rous sarcoma virus expresses a transcriptional activator that affects the LTR as well as other promoters. We discern this activity as a stimulation of the transient expression of an LTR-promoted hybrid transcriptional unit and also of the rat preproinsulin II gene in transfected NIH 3T3 cells. We map the activity to an alternate reading frame in the p19-p10 region of the gag gene and identify a mRNA whose spliced structure would direct translation of this reading frame from the Pr76gag initiation codon. This mRNA probably differs from genomic RNA only by the 282 nucleotide splice. The predicted translation product is a 124 residue polypeptide; the first six amino acids arise from gag. The target for the action of this transcriptional modulator at the LTR lies between 111 and 620 nucleotides upstream of the cap site.

Animals↗
PubMed1985

Intron/exon structure of the chicken pyruvate kinase gene.

The chicken pyruvate kinase gene is interrupted by at least ten introns, including nine introns within the coding region. We compare the structure of this gene with the three-dimensional protein structure of the homologous cat muscle enzyme. The introns are not randomly placed--they divide the coding sequence into fairly uniformly sized pieces encoding discrete elements of secondary structure. The introns tend to fall at interruptions between stretches of alpha-helix or beta-sheet residues, and each of the six exons that contribute to the barrel-shaped central domain include one or two repeats of a simple unit, an alpha-helix plus a beta strand. This structure suggests that introns were not inserted into a previously uninterrupted coding sequence, but instead are products of the evolution of the first pyruvate kinase gene. We have found some sequence homology between a segment of pyruvate kinase and the structurally homologous mononucleotide binding fold of alcohol dehydrogenase. The superposition of these two regions aligns an intron from the maize alcohol dehydrogenase gene four nucleotides from an intron in the chicken pyruvate kinase gene.

Amino Acid Sequence↗
PubMed1985

Chicken triosephosphate isomerase complements an Escherichia coli deficiency.

We present the sequence of full-length chicken triosephosphate isomerase (D-glyceraldehyde 3-phosphate ketol-isomerase, EC 5.3.1.1) mRNA based on the analysis of cDNA and genomic clones. To isolate cDNA clones encoding the enzyme, we screened a muscle cDNA library with radioactively labeled cDNA made from RNA that had been enriched by immunoselection of polysomes. We blocked the signal caused by contaminating species in the probe with cloned DNA corresponding to the contaminants. Screening a chicken genomic library with cDNA coding for triosephosphate isomerase led to the isolation of phage containing the entire gene, which we used to map the transcriptional start. When placed downstream from a hybrid trp-lac promoter, the cDNA encoding the chicken enzyme programs the synthesis of functional protein, as judged by enzymatic criteria and by complementation of an Escherichia coli mutant that is deficient in bacterial triosephosphate isomerase.

Animals↗
PubMed1985

Active site of triosephosphate isomerase: in vitro mutagenesis and characterization of an altered enzyme.

We have replaced the glutamic acid-165 at the active site of chicken triosephosphate isomerase with an aspartic acid residue using site-directed mutagenesis. Expression of the mutant protein in a strain of Escherichia coli that lacks the bacterial isomerase results in a complementation phenotype that is intermediate between strains that have no isomerase and strains that produce either the wild-type chicken enzyme or the native E. coli isomerase. The value of kcat for the purified mutant enzyme when glyceraldehyde 3-phosphate is the substrate is 1/1500th that of the wild-type enzyme, and the Km is decreased by a factor of 3.6. With dihydroxyacetone phosphate as substrate, the kcat value is 1/240th that of the wild-type enzyme, and Km is 2 times higher. The value of Ki for a competitive inhibitor, phosphoglycolate, is the same for the mutant and wild-type enzymes, at 2 X 10(-5) M. By treating the enzyme-catalyzed isomerization as a simple three step process and assuming that substrate binding is diffusion limited, it is evident that the mutation of glutamic acid-165 to aspartic acid principally affects the free energy of the transition state(s) for the catalytic reaction itself.

Animals↗
PubMed1984

Antibodies of the secondary response can be expressed without switch recombination in normal mouse B cells.

A few percent of mouse splenocytes express isotypes characteristic of the secondary response together with IgM, and some cells express these isotypes alone. We isolated populations of small memory cells that express (i) IgM but not IgG1, (ii) IgM but not IgA, (iii) IgM and IgG1, (iv) IgM and IgA, and (v) IgG1 but not IgM. We have analyzed their DNA to show that there has been no switch recombination or deletion in the Ig constant region (C) genes. Using sandwich RNA hybridizations, we have found that cells expressing IgG1 contain nuclear RNAs that have both C mu and C gamma 1 sequences, and that cells expressing IgA contain nuclear RNAs that have both C mu and C alpha sequences. We propose that the expression of an isotype characteristic of the secondary response in memory cells is accomplished by alternative RNA processing of large (up to 180 kilobases) nuclear RNA transcripts that span the heavy chain gene locus.

Animals↗