bluuebunny/crossref_metadata_2025 huggingface dataset:
Phencyclidine is a negative allosteric modulator of signal transduction at two subclasses of excitatory amino acid receptors.
Authors: Wroblewski J T , Nicoletti F , Fadda E , Costa E
Published: May 2006

Phencyclidine (PCP) and some of its pharmacological congeners inhibit the signal transduction at specific excitatory amino acid receptors of cerebellar granule cells in primary cultures. These drugs do not bind to the transmitter recognition sites, and affinity of this specific binding site is increased by the presence of the transmitter bound to its recognition sites. PCP inhibits phosphatidylinositol phosphate hydrolysis mediated by Mg2+-sensitive glutamate receptors (GP1) but not that mediated by Mg2+-insensitive glutamate receptors (GP2). In addition, PCP inhibits Ca2+ influx and cGMP formation mediated by the activation of Mg2+-sensitive glutamate receptors (GC1) but not that mediated by Mg2+-insensitive glutamate receptors (GC2). In this cell culture the activation of phosphatidylinositol phosphate hydrolysis by muscarinic receptor agonists is not affected by PCP. Since PCP inhibits noncompetitively GP1 and GC1 signal transduction it may act as a negative allosteric modulator of signal transduction at both receptors. The pharmacological profile of PCP and its congeners delimits a class of drugs modulating allosterically the action of the primary transmitter at GP1 and GC1 receptors. These drugs need the presence of the transmitter to act and they cannot be termed inverse agonists because they are devoid of activity in the absence of the transmitter; moreover, they do not bind to the transmitter recognition site nor do they prevent the transmitter binding to its recognition sites.
Eukaryotic Mr 83,000 heat shock protein has a homologue in Escherichia coli.
Authors: Bardwell J C , Craig E A
Published: May 2006

We have isolated a gene from Escherichia coli homologous to the gene encoding the Mr 83,000 Drosophila heat shock protein (hsp83). In E. coli the protein homologous to hsp83 is a heat shock protein called C62.5. The predicted amino acid sequence of C62.5 is 41% and 42% identical to the Drosophila and human hsp83 proteins, respectively. Selected regions of the protein have conservation as high as 90%. The gene encoding C62.5 (named htpG) is located between the dnaZ and adk genes at 11.1 minutes on the E. coli chromosome. The htpG gene appears to be a newly identified locus. The isolation of an E. coli homologue of hsp83 illustrates the remarkable conservation of heat shock proteins in evolution and will facilitate genetic and biochemical experiments aimed at determining the function of hsp83.
Preferential excision of the 5' proximal intron from mRNA precursors with two introns as mediated by the cap structure.
Authors: Ohno M , Sakamoto H , Shimura Y
Published: May 2006

We have studied the effect of the 5' cap structure on the splicing of precursor mRNAs containing three exons and two introns within a single molecule in a HeLa nuclear extract. When a precursor mRNA was capped, the upstream intron was spliced out more efficiently than the downstream intron. The differential splicing reactions of the two introns are not due to differences in the intrinsic efficiency of splicing of each intron, since the preferential excision of the upstream intron was also observed when the positions of the two introns relative to the cap structure were reversed. When uncapped precursor mRNA was used as substrate, the downstream intron was spliced out appreciably, but splicing of the upstream intron was greatly reduced. Preincubation of the extract with cap analogues inhibited splicing of the upstream intron but not the downstream intron. Thus, the cap structure exerts its effect primarily on the 5' proximal intron.
Protein kinase C activity and hexamethylenebisacetamide-induced erythroleukemia cell differentiation.
Authors: Melloni E , Pontremoli S , Michetti M , Sacco O , Cakiroglu A G , Jackson J F , Rifkind R A , Marks P A
Published: May 2006

Hexamethylenebisacetamide (HMBA) is a potent inducer of murine erythroleukemia (MEL) cell differentiation. The mechanism of action of HMBA is not known. In this study we provide evidence that protein kinase C has a role in inducer-mediated MEL cell differentiation: (i) HMBA induces the formation of a soluble, proteolytically activated form of protein kinase C that is catalytically active in the absence of Ca2+ and phospholipid; (ii) the protease inhibitor leupeptin blocks formation of this activated form of the kinase and inhibits HMBA-induced MEL cell hemoglobin accumulation; (iii) phorbol 12-myristate 13-acetate (PMA) inhibits HMBA-induced MEL differentiation and causes depletion of total protein kinase C activity; (iv) MEL cells depleted in protein kinase C activity by culture with PMA are resistant to induction by HMBA; (v) upon removal of PMA, restoration of MEL cell sensitivity to HMBA is correlated with reaccumulation of protein kinase C activity; and (vi) MEL cells grown to density arrest are both depleted of protein kinase C activity and resistant to HMBA. Together, these results suggest that HMBA-mediated MEL cell differentiation involves a protein kinase C-related mechanism and the proteolytically activated form of the kinase, which does not require Ca2+ or phospholipid for its catalytic activity.
Combined proton NMR imaging and spectral analysis of locust embryonic development
Authors: Gassner George , Lohman Joost A. B.
Published: May 2006

Images and spectra of desert locust [
Schistocerca gregaria
(Forskal)] embryos were collected
in vivo
using a 4.7-tesla proton (
1
H) NMR imaging spectrometer. The 100 × 100 × 500 μm image resolution and 125-nl localized spectral volumes were obtained within minutes of each other. The dynamics of embryonic development were slow enough that the time delay between imaging and spectral measurements is negligible. Thus, image and spectral data correspond closely and approximate real-time observations of embryonic changes. The above procedure was applied every 12 hr during the entire course of development. This analytical approach demonstrates that imaging and localized spectroscopy can be used to visualize and assess changes in embryonic water and lipid content in concert with the development of a living embryo.
HRAS1-selected chromosome transfer generates markers that colocalize aniridia- and genitourinary dysplasia-associated translocation breakpoints and the Wilms tumor gene within band 11p13.
Authors: Porteous D J , Bickmore W , Christie S , Boyd P A , Cranston G , Fletcher J M , Gosden J R , Rout D , Seawright A , Simola K O
Published: May 2006

We show that chromosome-mediated gene transfer can provide an enriched source of DNA markers for predetermined, subchromosomal regions of the human genome. Forty-four human DNA recombinants isolated from a HRAS1-selected chromosome-mediated gene transformant map exclusively to chromosome 11, with several sublocalizing to the Wilms tumor region at 11p13. We present a detailed molecular map of the deletion chromosomes 11 from five WAGR (Wilms tumor/aniridia/genitourinary abnormalities/mental retardation) syndrome patients, three of which are at the limits of cytogenetic resolution but shown here to be molecularly distinguishable and overlapping. We can define ten distinct regions of the short arm of chromosome 11, five of which subdivide band 11p13. We also map two independent 11p13 translocation breakpoints to within the smallest region of overlap defined by the WAGR deletions. The first comes from a patient with familial aniridia, and the second from a patient with Potter facies and genitourinary dysplasia. The close similarities in map location and affected cell lineage for Wilms tumor and genitourinary dysplasia suggest that they may be alternative manifestations of mutation at the same locus.
Dequalinium, a topical antimicrobial agent, displays anticarcinoma activity based on selective mitochondrial accumulation.
Authors: Weiss M J , Wong J R , Ha C S , Bleday R , Salem R R , Steele G D , Chen L B
Published: May 2006

Positively charged lipophilic compounds, such as rhodamine 123, localize in mitochondria and are selectively accumulated and retained by carcinoma cells. It has been suggested that this phenotype may be exploited for selective killing of carcinoma cells by lipophilic cations. Here we report that doubly positively charged dequalinium, which has been used for 30 years as an antimicrobial agent in over-the-counter mouthwashes, lozenges, ointments, and paints, exhibits significant anticarcinoma activity. Dequalinium is more effective than seven of eight established anticancer drugs in prolonging the survival of mice with intraperitoneally implanted mouse bladder carcinoma MB49. Dequalinium also inhibits the growth of subcutaneously implanted human colon carcinoma CX-1 in nude mice and recurrent rat colon carcinoma W163 in rats. Lipophilic cationic compounds, such as dequalinium, could comprise a unique class of anticarcinoma agents.
Anti-glycoprotein D antibodies that permit adsorption but block infection by herpes simplex virus 1 prevent virion-cell fusion at the cell surface.
Authors: Fuller A O , Spear P G
Published: May 2006

Certain monoclonal antibodies specific for glycoprotein D of herpes simplex virus have potent neutralizing activity but fail to block attachment of virus to cells. Here we have investigated the fate of neutralized and infectious virus after attachment to primate cells. Infectious virions fused with the cell surface such that naked nucleocapsids were detectable in the cytoplasm near or just under the plasma membrane. Neutralized virions did not fuse with the cell. They remained attached to the cell surface and could be rendered infectious by treatment with polyethylene glycol. We conclude that some anti-glycoprotein D neutralizing antibodies can inhibit the penetration of herpes simplex virus by blocking fusion of the virion envelope with the plasma membrane. These results identify a pathway of entry that initiates successful herpes simplex virus infection and a step in this pathway that is highly sensitive to neutralizing antibodies. A role for glycoprotein D in virion-cell fusion is indicated.
Phytoalexin synthesis by the sorghum mesocotyl in response to infection by pathogenic and nonpathogenic fungi
Authors: Nicholson Ralph L. , Kollipara Sharon S. , Vincent Jeffrey R. , Lyons Philip C. , Cadena-Gomez Gabriel
Published: May 2006

Infection of the sorghum mesocotyl by
Helminthosporium maydis
(a nonpathogen) and
Colletotrichum graminicola
(a pathogen) resulted in the rapid accumulation of a pigment complex by two sorghum cultivars. The components of the complex were fungitoxic. The principal compounds have been identified as the 3-deoxyanthocyanidins apigeninidin and luteolinidin. Apigeninidin accumulated in both sorghum cultivars in response to infection and was the predominant pigment. Luteolinidin accumulated in only one of the cultivars. Because of the speed of synthesis, occurrence only in response to inoculation, and fungitoxicity of the individual components, we propose that synthesis of the pigment complex constitutes a defense response and that the compounds apigeninidin and luteolinidin should be considered as phytoalexins.
Self-assembly of proglycinin and hybrid proglycinin synthesized
in vitro
from cDNA
Authors: Dickinson Craig D. , Floener Liliane A. , Lilley Glenn G. , Nielsen Niels C.
Published: May 2006

An
in vitro
system was developed that results in the self-assembly of subunit precursors into complexes that resemble those found naturally in the endoplasmic reticulum. Subunits of glycinin, the predominant seed protein of soybeans, were synthesized from modified cDNAs using a combination of the SP6 transcription and the rabbit reticulocyte translation systems. Subunits produced from plasmid constructions that encoded either Gy4 or Gy5 gene products, but modified such that their signal sequences were absent, self-assembled into trimers equivalent in size to those precursors found in the endoplasmic reticulum. In contrast, proteins synthesized
in vitro
from Gy4 constructs failed to self-assemble when the signal sequence was left intact (e.g., preproglycinin) or when the coding sequence was modified to remove 27 amino acids from an internal hydrophobic region, which is highly conserved among the glycinin subunits. Various hybrid subunits were also produced by trading portions of Gy4 and Gy5 cDNAs and all self-assembled in our system. The
in vitro
assembly system provides an opportunity to study the self-assembly of precursors and to probe for regions important for assembly. It will also be helpful in attempts to engineer beneficial nutritional changes into this important food protein.
Characterization of a mammalian mutant with a camptothecin-resistant DNA topoisomerase I.
Authors: Andoh T , Ishii K , Suzuki Y , Ikegami Y , Kusunoki Y , Takemoto Y , Okada K
Published: May 2006

DNA topoisomerase I was purified to near homogeneity from a clonal line of human lymphoblastic leukemia cells, RPMI 8402, that is resistant to camptothecin, a cytotoxic alkaloid from Camptotheca acuminata, and compared with that of the parent wild-type cells. As assayed by relaxation of the supercoiled plasmid DNA and by formation of enzyme-linked DNA breaks, the purified enzyme from the resistant cells was shown to be greater than 125-fold as resistant to camptothecin as the wild-type enzyme, comparable to a cellular resistance index of about 300. Therefore, the cellular resistance appears to be due to the resistance of the enzyme. The amount of the immunoreactive enzyme protein in whole extract appeared to be reduced to less than half that of the wild-type enzyme. These results establish that DNA topoisomerase I is the cellular target of camptothecin and that DNA topoisomerase I is essential for the survival of mammalian cells.
Bacteriorhodopsin mutants containing single tyrosine to phenylalanine substitutions are all active in proton translocation.
Authors: Mogi T , Stern L J , Hackett N R , Khorana H G
Published: May 2006

To study the possible role of the tyrosine residues in proton translocation by bacteriorhodopsin, we have replaced these residues individually by phenylalanine. The required codon changes were introduced in the bacterioopsin gene by replacement of appropriate restriction fragments by synthetic counterparts containing the desired nucleotide changes. The denatured opsin polypeptides obtained by expression of the mutant genes in Escherichia coli were purified and treated with a mixture of detergents, phospholipids, and retinal in a previously established renaturation procedure. All of the mutant proteins folded to regenerate bacteriorhodopsin-like chromophores. Three mutants with tyrosine to phenylalanine substitutions at positions 57, 83, and 185 regenerated the chromophore more slowly than the wild-type protein, and two of these mutants, Phe-57 and -83, showed slightly blue-shifted chromophores. When reconstituted into liposomes all of the mutant proteins with single Tyr----Phe substitutions pumped protons at rates and levels comparable to those of the wild-type bacteriorhodopsin. We conclude that single substitutions of tyrosine by phenylalanine do not affect folding, retinal binding, or light-dependent proton pumping in bacteriorhodopsin.
Thyroid hormone receptor binds to a site in the rat growth hormone promoter required for induction by thyroid hormone.
Authors: Koenig R J , Brent G A , Warne R L , Larsen P R , Moore D D
Published: May 2006

Transcription of the rat growth hormone (rGH) gene in pituitary cells is increased by addition of thyroid hormone (T3). This induction is dependent on the presence of specific sequences just upstream of the rGH promoter. We have partially purified T3 receptor from rat liver and examined its interaction with these rGH sequences. We show here that T3 receptor binds specifically to a site just upstream of the basal rGH promoter. This binding site includes two copies of a 7-base-pair direct repeat, the centers of which are separated by 10 base pairs. Deletions that specifically remove the T3 receptor binding site drastically reduce response to T3 in transient transfection experiments. These results demonstrate that T3 receptor can recognize specific DNA sequences and suggest that it can act directly as a positive transcriptional regulatory factor.
Effects of 8-bromo-cGMP on Ca2+ levels in vascular smooth muscle cells: possible regulation of Ca2+-ATPase by cGMP-dependent protein kinase.
Authors: Rashatwar S S , Cornwell T L , Lincoln T M
Published: May 2006

The effects of 8-bromo-cGMP on intracellular calcium concentrations in cultured rat aortic smooth muscle cells were studied. Both angiotensin II and depolarizing concentrations of K+ stimulated Ca2+ accumulation in the cytoplasm. The increase in Ca2+ due to angiotensin II was associated with an increase in inositol phosphates, while that due to K+ was not. Preincubation of cells with 8-bromo-cGMP (100 microM) caused an inhibition of peak Ca2+ accumulation to either angiotensin II or K+. To probe the mechanism of action of cGMP in vascular smooth muscle, the effects of cGMP-dependent protein kinase on Ca2+-ATPase from the cultured cell particulate material were investigated. Ca2+-activated ATPase was stimulated approximately equal to 2-fold by exogenous calmodulin and up to 4-fold by low concentrations of purified cGMP-dependent protein kinase. The inclusion of both calmodulin and cGMP-dependent protein kinase resulted in an additive stimulation of Ca2+-ATPase. Stimulation of Ca2+-ATPase activity was observed at all Ca2+ concentrations tested (0.01-1.0 microM). cAMP-dependent protein kinase catalytic subunit and protein kinase C were either ineffective or less effective than cGMP-dependent protein kinase in stimulating the Ca2+-ATPase from rat aortic smooth muscle cells. These results suggest a possible mechanism of action for cGMP in mediating decreases in cytosolic Ca2+ through activation of a Ca2+-ATPase and the subsequent removal of Ca2+ from the cell.
Three-dimensional structure of the bifunctional enzyme N-(5'-phosphoribosyl)anthranilate isomerase-indole-3-glycerol-phosphate synthase from Escherichia coli.
Authors: Priestle J P , Grütter M G , White J L , Vincent M G , Kania M , Wilson E , Jardetzky T S , Kirschner K , Jansonius J N
Published: May 2006

N-(5'-Phosphoribosyl)anthranilate isomerase-indole-3-glycerol-phosphate synthase from Escherichia coli is a monomeric bifunctional enzyme of Mr 49,500 that catalyzes two sequential reactions in the biosynthesis of tryptophan. The three-dimensional structure of the enzyme has been determined at 2.8-A resolution by x-ray crystallography. The two catalytic activities reside on distinct functional domains of similar folding, that of an eightfold parallel beta-barrel with alpha-helices on the outside connecting the beta-strands. Both active sites were located with an iodinated substrate analogue and found to be in depressions on the surface of the domains created by the outward-curving loops between the carboxyl termini of the beta-sheet strands and the subsequent alpha-helices. They do not face each other, making "channeling" of the substrate between active sites virtually impossible. Despite the structural similarity of the two domains, no significant sequence homology was found when topologically equivalent residues were compared.
Pituitary follicular cells produce basic fibroblast growth factor.
Authors: Ferrara N , Schweigerer L , Neufeld G , Mitchell R , Gospodarowicz D
Published: May 2006

Cultured monolayers of bovine pituitary follicular cells, which transport ions, contain high amounts of mitogenic activity for endothelial cells which, on the basis of gene expression analysis, heparin-Sepharose elution profile, bioassay, immunoblotting, radioimmunoassay, and radioreceptor assay, has been identified as basic fibroblast growth factor (bFGF). These data indicate that follicular cells may be a major source of bFGF in the pituitary gland. Considering that bFGF has been proposed to play a role in paracrine regulation of pituitary hormone secretion, the data also suggest that these cells may exert important local regulatory functions.
Reduced tyrosine kinase specific activity is associated with hypophosphorylation of pp60c-src in cells infected with avian erythroblastosis virus.
Authors: McCarley D J , Parsons S J
Published: May 2006

Avian erythroblastosis virus (AEV) is a replication-defective retrovirus that causes erythroblastosis and sarcomas in chickens and transforms immature erythroid cells and fibroblasts in culture. AEV encodes two oncogenes, v-erbA and v-erbB, whose products are closely related to the thyroxine receptor and the epidermal growth factor receptor, respectively. Since tyrosine protein kinases have been implicated in the process of normal growth signal transduction, we wished to study the possible consequences of the expression of these mutated, growth-regulating receptor genes on the activity of the cellular tyrosine kinase pp60c-src. A continuous cell line from AEV-infected quail embryo fibroblasts was derived that exhibited a typical transformed phenotype and expressed the viral oncogene products, p75gag-erbA and gp66-68erbB. Using an immune-complex kinase assay, we found that the specific activity of pp60c-src in AEV-transformed quail cells was decreased by a factor of 6-30 relative to that found in uninfected quail cells. A concomitant 50-80% reduction of 32Pi incorporation into the pp60c-src protein from radiolabeled, transformed cells was also observed, indicating a relationship between hypophosphorylation and diminished enzyme activity. Partial proteolytic phosphopeptide analysis revealed a decrease in phosphorylation of both serine- and tyrosine-containing peptides, suggesting an activation of specific phosphatases or inhibition of specific kinases in the AEV-transformed quail cells. Similar results were found in pp60c-src precipitated from AEV-transformed chicken and rat cells.
Vitamin K-dependent carboxylation of pulmonary surfactant-associated proteins.
Authors: Rannels S R , Gallaher K J , Wallin R , Rannels D E
Published: May 2006

Rat type II pneumocytes expressed vitamin K-dependent carboxylase activity that incorporated 14CO2 into microsomal protein precursors of molecular weights similar to those of surfactant-associated proteins (SAP). Compared to carboxylated precursor proteins present in the liver, these molecules appeared to be unique to the lung. Antibodies raised against purified rat surfactant reacted with SAP resolved by NaDodSO4/PAGE and with surfactant-containing lamellar bodies in type II pneumocyte cytoplasm. NaDodSO4/PAGE of microsomal proteins, after carboxylase-catalyzed incorporation of 14CO2, demonstrated radiolabeled, immunoreactive products identical to SAP. The presence of gamma-carboxyglutamic acid in these proteins was confirmed by HPLC analysis of SAP hydrolysates. Furthermore, lung carboxylase activity and SAP matured over similar time courses during fetal lung development. These results show that SAP are carboxylated by type II cells via a vitamin K-dependent pathway analogous to that for hepatic carboxylation of clotting factors. Further analogy to the clotting system suggests that gamma-carboxyglutamic acid residues in SAP polypeptides play a role in Ca2+ binding and thus in the known requirements for both the cation and SAP in the physiological function of pulmonary surfactant.
The RAD3 gene of Saccharomyces cerevisiae encodes a DNA-dependent ATPase.
Authors: Sung P , Prakash L , Weber S , Prakash S
Published: May 2006

The RAD3 gene of the yeast Saccharomyces cerevisiae is required for excision repair of damaged DNA and for cell viability. A protein of approximately equal to 89 kDa was purified to near homogeneity from yeast strains harboring multicopy plasmids that overproduce RAD3 protein; this protein corresponds closely to the expected size of the RAD3 protein and cross-reacts with the antiserum raised against a truncated RAD3 protein produced in Escherichia coli. The purified RAD3 protein shows a single-stranded DNA-dependent ATPase activity that catalyzes hydrolysis of ATP to ADP and Pi. The ATPase activity was coincident with the RAD3 protein during purification and is inhibited by anti-RAD3 antibodies, indicating that the RAD3 gene encodes this activity.
DNA methylation stabilizes X chromosome inactivation in eutherians but not in marsupials: evidence for multistep maintenance of mammalian X dosage compensation.
Authors: Kaslow D C , Migeon B R
Published: May 2006

In marsupials and eutherian mammals, X chromosome dosage compensation is achieved by inactivating one X chromosome in female cells; however, in marsupials, the inactive X chromosomes is always paternal, and some genes on the chromosome are partially expressed. To define the role of DNA methylation in maintenance of X chromosome inactivity, we examined loci for glucose-6-phosphate dehydrogenase and hypoxanthine phosphoribosyltransferase in a North American marsupial, the opossum Didelphis virginiana, by using genomic hybridization probes cloned from this species. We find that these marsupial genes are like their eutherian counterparts, with respect to sex differences in methylation of nuclease-insensitive (nonregulatory) chromatin. However, with respect to methylation of the nuclease-hypersensitive (regulatory) chromatin of the glucose-6-phosphate dehydrogenase locus, the opossum gene differs from those of eutherians, as the 5' cluster of CpG dinucleotides is hypomethylated in the paternal as well as the maternal gene. Despite hypomethylation of the 5' CpG cluster, the paternal allele, identified by an enzyme variant, is at best partially expressed; therefore, factors other than methylation are responsible for repression. In light of these results, it seems that the role of DNA methylation in eutherian X dosage compensation is to "lock in" the process initiated by such factors. Because of similarities between dosage compensation in marsupials and trophectoderm derivatives of eutherians, we propose that differences in timing of developmental events--rather than differences in the basic mechanisms of X inactivation--account for features of dosage compensation that differ among mammals.
Gated currents in isolated olfactory receptor neurons of the larval tiger salamander.
Authors: Firestein S , Werblin F S
Published: May 2006

The electrical properties of enzymatically isolated olfactory receptor cells were studied with whole-cell patch clamp. Voltage-dependent currents could be separated into three ionic components: a transient inward sodium current, a sustained inward calcium current, and an outward potassium current. Three components of the outward current could be identified by their gating and kinetics: a calcium-dependent potassium current [IK(Ca)], a voltage-dependent potassium current [IK(V)], and a transient potassium current (Ia). Typical resting potentials were near -54 mV, and typical input resistance was 3-6 G omega. Thus, only 3 pA of injected current was required to depolarize the cell to spike threshold near -45 mV. The response to a current step consisted of either a single spike regardless of stimulus strength, or a train of less than 8 spikes, decrementing in amplitude and frequency over approximately equal to 250 msec. Thus, the receptor response cannot be finely graded with stimulus intensity.
Inhibitor of eukaryotic initiation factor 4F activity in unfertilized sea urchin eggs.
Authors: Huang W I , Hansen L J , Merrick W C , Jagus R
Published: May 2006

Extracts from unfertilized sea urchin eggs contain an inhibitor of translation that inhibits protein synthesis in cell-free translation systems from sea urchin embryos or rabbit reticulocytes. The inhibitory effects of egg extracts can be reversed by the addition of mammalian eukaryotic initiation factor 4F (eIF-4F) in both sea urchin embryo and reticulocyte systems, suggesting that the inhibitor inactivates this initiation factor. The accumulated data suggest that the ability of eIF-4F to recycle may be compromised. The addition of eIF-4F to cell-free translation systems from unfertilized sea urchin eggs also stimulates protein synthesis. However, the stimulation does not increase protein synthetic activity in the egg cell-free translation system to the levels observed in those produced from 2-hr embryos. This suggests that, although the unfertilized egg contains an inhibitor of eIF-4F and reduced levels of eIF-4F activity, inactivation of this component is only one of the factors involved in the low rate of maternal mRNA utilization found prior to fertilization.
Oligonucleotide sequence signaling transcriptional termination of vaccinia virus early genes.
Authors: Yuen L , Moss B
Published: May 2006

In an in vitro system containing enzymes extracted from vaccinia virions, transcription of the vaccinia growth factor gene terminated approximately 50 base pairs downstream of a thymidine-rich sequence. Deletion mutagenesis suggested the presence of two tandem termination signals. The signal was identified by replacing the 3' end of the gene with the oligonucleotide AATTTTTAT that induced downstream termination. Further analysis of the transcripts formed with a series of templates containing 16 related synthetic oligonucleotides established the minimum functional termination signal as TTTTTNT, in which N represents any nucleotide. Termination efficiency may be increased, however, by the presence of an adenosine preceding the thymidine cluster. The general use of this signal at early times in infection but not at late times is supported by a survey of vaccinia virus gene sequences.
Human thrombomodulin gene is intron depleted: nucleic acid sequences of the cDNA and gene predict protein structure and suggest sites of regulatory control.
Authors: Jackman R W , Beeler D L , Fritze L , Soff G , Rosenberg R D
Published: May 2006

We have isolated a human thrombomodulin cDNA, and a human genomic clone containing the putative promoter domain, as well as the translated and untranslated regions of the endothelial cell receptor. The nucleotide sequence of the thrombomodulin cDNA allows us to provide a complete picture of the structure of this endothelial cell receptor, and to confirm its homology to the human low density lipoprotein receptor. The nucleotide sequence of the thrombomodulin gene suggests areas within the putative promoter domain that may be critical for regulating expression of the human endothelial cell receptor, indicates a potential signal peptide, and shows that no introns are present within the coding region. The overall organization of the human thrombomodulin gene is surprising because it represents an example of a gene that contains epidermal growth factor type B repeats and a membrane spanning region, which are not isolated within discrete exons.
A beta-galactosidase hybrid protein targeted to nuclei as a marker for developmental studies.
Authors: Bonnerot C , Rocancourt D , Briand P , Grimber G , Nicolas J F
Published: May 2006

The Escherichia coli lacZ gene has been used as an indicator gene for the study of cell lineage in vivo. To adapt this marker for gene expression studies, a sequence encoding a modified beta-galactosidase and including the simian virus 40 large tumor nuclear location signal (nls-beta-Gal) has been introduced into vectors. In differentiated cells, multipotential cells, and embryos, the constructs led to the expression of an enzymatically active protein. Its location was examined by its beta-galactosidase activity or by using antibodies and electron microscopy. The results show that the nls-beta-Gal protein remains mainly located at the nuclear periphery (probably at the nuclear pores) but does not reach the nucleoplasm. It suggests that an interaction with the nuclear membrane is necessary but not sufficient for protein uptake into the nucleus. In multipotential cells, the expression of nuclear location signal LacZ (nls-LacZ) interferes neither with cell growth nor with differentiation. Using various lacZ constructs, the transcriptional activity of embryos was studied. At the two-cell stage, the promoters of the Rous sarcoma virus, simian virus 40, and the beta-actin gene are functional but the Moloney murine leukemia virus long terminal repeat is not. Thus, transcriptional specificity must already be present at the stage of activation of the embryonic genome.
Accurate modification of a chromosomal plasmid by homologous recombination in human cells.
Authors: Song K Y , Schwartz F , Maeda N , Smithies O , Kucherlapati R
Published: May 2006

We have examined the consequences of modifying mammalian cellular DNA sequences by homologous recombination. A plasmid carrying a 248-base-pair deletion in the neomycin phosphotransferase (neo) gene was introduced into hamster and human cells. The integrated, defective neo gene was used as a target for modification by a second round of transfection with a plasmid carrying a different (283-base-pair) deletion in the neo gene. Recombinants resulting in an intact neo gene were selected by their G418 resistance phenotype. The best ratio of homologous to nonhomologous recombination events was about 1:80. Analyses of the functional neo genes in various independent cell lines establish that simple crossovers (single and double) generated the wild-type neo genes.
Ultrastructural evidence for the accumulation of insulin in nuclei of intact 3T3-L1 adipocytes by an insulin-receptor mediated process.
Authors: Smith R M , Jarett L
Published: May 2006

Monomeric ferritin-labeled insulin (Fm-Ins), a biologically active, electron-dense marker of occupied insulin receptors, was used to characterize the internalization of insulin in 3T3-L1 adipocytes. Fm-Ins bound specifically to insulin receptors and was internalized in a time- and temperature-dependent manner. Fm-Ins was found in cytoplasmic vesicles within 5-10 min at 37 degrees C and subsequently was observed in multivesicular bodies and lysosomes. In addition, small amounts of Fm-Ins were associated with nuclei after 30 min. The number of Fm-Ins particles observed in nuclei continued to increase in a time-dependent manner until at least 90 min. In the nucleus, several Fm-Ins particles usually were found in the same general location--near nuclear pores, associated with the periphery of the condensed chromatin. Addition of a 250-fold excess of unlabeled insulin or incubation at 15 degrees C reduced the number of Fm-Ins particles found in nuclei after 90 min by 99% or 92%, respectively. Nuclear accumulation of unlabeled ferritin was only 2% of that found with Fm-Ins after 90 min at 37 degrees C. Biochemical experiments utilizing 125I-labeled insulin and subcellular fractionation indicated that intact 3T3-L1 adipocytes internalized insulin rapidly and that approximately equal to 3% of the internalized ligand accumulated in nuclei after 1 hr. These data provide biochemical and high-resolution ultrastructural evidence that 3T3-L1 adipocytes accumulate potentially significant amounts of insulin in nuclei by an insulin receptor-mediated process. The transport of insulin or the insulin-receptor complex to nuclei in this cell or in others may be directly involved in the long-term biological effects of insulin--in particular, in the control of DNA and RNA synthesis.
Use of muscle contraction formalism for kinesin in fast axonal transport.
Authors: Hill T L
Published: May 2006

The general procedure is discussed for calculating the velocity of a vesicle along a microtubule. The formalism used previously for isotonic contraction in muscle (with multiple actin sites for a given cross-bridge) can be employed. However, some modifications must be made: (i) the kinetic diagram must include a state in which kinesin is absent from a vesicle binding site, (ii) an average must be taken over the locations of the vesicle binding sites relative to microtubule sites, and (iii) a self-consistency condition must be imposed that equates the mean force exerted by kinesin molecules on the vesicle with the frictional resisting force of the medium.
Sequence of an intestinal cDNA encoding human gastric inhibitory polypeptide precursor.
Authors: Takeda J , Seino Y , Tanaka K , Fukumoto H , Kayano T , Takahashi H , Mitani T , Kurono M , Suzuki T , Tobe T
Published: May 2006

Gastric inhibitory polypeptide (GIP) is a 42-amino acid hormone that stimulates insulin secretion in the presence of glucose. Complementary DNA clones encoding human GIP were isolated from a library prepared with RNA from duodenum. The predicted amino acid sequence indicates that GIP is derived by proteolytic processing of a 153-residue precursor, preproGIP. The GIP moiety is flanked by polypeptide segments of 51 and 60 amino acids at its NH2 and COOH termini, respectively. The former includes a signal peptide of about 21 residues and an NH2-terminal propeptide of 30 amino acids. GIP is released from the precursor by processing at single arginine residues. There is a region of nine amino acids in the COOH-terminal propeptide of the GIP precursor that has partial homology with a portion of chromogranin A as well as pancreastatin.
Light-stimulated inositolphospholipid turnover in
Samanea saman
leaf pulvini
Authors: Morse M. J. , Crain Richard C. , Satter Ruth L.
Published: May 2006

Leaflets of
Samanea saman
open and close rhythmically, driven by an endogenous circadian clock. Light has a rapid, direct effect on the movements and also rephases the rhythm. We investigated whether light signals might be mediated by increased inositolphospholipid turnover, a mechanism for signal transduction that is widely utilized in animal systems.
Samanea
motor organs (pulvini) labeled with [
3
H]inositol were irradiated briefly (5-30 sec) with white light, and membrane-localized phosphatidylinositol phosphates and their aqueous breakdown products, the inositol phosphates, were examined. After a 15-sec or longer light pulse, labeled phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate decreased and their labeled metabolic products inositol 1,4-bisphosphate and inositol 1,4,5-trisphosphate increased, changes characteristic of inositolphospholipid turnover. We conclude that inositolphospholipid turnover may act as a phototransduction mechanism in
Samanea
pulvini in a manner that is similar to that reported in animal systems.
Binary liquid phase separation and critical phenomena in a protein/water solution.
Authors: Thomson J A , Schurtenberger P , Thurston G M , Benedek G B
Published: May 2006

We have investigated the phase diagram of aqueous solutions of the bovine lens protein gamma II-crystallin. For temperatures T less than Tc = 278.5 K, we find that these solutions exhibit a reversible coexistence between two isotropic liquid phases differing in protein concentration. The dilute and concentrated branches of the coexistence curve were characterized, consistently, both by measurements of the two coexisting concentrations, c(T), and by measuring the cloud temperatures for various initial concentrations. We estimate that the critical concentration, cc, is 244 mg of protein per ml solution. The coexistence curve is well represented by the absolute value of (c - cc)/cc = 5.2 square root (Tc - T)/Tc. Using the temperature dependence of the scattered light intensity along isochores parallel to the critical isochore, we estimated the location of the spinodal line and found it to have the form (c - cc)/cc = 3.0 square root (Tc - T)/Tc. The ratio of the widths of the coexistence curve and the spinodal line, (5.2/3.0), is close to the mean-field value square root 3. We have also observed the growth of large crystals of gamma II-crystallin in some of these aqueous solutions and have made preliminary observations as to the factors that promote or delay the onset of crystallization. These findings suggest that selected protein/water systems can serve as excellent model systems for the study of phase transitions and critical phenomena.
Isolation of osteogenin, an extracellular matrix-associated, bone-inductive protein, by heparin affinity chromatography.
Authors: Sampath T K , Muthukumaran N , Reddi A H
Published: May 2006

Implantation of demineralized diaphyseal bone matrix in subcutaneous sites induces a sequence of events resulting in the local differentiation of endochondral bone. Demineralized bovine bone matrix was dissociatively extracted in 4.0 M guanidine hydrochloride and the bone-inductive proteins were purified greater than 12,000-fold. The purification steps include affinity chromatography on heparin-Sepharose, hydroxyapatite chromatography, gel filtration, and C18 reverse-phase HPLC. Since the purified protein in conjunction with insoluble collagenous bone matrix induced new bone differentiation in vivo we have designated this component osteogenin. The osteogenic potential is specific for osteogenin and is not exhibited by previously isolated growth factors.
Toward a theory for the evolution of cultural communication: coevolution of signal transmission and reception.
Authors: Aoki K , Feldman M W
Published: May 2006

A haploid sexual two-locus model of gene-culture coevolution is examined, in which a dichotomous phenotype subject to natural selection is transmitted vertically with probabilities dependent on the chosen parent's genotype and phenotype and the offspring's genotype. Stability conditions for the genetically monomorphic corner equilibria are obtained. In a specialization of this general model, one locus controls the transmission and the other controls the reception of adaptive information. The corner and edge equilibria of this doubly coevolutionary model are fully analyzed, and conditions for transmission and reception to coevolve are derived in terms of the efficiency of vertical transmission, the selective advantage gained from possessing the information, the costs of transmission and reception, and the recombination fraction between the two loci. Possible applications of the model are to the evolution of semantic alarm calls in vervet monkeys and the phonetic aspects of human language. In a third model with diploid genetics, we consider the initial increase of cultural transmission from a mutation-selection balance in which the adaptive phenotype is the consequence of a dominant gene at one locus. A second gene controls the transmission of the phenotype in such a way that a new mutant at this second locus permits learning of the adaptive phenotype from a parent who has it. This new mutant cannot increase when rare.
Transposon mutagenesis of type III group B Streptococcus: correlation of capsule expression with virulence.
Authors: Rubens C E , Wessels M R , Heggen L M , Kasper D L
Published: May 2006

The capsular polysaccharide of type III group B Streptococcus (GBS) is thought to be a major factor in the virulence of this organism. Transposon mutagenesis was used to obtain isogenic strains of a GBS serotype III clinical isolate (COH 31r/s) with site-specific mutations in the gene(s) responsible for capsule production. The self-conjugative transposon Tn916 was transferred to strain COH 31r/s during incubation with Streptococcus faecalis strain CG110 on membrane filters. Eleven transconjugant clones did not bind type III GBS antiserum by immunoblot. Immunofluorescence, competitive ELISA, and electron microscopy confirmed the absence of detectable GBS type III capsular polysaccharide in one of the transconjugants, COH 31-15. Southern hybridization analysis with a Tn916 probe confirmed the presence of the transposon sequence within each mutant. A 3.0-kilobase EcoRI fragment that flanked the Tn916 sequence was subcloned from mutant COH 31-15. This fragment shared homology with DNA from the other GBS serotypes, suggesting a common sequence for capsulation shared by organisms of different capsular types. Loss of capsule expression resulted in loss of virulence in a neonatal rat model. We conclude that a gene common to all capsular types of GBS is required for surface expression of the type III capsule and that inactivation of this gene by Tn916 results in the loss of virulence.
Flow cytometric resonance energy transfer measurements support the association of a 95-kDa peptide termed T27 with the 55-kDa Tac peptide.
Authors: Szöllösi J , Damjanovich S , Goldman C K , Fulwyler M J , Aszalos A A , Goldstein G , Rao P , Talle M A , Waldmann T A
Published: May 2006

Two monoclonal antibodies (OKT27 and OKT27b) have been produced that react with distinct epitopes of a 95-kDa peptide. The T27 antigen is widely distributed, being expressed on B lymphocytes, monocytes, and adult T-leukemic cells but not on polymorphonuclear leukocytes or platelets. There was a low level of T27 expression on resting T cells that increased on T-cell activation. In preliminary studies, the OKT27b antibody coprecipitated a 55-kDa peptide, as well as the 95-kDa peptide, from the radiolabeled cells of the HuT 102B2 cell line. Preclearance with anti-Tac, a monoclonal antibody to the 55-kDa peptide of the multichain interleukin 2 receptor, removed the 55-kDa but not the 95-kDa peptide from subsequent OKT27b immunoprecipitates of HuT 102B2 extracts, suggesting the possibility that the T27 peptide was associated with the Tac peptide. However, the precipitation of the p55 Tac peptide by OKT27b was quite inconsistent. Thus, additional information was sought using a flow cytometric energy transfer technique to provide a physical estimation of the proximity between the Tac and the T27 peptides. The flow cytometric version of the fluorescence resonance energy transfer technique permits the determination of inter- and intramolecular distances at 2- to 10-nm levels on a cell-by-cell basis. Using this approach, there was a mean energy transfer of 7.3% with HuT 102B2 cells when fluorescein isothiocyanate anti-Tac served as the donor and tetramethylrhodamine isothiocyanate OKT27 served as the acceptor. In contrast, there was no energy transfer in comparable studies observed when fluorescein anti-Tac and rhodamine anti-transferrin receptor antibodies were used. These observations support the conclusion that there is a close nonrandom proximity in HuT 102B2 cells between the 95-kDa peptide identified by the OKT27 monoclonal antibody and the p55 Tac peptide of the multichain interleukin 2 receptor.