bluuebunny/crossref_metadata_2025 huggingface dataset:
Plasmid Detection and Sizing in Single Colony Lysates
Authors: Barnes Wayne M.
Published: October 2006

A simple and contained procedure for the rapid assay of the presence and size of plasmids similar to Col E1 is described. Bacteria are picked from an agar plate with a toothpick, lysed with dodecyl sulfate and heat, and placed directly on an agarose gel for electrophoresis.
Autologous Peptides Constitutively Occupy the Antigen Binding Site on Ia
Authors: Buus Søren , Sette Alessandro , Colon Sonia M. , Grey Howard M.
Published: October 2006

Low molecular weight material associated with affinity-purified class II major histocompatibility complex (MHC) molecules of mouse (Ia) had the expected properties of peptides bound to the antigen binding site of Ia. Thus, the low molecular weight material derived from the I-A
d
isotype was efficient in inhibiting the binding of
125
I-labeled I-A
d
-specific peptide to I-A
d
, but did not significantly inhibit the binding of an I-E
d
-specific peptide to I-E
d
; the reciprocal isotype-specific inhibition was demonstrated with low molecular weight material derived from I-E
d
. The inhibitory material was predominantly peptide in nature, as shown by its susceptibility to protease digestion. It was heterogeneous as measured by gel filtration (mean molecular weight ∼3000), and when characterized by high-performance liquid chromatography, it eluted over a wide concentration of solvent. Such self peptide-MHC complexes may have broad significance in the biology of T cell responses, including generation of the T cell repertoire, the specificity of mixed lymphocyte responses, and the immune surveillance of self and nonself antigens in peripheral lymphoid tissues.
A Specific, Highly Active Malate Dehydrogenase by Redesign of a Lactate Dehydrogenase Framework
Authors: Wilks Helen M. , Hart Keith W. , Feeney Raymond , Dunn Cameron R. , Muirhead Hilary , Chia William N. , Barstow David A. , Atkinson Tony , Clarke Anthony R. , Holbrook J. John
Published: October 2006

Three variations to the structure of the nicotinamide adenine dinucleotide (NAD)-dependent L-lactate dehydrogenase from
Bacillus stearothermophilus
were made to try to change the substrate specificity from lactate to malate: Asp
197
→ Asn, Thr
246
→ Gly, and Gln
102
→ Arg). Each modification shifts the specificity from lactate to malate, although only the last (Gln
102
→ Arg) provides an effective and highly specific catalyst for the new substrate. This synthetic enzyme has a ratio of catalytic rate (
k
cat
) to Michaelis constant (
K
m
) for oxaloacetate of 4.2 x 10
6
M
-1
s
-1
, equal to that of native lactate dehydrogenase for its natural substrate, pyruvate, and a maximum velocity (250 s
-1
), which is double that reported for a natural malate dehydrogenase from
B. stearothermophilus
.
Suppression of the Neoplastic Phenotype by Replacement of the RB Gene in Human Cancer Cells
Authors: Huang Huei-Jen Su , Yee Jiing-Kuan , Shew Jin-Yuh , Chen Phang-Lang , Bookstein Robert , Friedmann Theodore , Lee Eva Y.-H. P. , Lee Wen-Hwa
Published: October 2006

Mutational inactivation of the retinoblastoma susceptibility (RB) gene has been proposed as a crucial step in the formation of retinoblastoma and other types of human cancer. This hypothesis was tested by introducing, via retroviral-mediated gene transfer, a cloned RB gene into retinoblastoma or osteosarcoma cells that had inactivated endogenous RB genes. Expression of the exogenous RB gene affected cell morphology, growth rate, soft agar colony formation, and tumorigenicity in nude mice. This demonstration of suppression of the neoplastic phenotype by a single gene provides direct evidence for an essential role of the RB gene in tumorigenesis.
Grafting Genetically Modified Cells to the Damaged Brain: Restorative Effects of NGF Expression
Authors: Rosenberg Michael B. , Friedmann Theodore , Robertson Robin C. , Tuszynski Mark , Wolff Jon A. , Breakefield Xandra O. , Gage Fred H.
Published: October 2006

Fibroblasts were genetically modified to secrete nerve growth factor (NGF) by infection with a retroviral vector and then implanted into the brains of rats that had surgical lesions of the fimbria-fornix. The grafted cells survived and produced sufficient NGF to prevent the degeneration of cholinergic neurons that would die without treatment. In addition, the protected cholinergic cells sprouted axons that projected in the direction of the cellular source of NGF. These results indicate that a combination of gene transfer and intracerebral grafting may provide an effective treatment for some disorders of the central nervous system.
Localization of an Arg-Gly-Asp Recognition Site Within an Integrin Adhesion Receptor
Authors: D'Souza Stanley E. , Ginsberg Mark H. , Burke Timothy A. , Lam Stephen C.-T. , Plow Edward F.
Published: October 2006

Many adhesive interactions are mediated by Arg-Gly-Asp (RGD) sequences within adhesive proteins. Such RGD sequences are frequently recognized by structurally related heterodimers that are members of the integrin family of adhesion receptors. A region was found in the platelet RGD receptor, gpIIb/IIIa, to which an RGD peptide becomes chemically cross-linked. This region corresponds to residues 109 to 171 of gpIIIa. This segment is conserved among the β subunits of the integrins (76 percent identity of sequence), indicating that it may play a role in the adhesive functions of this family of receptors.
Perivascular Microglial Cells of the CNS Are Bone Marrow-Derived and Present Antigen in Vivo
Authors: Hickey William F. , Kimura Hiromitsu
Published: October 2006

A crucial question in the study of immunological reactions in the central nervous system (CNS) concerns the identity of the parenchymal cells that function as the antigen-presenting cells in that organ. Rat bone marrow chimeras and encephalitogenic, major histocompatability-restricted T-helper lymphocytes were used to show that a subset of endogenous CNS cells, commonly termed "perivascular microglial cells," is bone marrow-derived. In addition, these perivascular cells are fully competent to present antigen to lymphocytes in an appropriately restricted manner. These findings are important for bone marrow transplantation and for neuroimmunological diseases such as multiple sclerosis.
Evidence from Cassette Mutagenesis for a Structure-Function Motif in a Protein of Unknown Structure
Authors: Clarke Neil D. , Lien Donald C. , Schimmel Paul
Published: October 2006

The three-dimensional structure of most enzymes is unknown; however, many enzymes may have structural motifs similar to those in the known structures of functionally related enzymes. Evidence is presented that an enzyme of unknown structure [Ile-transfer RNA (tRNA) synthetase] may share a functionally important structural motif with an enzyme of related function (Tyr-tRNA synthetase). This approach involves (i) identifying segments of Ile-tRNA synthetase that have been unusually conserved during evolution, (ii) predicting the function of one such segment by assuming a structural relation between Ile-tRNA synthetase and Tyr-tRNA synthetase, and (iii) testing the predicted function by mutagenesis and subsequent biochemical analysis. Random mutations were introduced by cassette mutagenesis into a ten-amino-acid segment of Ile-tRNA synthetase that was predicted to be involved in the formation of the binding site for isoleucine. Few amino acid substitutions appear to be tolerated in this region. However, one substitution (independently isolated twice) increased the Michaelis constant
K
m
for isoleucine in the adenylate synthesis reaction by greater than 6000-fold, but had little effect on the
K
m
for adenosine triphosphate, the apparent
K
m
for tRNA, or the rate constant
k
cat
.
Localization of Cognitive Operations in the Human Brain
Authors: Posner Michael I. , Petersen Steven E. , Fox Peter T. , Raichle Marcus E.
Published: October 2006

The human brain localizes mental operations of the kind posited by cognitive theories. These local computations are integrated in the performance of cognitive tasks such as reading. To support this general hypothesis, new data from neural imaging studies of word reading are related to results of studies on normal subjects and patients with lesions. Further support comes from studies in mental imagery, timing, and memory.
Regulation of Inducible and Tissue-Specific Gene Expression
Authors: Maniatis Tom , Goodbourn Stephen , Fischer Janice A.
Published: October 2006

Molecular genetics approaches have been used to identify and characterize cis-acting DNA sequences required for eukaryotic gene regulation. These sequences are modular in nature, consisting of arrays of short (10- to 12-base pair) recognition elements that interact with specific transcription factors. Some transcription factors have been extensively purified and the corresponding genes have been cloned, but the mechanisms by which they promote transcription are not yet understood. Positive and negative regulatory elements that function only in specific cell types or in response to extracellular inducers have been identified. A number of cases of inducible and tissue-specific gene expression involve the activation of preexisting transcription factors, rather than the synthesis of new proteins. This activation may involve covalent modification of the protein or an allosteric change in its structure. The modification of regulatory proteins may play a central role in the mechanisms of eukaryotic gene regulation.
Localization of Amyloid β Protein Messenger RNA in Brains from Patients with Alzheimer's Disease
Authors: Bahmanyar Sina , Higgins Gerald A. , Goldgaber Dmitry , Lewis David A. , Morrison John H. , Wilson Michael C. , Shankar Susarla K. , Gajdusek D. Carleton
Published: October 2006

The distribution of cells containing messenger RNA that encodes amyloid β protein was determined in hippocampi and in various cortical regions from cynomolgus monkeys, normal humans, and patients with Alzheimer's disease by in situ hybridization. Both
35
S-labeled RNA antisense and sense probes to amyloid β protein messenger RNA were used to ensure specific hybridization. Messenger RNA for amyloid β protein was expressed in a subset of neurons in the prefrontal cortex from monkeys, normal humans, and patients with Alzheimer's disease. This messenger RNA was also present in the neurons of all the hippocampal fields from monkeys, normal humans and, although to a lesser extent in cornu ammonis 1, patients with Alzheimer's disease. The distribution of amyloid β protein messenger RNA was similar to that of the neurofibrillary tangles of Alzheimer's disease in some regions, but the messenger RNA was also expressed in other neurons that are not usually involved in the pathology of Alzheimer's disease.
Adipsin: A Circulating Serine Protease Homolog Secreted by Adipose Tissue and Sciatic Nerve
Authors: Cook Kathleen Sue , Min Hye Yeong , Johnson David , Chaplinsky Robert J. , Flier Jeffrey S. , Hunt Clayton R. , Spiegelman Bruce M.
Published: October 2006

Adipsin is a serine protease homolog whose primary structure was predicted from the nucleotide sequence of a differentiation-dependent adipocyte messenger RNA. Immunoblots probed with antisera to synthetic peptides identify two forms of adipsin that are synthesized and secreted by 3T3 adipocytes. These proteins of 44 and 37 kilodaltons are converted to 25.5 kilodaltons by enzymatic deglycosylation. Although adipsin is principally synthesized in adipose tissue, it is also produced by sciatic nerve and is found in the bloodstream. Because of the apparent restriction of adipsin synthesis to tissues highly active in lipid metabolism, its presence in serum, and its modulation in altered metabolic states, this molecule may play a previously unrecognized role in systemic lipid metabolism or energy balance.
Multiple Global Regulators Control
HIS4
transcription in Yeast
Authors: Arndt Kim T. , Styles Cora , Fink Gerald R.
Published: October 2006

Gene expression is dependent on the interaction of DNA binding factors with distinct promoter control elements to activate RNA synthesis. The expression of the
HIS4
gene in yeast is under two different control systems. One of these, general amino acid control, involves a DNA binding protein, GCN4, that stimulates transcription in response to amino acid starvation by binding to 5′-TGACTC-3′ sequences in the
HIS4
promoter region. A second system, the basal level control, stimulates
HIS4
transcription in the absence of amino acid starvation. The basal level transcription of the
HIS4
gene is under the control of two genes,
BAS1
and
BAS2
, which are also required for the control of purine biosynthesis. In addition,
BAS2
is required for the utilization of organic phosphates in the growth medium. Genetic mapping and DNA sequence analysis show that
BAS2
is
PHO2
, a gene previously identified as a regulator of phosphate metabolism. Direct biochemical analysis shows that the
BAS2
gene encodes a protein that binds to both the
HIS4
and
PHO5
promoters. The involvement of a single DNA binding protein in the regulation of histidine, adenine, and phosphate metabolism suggests that yeast may use a few key DNA binding proteins to coordinate the regulation of diverse metabolic pathways.
The Structure of Sister Minichromosome DNA Before Anaphase in
Saccharomyces Cerevisiae
Authors: Koshland Douglas , Hartwell Leland H.
Published: October 2006

The role of DNA topology in holding sister chromatids together before anaphase was investigated by analyzing the structure of a small circular minichromosome in cell cycle (cdc) mutants of the yeast
Saccharomyces cerevisiae
. In the majority of cells arrested after S phase but before anaphase, sister minichromosome molecules are not topologically interlocked with each other. The analysis of the ploidy of minichromosomes in cells that are released from arrest demonstrates that the sister molecules are properly segregated when the cell cycle block is removed. Therefore, sister minichromosome molecules need not remain topologically interlocked until anaphase in order to be properly segregated, and topological interlocking of sister DNA molecules apparently is not the primary force holding sister chromatids together.
Nicotinic Antagonists Enhance Process Outgrowth by Rat Retinal Ganglion Cells in Culture
Authors: Lipton Stuart A. , Frosch Matthew P. , Phillips Micheal D. , Tauck David L. , Aizenman Elias
Published: October 2006

Functional nicotinic cholinergic receptors are found on mammalian retinal ganglion cell neurons in culture. The neurotransmitter acetylcholine (ACh) can be detected in the medium of many of these retinal cultures, after release presumably from the choline acetyltransferase-positive amacrine cells. The postsynaptic effect of endogenous or applied ACh on the ganglion cells can be blocked with specific nicotinic antagonists. Here it is shown that within 24 hours of producing such a pharmacologic blockade, the retinal ganglion cells begin to sprout or regenerate neuronal processes. Thus, the growth-enhancing effect of nicotinic antagonists may be due to the removal of inhibition to growth by tonic levels of ACh present in the culture medium. Since there is a spontaneous leak of ACh in the intact retina, the effects of nicotinic cholinergic drugs on process outgrowth in culture may reflect a normal control mechanism for growth or regeneration of retinal ganglion cell processes that is exerted by ACh in vivo.
The Nasotemporal Division in Primate Retina: The Neural Bases of Macular Sparing and Splitting
Authors: Leventhal Audie G. , Ault Steven J. , Vitek Dagmar J.
Published: October 2006

In primates, each hemisphere contains a representation of the contralateral visual hemifield; unilateral damage to the visual pathways results in loss of vision in half of the visual field. Apparently similar severe, unilateral lesions to the central visual pathways can result in two qualitatively different central visual field defects termed macular sparing and macular splitting. In macular sparing a 2° to 3° region around the fovea is spared from the effects of unilateral damage to the visual pathways. In macular splitting there is no such spared region and the scotoma produced by unilateral brain damage bisects the fovea. The patterns of decussation of the different classes of retinal ganglion cells in both New World (
Saimiri sciureus
) and Old World (
Macaca fascicularis
) monkeys have been determined by horseradish peroxidase injection. In both species the distributions of ipsilaterally and contralaterally projecting ganglion cells in the central retina are different from those in other mammals and suggest neural bases for macular sparing and splitting, respectively.
Three-Dimensional Solution Structure of Plastocyanin from the Green Alga
Scenedesmus obliquus
Authors: Moore Jonathan M. , Case David A. , Chazin Walter J. , Gippert Garry P. , Havel Timothy F. , Powls Roy , Wright Peter E.
Published: October 2006

The solution conformation of plastocyanin from the green alga
Scenedesmus obliquus
has been determined from distance and dihedral angle constraints derived by nuclear magnetic resonance (NMR) spectroscopy. Structures were generated with distance geometry and restrained molecular dynamics calculations. A novel molecular replacement method was also used with the same NMR constraints to generate solution structures of
S. obliquus
plastocyanin from the x-ray structure of the homologous poplar protein.
Scenedesmus obliquus
plastocyanin in solution adopts a β-barrel structure. The backbone conformation is well defined and is similar overall to that of poplar plastocyanin in the crystalline state. The distinctive acidic region of the higher plant plastocyanins, which functions as a binding site for electron transfer proteins and inorganic complexes, differs in both shape and charge in
S. obliquus
plastocyanin.
Inhibition of Self-Binding Antibodies (Autobodies) by a V
H
-Derived Peptide
Authors: Kang Chang-Yuil , Brunck Terry K. , Kieber-Emmons Thomas , Blalock J. Edwin , Kohler Heinz
Published: October 2006

The self-binding properties of a dominant idiotypic antibody (T15) and a minor idiotypic antibody (M603), both specific for phosphorylcholine, were examined as models of self-binding antibodies (autobodies). Observed differences in the self-binding affinity of T15 and M603 relate to variable sequence differences in their respective heavy and light chains. A molecular recognition theory based on the translation of coding and noncoding DNA strands was used to identify complementary amino acid sequences responsible for self-binding. The second hypervariable region of the heavy chain domain, extending into the third framework region, was predicted as the primary self-binding locus. Among peptides synthesized with different variable heavy and light chain regions, a 24-residue peptide spanning the second hypervariable and third framework regions of the heavy chain of T15 was nearly as effective as phosphorylcholine in inhibiting the self-binding complexes.
Kin Selection and the Evolution of Monogamy
Authors: Peck Joel R. , Feldman Marcus W.
Published: October 2006

A two-locus genetic model is studied in which one locus controls the tendency of individuals to act altruistically toward siblings and the other locus controls the mating habits of females. It is demonstrated that genetic variation at the altruism locus is often sufficient to induce an increase in the frequency of genes that cause females to produce all of their offspring with a single mate. This occurs because of nonrandom associations that develop between genes that cause altruism and those that affect female mating behavior. The results provide a new explanation for the evolution of monogamy, and they suggest a previously unexplored mechanism for the evolution of a variety of other behavioral traits as well.
RH 5849, a Nonsteroidal Ecdysone Agonist: Effects on a
Drosophila
Cell Line
Authors: Wing Keith D.
Published: October 2006

The steroid molting hormone 20-hydroxyecdysone is the physiological inducer of molting and metamorphosis in insects. In ecdysone-sensitive
Drosophila
K
c
cells, the insecticide RH 5849 (1,2-dibenzoyl-1-
tert
-butylhydrazine) mimics the action of 20-hydroxyecdysone by causing the formation of processes, an inhibition of cell proliferation, and induction of acetylcholinesterase. RH 5849 also competes with [
3
H]ponasterone A for high-affinity ecdysone receptor sites from K
c
cell extracts. Resistant cell populations selected by growth in the continued presence of either RH 5849 or 20-hydroxyecdysone are insensitive to both compounds and exhibit a decreased titer of measurable ecdysone receptors. Although it is less potent than 20-hydroxyecdysone in both whole-cell and cell-free receptor assays, RH 5849 is the first nonsteroidal ecdysone agonist.
Autoregulation of Enzymes by Pseudosubstrate Prototopes: Myosin Light Chain Kinase
Authors: Pearson Richard B. , Wettenhall Richard E. H. , Means Anthony R. , Hartshorne David J. , Kemp Bruce E.
Published: October 2006

The myosin light chain kinase requires calmodulin for activation. Tryptic cleavage of the enzyme generates an inactive 64-kilodalton (kD) fragment that can be further cleaved to form a constitutively active, calmodulin-independent, 61-kD fragment. Microsequencing and amino acid analysis of purified peptides after proteolysis of the 61- and 64-kD fragments were used to determine the amino-terminal and carboxyl-terminal sequences of the 64-kD fragment. Cleavage within the calmodulin-binding region at Arg
505
generates the catalytically inactive 64-kD fragment, which is incapable of binding calmodulin. Further digestion removes a carboxyl-terminal fragment, including the pseudosubstrate sequence Ser
484
-Lys-Asp-Arg-Met-Lys-Lys-Tyr-Met- Ala-Arg-Arg-Lys-Trp-Gln-Lys-Thr-Gly-His-Ala-Val-Arg
505
and results in a calmodulin-independent 61-kD fragment. Both the 61- and 64-kD fragments have the same primary amino-terminal sequences. These results provide direct support for the concept that the pseudosubstrate structure binds the active site and that the role of calmodulin is to modulate this interaction. Pseudosubstrates may be utilized in analogous ways by other allosterically regulated enzymes.
Imaging of Phosphorescence: A Novel Method for Measuring Oxygen Distribution in Perfused Tissue
Authors: Rumsey William L. , Vanderkooi Jane M. , Wilson David F.
Published: October 2006

The imaging of phosphorescence provides a method for monitoring oxygen distribution within the vascular system of intact tissues. Isolated rat livers were perfused through the portal vein with media containing palladium coproporphyrin, which phosphoresced and was used to image the liver at various perfusion rates. Because oxygen is a powerful quenching agent for phosphors, the transition from well-perfused liver to anoxia (no flow of oxygen) resulted in large increases of phosphorescence. During stepwise restoration of oxygen flow, the phosphorescence images showed marked heterogeneous patterns of tissue reoxygenation, which indicated that there were regional inequalities in oxygen delivery.
Direct Cloning and Sequence Analysis of Enzymatically Amplified Genomic Sequences
Authors: Scharf Stephen J. , Horn Glenn T. , Erlich Henry A.
Published: October 2006

A method is described for directly cloning enzymatically amplified segments of genomic DNA into an M13 vector for sequence analysis. A 110-base pair fragment of the human β-globin gene and a 242-base pair fragment of the human leukocyte antigen DQα locus were amplified by the polymerase chain reaction method, a procedure based on repeated cycles of denaturation, primer annealing, and extension by DNA polymerase I. Oligonucleotide primers with restriction endonuclease sites added to their 5′ ends were used to facilitate the cloning of the amplified DNA. The analysis of cloned products allowed the quantitative evaluation of the amplification method's specificity and fidelity. Given the low frequency of sequence errors observed, this approach promises to be a rapid method for obtaining reliable genomic sequences from nanogram amounts of DNA.
The Gene for Familial Polyposis Coli Maps to the Long Arm of Chromosome 5
Authors: Leppert M. , Dobbs M. , Scambler P. , O'Connell P. , Nakamura Y. , Stauffer D. , Woodward S. , Burt R. , Hughes J. , Gardner E. , Lathrop M. , Wasmuth J. , Lalouel J.-M. , White R.
Published: October 2006

The inherited genetic defect in adenomatous polyposis has been localized to a small region on the long arm of chromosome 5. Sixteen DNA marker loci were used to construct a linkage map of the chromosome. When five kindreds segregating a gene for adenomatous polyposis coli were characterized with a number of the markers, significant linkage was found between one marker and the disease gene. Linkage analysis determined the location of the defective gene within a primary genetic map of chromosome 5.
Aminoguanidine Prevents Diabetes-Induced Arterial Wall Protein Cross-Linking
Authors: Brownlee M. , Vlassara H. , Kooney A. , Ulrich P. , Cerami A.
Published: October 2006

Age-associated increases in collagen cross-linking and accumulation of advanced glycosylation products are both accelerated by diabetes, suggesting that glucose-derived cross-link formation may contribute to the development of chronic diabetic complications as well as certain physical changes of aging. Aminoguanidine, a nucleophilic hydrazine compound, prevented both the formation of fluorescent advanced nonenzymatic glycosylation products and the formation of glucose-derived collagen cross-links in vitro. Aminoguanidine administration to rats was equally effective in preventing diabetes-induced formation of fluorescent advanced nonenzymatic glycosylation products and cross-linking of arterial wall connective tissue protein in vivo. The identification of aminoguanidine as an inhibitor of advanced nonenzymatic glycosylation product formation now makes possible precise experimental definition of the pathogenetic significance of this process and suggests a potential clinical role for aminoguanidine in the future treatment of chronic diabetic complications.
Transforming Growth Factor-β: Biological Function and Chemical Structure
Authors: Sporn Michael B. , Roberts Anita B. , Wakefield Lalage M. , Assoian Richard K.
Published: October 2006

Transforming growth factor-β (TGF-β) is a multifunctional peptide that controls proliferation, differentiation, and other functions in many cell types. Many cells synthesize TGF-β and essentially all of them have specific receptors for this peptide. TGF-β regulates the actions of many other peptide growth factors and determines a positive or negative direction of their effects. Its marked ability to enhance formation of connective tissue in vivo suggests several therapeutic applications.
A Chimeric, Ligand-Binding v-
erb
B/EGF Receptor Retains Transforming Potential
Authors: Riedel Heimo , Schlessinger Joseph , Ullrich Axel
Published: October 2006

Comparison of amino acid sequences from human epidermal growth factor (EGF) receptor and avian erythroblastosis virus
erb
B oncogene product suggests that v-
erb
B represents a truncated avian EGF receptor gene product. Although both proteins are transmembrane tyrosine kinases, the v-
erb
B protein lacks most of the extracellular ligand-binding domain and a 32-amino acid cytoplasmic sequence present in the human EGF receptor. To test the validity of the proposed origin of v-
erb
B and to investigate the functional significance of the deleted extracellular sequences, a chimeric gene encoding the extracellular and the transmembrane domain of the human EGF receptor joined to sequences coding for the cytoplasmic domain of the avian
erb
B oncogene product was constructed. When expressed in Rat1 fibroblasts, this reconstituted gene product (HER-
erb
B) was transported to the cell surface and bound EGF. Its autophosphorylation activity was stimulated by interaction with the ligand. Expression of the HER-
erb
B chimera led to anchorage-independent cell growth in soft agar and EGF-induced focus formation in Rat1 monolayers. Thus, it appears that v-
erb
B protein sequences in the chimeric receptor retain their transforming activity under the influence of the human extracellular EGF-binding domain.
Trophic Stimulation of Cultured Neurons from Neonatal Rat Brain by Epidermal Growth Factor
Authors: Morrison Richard S. , Kornblum Harley I. , Leslie Frances M. , Bradshaw Ralph A.
Published: October 2006

Epidermal growth factor (EGF) is a potent polypeptide mitogen originally isolated from the adult male mouse submaxillary gland. It also acts as a gastrointestinal hormone. EGF-immunoreactive material has recently been identified within neuronal fibers and terminals in rodent brain. In the present study, EGF was found to enhance survival and process outgrowth of primary cultures of subneocortical telencephalic neurons of neonatal rat brain in a dose-dependent manner. This effect was observed with EGF concentrations as low as 100 picograms per milliliter (0.016 nanomolar) and was dependent on the continuous presence of EGF in the medium. Similar effects were observed with basic fibroblast growth factor, but several other growth-promoting substances, including other mitogens for glial elements, were without effect. Thus EGF, in addition to its mitogenic and hormonal activities, may act as a neurite elongation and maintenance factor for select neurons of the rodent central nervous system.
Mutations in Diphtheria Toxin Separate Binding from Entry and Amplify Immunotoxin Selectivity
Authors: Greenfield Larry , Johnson Virginia Gray , Youle Richard J.
Published: October 2006

Monoclonal antibodies linked to toxic proteins (immunotoxins) can selectively kill some tumor cells in vitro and in vivo. However, reagents that combine the full potency of the native toxins with the high degree of cell type selectivity of monoclonal antibodies have not previously been designed. Two heretofore inseparable activities on one polypeptide chain of diphtheria toxin and ricin account for the failure to construct optimal reagents. The B chains (i) facilitate entry of the A chain to the cytosol, which allows immunotoxins to efficiently kill target cells, and (ii) bind to receptors present on most cells, which imparts to immunotoxins a large degree of non-target cell toxicity. This report identifies point mutations in the B polypeptide chain of diphtheria toxin that block binding but allow cytosol entry. Three mutants of diphtheria toxin have 1/1,000 to 1/10,000 the toxicity and 1/100 to 1/8,000 the binding activity of diphtheria toxin. Linking of either of two of the inactivated mutant toxins (CRM103, Phe
508
; CRM107, Phe
390
, Phe
525
) to a monoclonal antibody specific for human T cells reconstitutes full target-cell toxicity—indistinguishable from that of the native toxin linked to the same antibody—without restoring non-target cell toxicity. This separation of the entry function from the binding function generates a uniquely potent and cell type-specific immunotoxin that retains full diphtheria toxin toxicity, yet is four to five orders of magnitude less toxic than the native toxin is to nontarget cells.
Functional Role of Aspartic Acid-27 in Dihydrofolate Reductase Revealed by Mutagenesis
Authors: Howell Elizabeth E. , Villafranca Jesus E. , Warren Mark S. , Oatley Stuart J. , Kraut Joseph
Published: October 2006

The crystal structures and enzymic properties of two mutant dihydrofolate reductases (
Escherichia coli
) were studied in order to clarify the functional role of an invariant carboxylic acid (aspartic acid at position 27) at the substrate binding site. One mutation, constructed by oligonucleotide-directed mutagenesis, replaces Asp
27
with asparagine; the other is a primary-site revertant to Ser
27
. The only structural perturbations involve two internally bound water molecules. Both mutants have low but readily measurable activity, which increases rapidly with decreasing
p
H. The mutant enzymes were also characterized with respect to relative folate: dihydrofolate activities and kinetic deuterium isotope effects. It is concluded that Asp
27
participates in protonation of the substrate but not in electrostatic stabilization of a positively charged, protonated transition state.
Self-Assembling Cytotoxins
Authors: Rideout Darryl
Published: October 2006

Decanal and
N
-amino-
N
′-1-octylguanidine (AOG), combined at 28 μ
M
each, mediated erythrocyte lysis within 80 minutes under physiological conditions. By contrast, no lysis was observed after 20 hours with either decanal (56 μ
M
) or AOG (100 μ
M
) alone. The pronounced synergism observed for these chemicals and similar reactive pairs of chemicals is due to the self-assembly of more cytotoxic hydrazones in situ. Decanal and AOG also exhibit synergistic activity against cultured human cells (HeLa) and bacteria (
Escherichia coli
J96). This synergism may be useful in the design of cytotoxins that would self-assemble selectively from nontoxic precursors within tumors, while sparing normal tissue.
A New Approach to the Oral Administration of Insulin and Other Peptide Drugs
Authors: Saffran Murray , Kumar G. Sudesh , Savariar Celin , Burnham Jeffrey C. , Williams Frederick , Neckers Douglas C.
Published: October 2006

The oral administration of peptide drugs is well known to be precluded by their digestion in the stomach and small intestine. As a new approach to oral delivery, peptide drugs were coated with polymers cross-linked with azoaromatic groups to form an impervious film to protect orally administered drugs from digestion in the stomach and small intestine. When the azopolymer-coated drug reached the large intestine, the indigenous microflora reduced the azo bonds, broke the cross-links, and degraded the polymer film, thereby releasing the drug into the lumen of the colon for local action or for absorption. The ability of the azopolymer coating to protect and deliver orally administered peptide drugs was demonstrated in rats with the peptide hormones vasopressin and insulin.
Detoxification of Bacterial Lipopolysaccharides (Endotoxins) by a Human Neutrophil Enzyme
Authors: Munford Robert S. , Hall Catherine L.
Published: October 2006

Lipopolysaccharides in the cell walls of Gram-negative bacteria elicit toxic as well as potentially beneficial inflammatory responses in animals. It is now reported that tissue toxicity caused by lipopolysaccharides is preferentially reduced by an enzymatic activity in human neutrophils. Acyloxyacyl hydrolysis removes fatty acyl chains that are linked to the hydroxyl groups of 3-hydroxytetradecanoyl residues in the bioactive lipid A moiety of the lipopolysaccharides. Maximal acyloxyacyl hydrolysis reduced lipopolysaccharide tissue toxicity, as measured in the dermal Shwartzman reaction, by a factor of 100 or more. In contrast, the ability of the deacylated lipopolysaccharides to stimulate B lymphocytes to divide was decreased only by a factor of 12. It is suggested that during tissue invasion by Gram-negative bacteria acyloxyacyl hydrolysis may be a defense mechanism that reduces the toxicity of lipopolysaccharides while preserving some of their potentially beneficial inflammatory and immune stimuli.
Saturation Mutagenesis of the Yeast
his3
Regulatory Site: Requirements for Transcriptional Induction and for Binding by GCN4 Activator Protein
Authors: Hill David E. , Hope Ian A. , Macke Jennifer P. , Struhl Kevin
Published: October 2006

Expression of the yeast
his3
and other amino acid biosynthetic genes is induced during conditions of amino acid starvation. The coordination of this response is mediated by a positive regulatory protein called GCN4, which binds specifically to regulatory sites upstream of all coregulated genes and stimulates their transcription. The nucleotide sequence requirements of the
his3
regulatory site were determined by analysis of numerous point mutations obtained by a novel method of cloning oligonucleotides. Almost all single base pair mutations within the nine base pair sequence ATGACTCTT significantly reduce
his3
induction in vivo and GCN4 binding in vitro, whereas changes outside this region have minimal effects. One mutation, which generates a sequence that most closely resembles the consensus for 15 coregulated genes, increases both the level of induction and the affinity for GCN4 protein. The palindromic nature of the optimal sequence, ATGACTCAT, suggests that GCN4 protein binds as a dimer to adjacent half-sites that possibly overlap.
An Alternative Pathway for Meiotic Chromosome Segregation in Yeast
Authors: Dawson Dean S. , Murray Andrew W. , Szostak Jack W.
Published: October 2006

In meiosis I of most organisms, homologous chromosomes pair, recombine, and then segregate to opposite poles of the cell. Crossing-over is normally necessary to ensure the proper segregation of the homologs. Recently developed techniques have made it possible to study meiosis with highly defined artificial chromosomes. These techniques were used to demonstrate the existence of a system capable of segregating pairs of nonrecombined artificial chromosomes, regardless of the extent of their sequence homology. This system may contribute to the high fidelity of meiosis by mediating the segregation of pairs of natural chromosomes that have failed to recombine.
Research Involving Human Subjects
Authors: Gray Bradford H. , Cooke Robert A. , Tannenbaum Arnold S.
Published: October 2006

This article reports a study of the activities and performance of institutional review boards to protect human research subjects. Researchers and institutional review board members were generally supportive of the review system, although substantial criticisms were also heard. Institutional review boards had some direct impact on half of the proposals reviewed by requiring either modification of or additional information about proposed research. The data, however, raise questions about the effectiveness of some review board actions, for example, with regard to informed consent. Some policy implications of the study are presented.
Identification of an Amplified, Highly Expressed Gene in a Human Glioma
Authors: Kinzler Kenneth W. , Bigner Sandra H. , Bigner Darell D. , Trent Jeffrey M. , Law Martha L. , O'Brien Stephen J. , Wong Albert J. , Vogelstein Bert
Published: October 2006

A gene, termed
gli
, was identified that is amplified more than 50-fold in a malignant glioma. The gene is expressed at high levels in the original tumor and its derived cell line and is located at chromosome 12 position (q13 to q14.3). The
gli
gene is a member of a select group of cellular genes that are genetically altered in primary human tumors.
Steroidogenesis-Activator Polypeptide Isolated from a Rat Leydig Cell Tumor
Authors: Pedersen Robert C. , Brownie Alexander C.
Published: October 2006

A cycloheximide-sensitive protein responsive to adenosine 3′,5′-monophosphate has been postulated to participate in the regulation of cholesterol side-chain cleavage activity in steroidogenic tissues. Such a steroidogenesis activator polypeptide (SAP) had been isolated from rat adrenocortical tissue and partially characterized. Now a polypeptide with comparable chromatographic behavior and biological activity has been purified from the rat H-540 Leydig cell tumor in quantities sufficient for amino acid sequencing. The activator contains 30 amino acid residues and has a molecular weight of 3215. The synthetic construct based on this sequence is virtually equipotent with native H-540 tumor SAP in an adrenal mitochondrial cholesterol side-chain cleavage assay. Hormonal regulation of the intracellular concentration of this activator may control the rate of cholesterol metabolism in steroidogenic organs.
Identification and Localization of Mutations at the Lesch-Nyhan Locus by Ribonuclease A Cleavage
Authors: Gibbs Richard A. , Caskey C. Thomas
Published: October 2006

Many mutations leading to human disease are the result of single DNA base pair changes that cannot be identified by Southern analysis. This has prompted the development of alternative assays for point mutation detection. The recently described ribonuclease A cleavage procedure, with a polyuridylic acid-paper affinity chromatography step, has been used to identify the mutational lesions in the hypoxanthine phosphoribosyltransferase (HPRT) messenger RNAs of patients with Lesch-Nyhan syndrome. Distinctive ribonuclease A cleavage patterns were identified in messenger RNA from 5 of 14 Lesch-Nyhan patients who were chosen because no HPRT Southern or Northern blotting pattern changes had been found. This approach now allows HPRT mutation detection in 50 percent of the cases of Lesch-Nyhan syndrome. The polyuridylic acid-paper affinity procedure provides a general method for analysis of low abundance messenger RNAs.
Newly Identified 'Glutamate Interneurons' and Their Role in Locomotion in the Lamprey Spinal Cord
Authors: Buchanan James T. , Grillner Sten
Published: October 2006

A new class of excitatory premotor interneurons that are important in the generation of locomotion in the lamprey has now been described. In the isolated spinal cord, these neurons act simultaneously with their postsynaptic motoneurons during fictive swimming. They are small and numerous, and they monosynaptically excite both motoneurons and inhibitory premotor interneurons. The excitatory postsynaptic potentials are depressed by an antagonist of excitatory amino acids. These interneurons receive reticulospinal input from the brain stem and polysynaptic input from skin afferents. A model of the network underlying locomotion based on the synaptic interactions of these neurons can now be proposed for the lamprey.
Free Energy Calculations by Computer Simulation
Authors: Bash P. A. , Singh U. C. , Langridge R. , Kollman P. A.
Published: October 2006

A fundamental problem in chemistry and biochemistry is understanding the role of solvation in determining molecular properties. Recent advances in statistical mechanical theory and molecular dynamics methodology can be used to solve this problem with the aid of supercomputers. By using these advances the free energies of solvation of all the chemical classes of amino acid side chains, four nucleic acid bases and other organic molecules can be calculated. The effect of a site-specific mutation on the stability of trypsin is predicted. The results are in good agreement with available experiments.
Radioactive Cesium from the Chernobyl Accident in the Greenland Ice Sheet
Authors: Davidson C. I. , Harrington J. R. , Stephenson M. J. , Monaghan M. C. , Pudykiewicz J. , Schell W. R.
Published: October 2006

Measurements of cesium-134 and cesium-137 in Greenland snow together with models of long-range transport have been used to assess radionuclide deposition in the Arctic after the Chernobyl accident. The results suggest that a well-defined layer of radioactive cesium is now present in polar glaciers, providing a new reference for estimating snow accumulation rates and dating ice core samples.
Myocardial Failure in Cats Associated with Low Plasma Taurine: a Reversible Cardiomyopathy
Authors: Pion P. D. , Kittleson M. D. , Rogers Q. R. , Morris J. G.
Published: October 2006

Thousands of pet cats die each year with dilated cardiomyopathy, the cause of which is unknown. Although taurine is present in millimolar concentrations in the myocardium of all mammals, taurine depletion has not previously been associated with a decrease in myocardial function in any species. In this study, low plasma taurine concentrations associated with echocardiographic evidence of myocardial failure were observed in 21 cats fed commercial cat foods and in 2 of 11 cats fed a purified diet containing marginally low concentrations of taurine for 4 years. Oral supplementation of taurine resulted in increased plasma taurine concentrations and was associated with normalization of left ventricular function in both groups of cats. Since myocardial concentrations of taurine are directly related to plasma concentrations and low plasma concentrations were found to be associated with myocardial failure in cats, a direct link between decreased taurine concentration in the myocardium and decreased myocardial mechanical function is proposed.
A Parathyroid Hormone-Related Protein Implicated in Malignant Hypercalcemia: Cloning and Expression
Authors: Suva L. J. , Winslow G. A. , Wettenhall R. E. H/. , Hammonds R. G. , Moseley J. M. , Diefenbach-Jagger H. , Rodda C. P. , Kemp B. E. , Rodriguez H. , Chen E. Y. , Hudson P. J. , Martin T. J. , Wood W. I.
Published: October 2006

Humoral hypercalcemia of malignancy is a common complication of lung and certain other cancers. The hypercalcemia results from the actions of tumor factors on bone and kidney. We report here the isolation of full-length complementary DNA clones of a putative hypercalcemia factor, and the expression from the cloned DNA of the active protein in mammalian cells. The clones encode a prepro peptide of 36 amino acids and a mature protein of 141 amino acids that has significant homology with parathyroid hormone in the amino-terminal region. This previously unrecognized hormone may be important in normal as well as abnormal calcium metabolism.
The
raf
Oncogene Is Associated with a Radiation-Resistant Human Laryngeal Cancer
Authors: Kasid U. , Pfeifer A. , Weichselbaum R. R. , Dritschilo A. , Mark G. E.
Published: October 2006

In order to identify the genetic factors associated with the radiation-resistant human laryngeal carcinoma cell line (SQ-20B), tumor cell DNA was transfected into NIH/3T3 cells. A high incidence (six out of six) of
raf
sequences was found in transfected NIH/3T3 clones and the tumorigenic potential of SQ-20B DNA could be linked to genomic fragments that represent most of the kinase domain of human c-
raf
-1. An apparently unaltered 3.5-kilobase pair (kb) human c-
raf
transcript was identified in SQ-20B cells but was not observed in the transfected NIH/3T3 cell clones. Two new transcripts (4.2 kb and 2.6 kb) were found in tumorigenic clones; the large transcript was missing in a very poorly tumorigenic clone. Cytogenetic analysis indicated that the normal autosomes of chromosome 3 were absent in SQ-20B karyotypes and had formed apparently stable marker chromosomes. Unlike the recipient NIH/3T3 cell line, 30 percent of the transformed clone-1 metaphases had minute and double-minute chromosomes representative of amplified DNA sequences. The frequency of the c-
raf
-1 identification by NIH/3T3 transfection of SQ-20B DNA suggests the presence of some genetic abnormality within this locus.
D-Alanine in the Frog Skin Peptide Dermorphin Is Derived from L-Alanine in the Precursor
Authors: Richter Klaus , Egger Richard , Kreil Günther
Published: October 2006

A D-alanine-containing peptide termed dermorphin, with potent opiate-like activity, has been isolated from skin of the frog
Phyllomedusa sauvagei
. Complementary DNA (cDNA) libraries were constructed from frog skin messenger RNA and screened with a mixture of oligonucleotides that contained the codons complementary to five amino acids of dermorphin. Clones were detected with inserts coding for different dermorphin precursors. The predicted amino acid sequences of these precursors contained homologous repeats of 35 amino acids that included one copy of the heptapeptide dermorphin. In these cloned cDNAs, the alanine codon GCG occurred at the position where D-alanine is present in the end product. This suggests the existence of a novel post-translational reaction for the conversion of an L-amino acid to its D-isomer.
Behavioral Recovery Induced by Applied Electric Fields After Spinal Cord Hemisection in Guinea Pig
Authors: Borgens Richard B. , Blight Andrew R. , McGinnis M. E.
Published: October 2006

Applied electric fields were used to promote axonal regeneration in spinal cords of adult guinea pigs. A propriospinal intersegmental reflex (the cutaneous trunci muscle reflex) was used to test lateral tract function after hemisection of the thoracic spinal cord. An electrical field (200 microvolts per millimeter, cathode rostral) applied across the lesion led to functional recovery of the cutaneous trunci muscle reflex in 25 percent of experimental animals, whereas the functional deficit remained in control animals, which were implanted with inactive stimulators.