bluuebunny/crossref_metadata_2025 huggingface dataset:
Partial suppression of anchorage-independent growth and tumorigenicity in immunodeficient mice by transfection of the H-2 class I gene H-2Ld into a human colon cancer cell line (HCT).
Authors: Gattoni-Celli S , Willett C G , Rhoads D B , Simon B , Strauss R M , Kirsch K , Isselbacher K J
Published: May 2006

Many human tumors, particularly those of epithelial origin, appear to express greatly reduced levels of major histocompatibility complex class I antigens on their surface. It has been previously reported that the class I gene H-2Ld, introduced into adenovirus type 12-transformed mouse cells, induces reversal of oncogenesis in immunocompetent BALB/c mice. We have tested the hypothesis that the H-2Ld gene, when transfected into HCT colon cancer cells, may alter their transformed phenotype. Two H-2Ld transfectants, HCT-Ii and HCT-If, were found to exhibit a markedly reduced-to-virtually suppressed ability to form colonies in soft agar in comparison to a transfectant (HCTh) carrying only the neomycin-resistance gene. We also compared the tumorigenicity of HCTh vs. HCT-If cells in two different strains of immunodeficient mice: nude (T-) and triple-deficient mutants (T-, NK-, B-). At 28 days postinjection of 10(7) and 10(6) cells, the size and growth rate of HCT-If tumors were greatly reduced compared to HCTh cells. Therefore, as assayed in immunodeficient animals, expression of the class I H-2Ld gene in HCT cells appears to correlate with partial suppression of the tumorigenic phenotype, suggesting that the expression of a transfected class I gene may by itself alter the phenotype of the recipient cell and that such phenotypic changes may be independent of the immune system.
Physical linkage of the genes for platelet membrane glycoproteins IIb and IIIa.
Authors: Bray P F , Barsh G , Rosa J P , Luo X Y , Magenis E , Shuman M A
Published: May 2006

The fibrinogen receptor on human platelets is a prototypic member of the integrin family and is composed of subunit glycoproteins IIb (gpIIb) and IIIa (gpIIIa) in a 1:1 stoichiometric ratio. We have isolated cDNA clones for gpIIb and gpIIIa and localized both genes to chromosome 17. In the current study, several approaches were used to localize and map the genes for gpIIb and gpIIIa. A preliminary evaluation of subchromosomal localization was performed by using a panel of mouse-human somatic cell hybrids that contain different amounts of the long arm of human chromosome 17. Southern hybridization to the DNA of these hybrids shows that both genes map near the thymidine kinase gene. In situ hybridization to intact human chromosomes localized both genes to the 17q21-22 region. To better define the physical distance between the two genes, we examined the genomic hybridization pattern of each cDNA probe to high molecular weight restriction fragments separated by pulsed-field gel electrophoresis. Serial hybridizations of the same filter have allowed construction of long-range Mlu I and Sfi I restriction maps spanning more than 500 kilobases. Finally, nonoverlapping portions of the cDNAs for both gpIIb and gpIIIa were used to probe Sfi I digests of genomic DNA separated by field-inversion gels. This confirmed that the genes are physically linked within the same 260-kilobase Sfi I fragment and suggests that the gene for gpIIb is located on the 3' side of the gene for gpIIIa. These results suggest that coordinate expression of gpIIb and gpIIIa may depend on physical proximity.
Eradication of Epstein-Barr virus by allogeneic bone marrow transplantation: implications for sites of viral latency.
Authors: Gratama J W , Oosterveer M A , Zwaan F E , Lepoutre J , Klein G , Ernberg I
Published: May 2006

Wild-type strains of Epstein-Barr virus (EBV) can be distinguished on the basis of variations in the molecular weight of virus-encoded, growth transformation-associated proteins. This approach was used to study the persistence of EBV in two seropositive recipients of allogeneic bone marrow transplants. The first patient received marrow from her EBV-seronegative brother, became EBV seronegative after grafting, and remained so for greater than 1200 days. Subsequently, she became infected with a new EBV strain that differed from her pretransplant strain but was indistinguishable from the virus isolated from her husband. The second patient received marrow from his EBV-seropositive brother. This patient showed only a transient decrease in IgG antibodies to EBV capsid antigen. His pretransplant strain differed from the virus of his donor. On days 252 and 915 after transplantation, lymphoblastoid cell lines were grown from the peripheral blood of the patient and were found to carry exclusively the virus of the donor. These results suggest that the latently EBV-infected host cells reside in a cellular compartment that can be destroyed by graft-versus-host reactivity, irradiation, or cytotoxic drugs. Hemopoietic tissue is the most likely candidate.
Metal-specific synthesis of two metallothioneins and gamma-glutamyl peptides in Candida glabrata.
Authors: Mehra R K , Tarbet E B , Gray W R , Winge D R
Published: May 2006

Cellular resistance to heavy metal cytotoxicity in most species is mediated by the binding of metal ions either to a cysteine-rich polypeptide in the metallothionein family or to short cysteine-containing gamma-glutamyl peptides. One of these metal binding systems has been found in most organisms studied. However, the yeast Candida (Torulopsis) glabrata expresses both metallothionein and the gamma-glutamyl peptides for metal detoxification, and each system is regulated in a metal-specific manner. Exposure of C. glabrata to copper salts stimulates formation of two metallothionein-like polypeptides with a cysteine content of 30 mol% and the repeated sequence Cys-Xaa-Cys. The cells synthesize gamma-glutamyl peptides upon exposure to cadmium salts. Penta- and tetrapeptides that form a cadmium-thiolate cluster in a peptide oligomer containing labile sulfur are synthesized.
Two-dimensional protonic percolation on lightly hydrated purple membrane
Authors: Rupley John A. , Siemankowski Linda , Careri Giorgio , Bruni Fabio
Published: May 2006

The capacitance and dielectric loss factor were measured for a sample of purple membrane of
Halobacterium halobium
as a function of hydration level (0.017 to >0.2 g of water/g of membrane) and frequency (10 kHz to 10 MHz). The capacitance and the derived conductivity show explosive growth above a threshold hydration level,

h
c

≈ 0.0456. The conductivity shows a deuterium isotope effect, H/
2
H = 1.38, in close agreement with expectation for a protonic process. The level

h
c

is frequency independent and shows no deuterium isotope effect. These properties are analogous to those found for lysozyme in a related study. Protonic conduction for the purple membrane can be considered, as for lysozyme, within the framework of a percolation model. The critical exponent,
t
, which describes the conductivity of a percolative system near the threshold, has the value 1.23. This number is in close agreement with expectation from theory for a two-dimensional percolative process. The dielectric properties of the purple membrane are more complex than those of lysozyme, seen in the value of

h
c

and in the frequency and hydration dependence of the loss factor. There appear to be preferred regions of proton conduction. The percolation model is based upon stochastic behavior of a system partially populated with conducting elements. This model suggests that ion transport in membranes and its control can be based on pathways formed of randomly connected conducting elements and that a fixed geometry (a proton wire) is not the only possible basis for a mechanism of conduction.
Antibody to phosphatidylinositol 4,5-bisphosphate inhibits oncogene-induced mitogenesis.
Authors: Fukami K , Matsuoka K , Nakanishi O , Yamakawa A , Kawai S , Takenawa T
Published: May 2006

The hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) has been shown to be enhanced in cells transformed by some types of oncogenes and tumor viruses, but it is still unknown whether the breakdown of PIP2 plays a role in oncogene-induced cell proliferation. For examination of this problem, monoclonal antibody specifically directed to PIP2 was injected into cells. This antibody bound to endogenous PIP2 and so inhibited its intracellular breakdown. Injection of this antibody into ras-transformed cells cultured in the presence of serum caused reversible and dose-dependent decrease in proliferation and reversion of the cell morphology to that of the normal phenotype. The antibody also inhibited the proliferation of src- and erbB-transformed cells but had no effect on the proliferation of untransformed or myc-transformed cells. These results show that the breakdown of PIP2 is involved in the signaling pathways for mitogenesis in cells transformed by oncogenes such as ras, src, and erbB.
Conditional enhancement of liver-specific gene transcription.
Authors: Zaret K S , DiPersio C M , Jackson D A , Montigny W J , Weinstat D L
Published: May 2006

We sought to develop a cell line in which liver-specific transcription could be induced at will, to facilitate the study of factors that cause hepatocyte-specific transcription of the serum albumin gene in mice. We therefore created the H2.35 cell line from mouse hepatocytes infected with a temperature-sensitive strain of simian virus 40. During routine propagation at the permissive temperature, H2.35 cells exhibit extremely low levels of albumin transcription and mRNA. Albumin mRNA increases at least 100-fold when H2.35 cells are cultured at the restrictive temperature and in serum-free medium on a collagen substratum; the two latter conditions maintain the differentiated state of primary hepatocyte cultures. Although a major cause of the mRNA increase is posttranscriptional, the transcription rates of albumin and other liver-specific genes increase significantly. Transient-transfection experiments demonstrated that an induction of transcription is caused by activation of an albumin upstream sequence that was previously shown to enhance liver-specific transcription in transgenic mice. Thus, hepatocyte differentiation appears to be maintained in part by extracellular signals that stimulate the activity of a tissue-specific enhancer element.
Defining cellular senescence in IMR-90 cells: a flow cytometric analysis.
Authors: Sherwood S W , Rush D , Ellsworth J L , Schimke R T
Published: May 2006

Using multiparameter flow cytometric analysis, we find that senescent cells accumulate in a unique cell-cycle compartment characterized in cell-cycle arrest in G1 and a significantly reduced nucleocytoplasmic ratio (genome size/cell mass) relative to cycling cells. With respect to gross cellular phenotype, the quiescent state of senescent cells differs from quiescence induced by density inhibition; the former is associated with a reduction in the nucleocytoplasmic ratio, while the latter is associated with an increase in the nucleocytoplasmic ratio. Senescent cells were present at all passages examined. The frequency of senescent cells was low in early-passage cultures and increased with passage number. Senescence of populations of IMR-90 cells reflects change in the relative frequency of these cells. The frequency of cells with karyotypic changes increased with the progressive accumulation of out-of-cycle cells.
Recombinant tumor necrosis factor increases pulmonary vascular permeability independent of neutrophils.
Authors: Horvath C J , Ferro T J , Jesmok G , Malik A B
Published: May 2006

We studied the effects of intravenous infusion of recombinant human tumor necrosis factor type alpha (rTNF-alpha; 12 micrograms/kg) on lung fluid balance in sheep prepared with chronic lung lymph fistulas. The role of neutrophils was examined in sheep made neutropenic with hydroxyurea (200 mg/kg for 4 or 5 days) before receiving rTNF-alpha. Infusion of rTNF-alpha resulted in respiratory distress and 3-fold increases in pulmonary arterial pressure and pulmonary vascular resistance within 15 min, indicating intense pulmonary vasoconstriction. Pulmonary lymph flow (i.e., net transvascular fluid filtration rate) and transvascular protein clearance rate (a measure of vascular permeability to protein) increased 2-fold within 30 min. The increased permeability was associated with leukopenia and neutropenia. The pulmonary hypertension and vasoconstriction subsided but fluid filtration and vascular permeability continued to increase. Sheep made neutropenic had similar increases in pulmonary transvascular fluid filtration and vascular permeability. rTNF-alpha also produced concentration-dependent increases in permeability of 125I-labeled albumin across ovine endothelial cell monolayers in the absence of neutrophils or other inflammatory mediators. The results indicate that rTNF-alpha increases pulmonary vascular permeability to protein by an effect on the endothelium.
Binding region for human immunodeficiency virus (HIV) and epitopes for HIV-blocking monoclonal antibodies of the CD4 molecule defined by site-directed mutagenesis.
Authors: Mizukami T , Fuerst T R , Berger E A , Moss B
Published: May 2006

The binding region for human immunodeficiency virus (HIV) and epitopes for a panel of HIV-blocking anti-CD4 monoclonal antibodies of the CD4 molecule were defined by using in vitro site-directed mutagenesis. Codons for two amino acid residues (Ser-Arg) were inserted at selected positions within the region encoding the first and second immunoglobulin-like domains of CD4. A vaccinia virus-based expression system was used to produce soluble full-length extracellular CD4 fragments containing the insertions. The mutant proteins were tested for direct binding to soluble gp120 (the CD4-binding subunit of the viral envelope glycoprotein) and to a series of HIV-blocking anti-CD4 monoclonal antibodies. Impaired gp120 binding activity resulted from insertions after amino acid residues 31, 44, 48, 52, 55, and 57 in the first immunoglobulin-like domain. The epitopes for two HIV-blocking monoclonal antibodies, OKT4A and OKT4D, were also mapped in the gp120-binding region in the first domain. Insertions after amino acid residues 21 and 91 in the first domain had no effect on gp120 binding but impaired the binding of OKT4E, suggesting that this antibody recognizes a discontinuous epitope not directly involved in gp120 binding. Moderate impairment of gp120 binding resulted from the insertion after amino acid residues 164 in the second immunoglobulin-like domain, where the epitopes for monoclonal antibodies MT151 and OKT4B were also mapped.
High-level expression of a bioengineered, cysteine-free hepatocyte-stimulating factor (interleukin 6)-like protein.
Authors: Jambou R C , Snouwaert J N , Bishop G A , Stebbins J R , Frelinger J A , Fowlkes D M
Published: May 2006

Hepatocyte-stimulating factor, interferon-beta 2, B-cell stimulation factor 2, and hybridoma/plasmacytoma growth factor are identical proteins presently referred to as interleukin 6 (IL-6). Through the use of synthetic oligonucleotide technology, we have constructed a biologically active recombinant IL-6 (rIL-6) gene based on the sequence of a human IL-6 cDNA. The synthetic gene encodes a cysteine-free, bioengineered rIL-6 protein that is expressed at high levels in Escherichia coli as a tripartite fusion protein. Cleavage of the fusion protein with collagenase releases a 23-kDa rIL-6 protein that can be easily purified to homogeneity. We show that the rIL-6 protein displays a range of biological activities similar to those of natural human IL-6, as demonstrated by its ability to (i) protect cells from viral infection, (ii) stimulate the synthesis of fibrinogen in rat FAZA 967 cells, and (iii) induce the terminal differentiation of B cells, resulting in elevated secretion of immunoglobulin.
A poxvirus-derived vector that directs high levels of expression of cloned genes in mammalian cells.
Authors: Patel D D , Ray C A , Drucker R P , Pickup D J
Published: May 2006

High levels of expression of cloned genes have been obtained in mammalian cells by using poxvirus-derived insertion/expression vectors. These vectors employ the cis-acting element (CAE I) that directs the transcription of one of the most strongly expressed genes of cowpox virus. This gene (the 160K gene) encodes the 160-kDa protein that is the major component of the A-type cytoplasmic inclusions. Its counterpart in vaccinia virus (VV) is the 94K gene contained in the HindIII A fragment of the viral DNA. Two insertion vectors have been constructed; each is designed to allow cloned genes to be placed immediately downstream of a modified version of CAE I within a poxvirus genome. One vector, p1200, enables the CAE I-cloned-gene constructs to be inserted into the thymidine kinase gene of VV. This vector was used to create a VV recombinant that directed expression of the chloramphenicol acetyltransferase (CAT) gene. The other vector, p2101, enables the CAE I-cloned-gene constructs to be inserted into the VV 94K gene. The prototype of this vector was used to create a VV recombinant that directed expression of a hybrid CAT-lacZ gene. Infection of cultured human cells with these recombinants led to high levels of synthesis of either the CAT gene product or the CAT-lacZ gene product. Each of these proteins was produced in quantities that were easily detected by Coomassie blue staining of total cell proteins resolved by polyacrylamide gel electrophoresis. We estimate that these vectors are capable of directing the synthesis of milligram amounts of gene product per 10(9) mammalian cells.
Monomeric and aggregated bacteriorhodopsin: Single-turnover proton transport stoichiometry and photochemistry
Authors: Grzesiek Stephan , Dencher Norbert A.
Published: May 2006

The question of the basic functional transport unit of bacteriorhodopsin (BR) has been addressed by comparing the proton pumping stoichiometry as well as the photocycle kinetics of monomeric and aggregated BR in phospholipid vesicles. When time-resolved laser spectroscopy was used in combination with the optical pH-indicator pyranine, single-turnover experiments revealed approximately 0.5-0.8 and 0.8-1.2 protons vectorially translocated per photocycling monomeric and aggregated BR molecule, respectively. Since both these values are akin and very similar to the pumping stoichiometry of crystalline BR molecules in the purple membrane, the BR monomer has been proven to be the essential transport unit. The natural arrangement of the photopigments in a crystalline array of immobilized trimers is not required for efficient vectorial proton translocation.
Polarized apical distribution of glycosyl-phosphatidylinositol-anchored proteins in a renal epithelial cell line.
Authors: Lisanti M P , Sargiacomo M , Graeve L , Saltiel A R , Rodriguez-Boulan E
Published: May 2006

Polarized epithelial cell monolayers contain two distinct plasma membrane domains as delineated by the presence of tight junctions--i.e., an apical surface that faces the external environment and a basolateral surface that functions both in cell-cell contact and cell-substrate attachment. Central to the understanding of epithelial cell polarity is the question of how such cell-surface specializations are generated. A different class of membrane glycoproteins has recently emerged that may yield new insight into the mechanism underlying the biogenesis of this polarity. Members of this class contain a large extracellular protein domain linked to the membrane via glycosyl-phosphatidylinositol. Using a polarized renal epithelial cell line (Madin-Darby canine kidney), we identified endogenous glycosyl-phosphatidylinositol-anchored proteins through release by a phosphatidylinositol-specific phospholipase C. Six glycosyl-phosphatidylinositol-anchored proteins of 110, 85, 70, 55, 38, and 35 kDa were identified and appeared to be restricted to the apical surface. Our data are consistent with the notion that the glycosyl-phosphatidylinositol membrane anchor may contain the necessary information for "targeting" to the apical surface.
Functional diversity of gro gene expression in human fibroblasts and mammary epithelial cells.
Authors: Anisowicz A , Zajchowski D , Stenman G , Sager R
Published: May 2006

Previous studies of gro and related genes that are overexpressed in transformed fibroblasts suggest that gro may encode a specific growth regulator. However, DNA and protein sequence comparisons reveal relatedness to platelet factor 4 and other proteins involved in the inflammatory response. In this paper, both growth-related and cytokine-induced responses in gro gene expression are described. Human foreskin fibroblasts are shown to express approximately 10-fold elevated gro, myc, and fos mRNAs in response to serum and to phorbol 12-myristate 13-acetate stimulation, with early response kinetics indicative of growth regulation. In response to interleukin 1, however, in growing cells gro mRNA is elevated at least 100-fold but myc remains constant and fos is not expressed, suggesting a second regulatory pathway. In normal cultured mammary epithelial cells, gro is constitutively expressed, and elevated mRNA levels are induced by phorbol 12-myristate 13-acetate, but not by interleukin 1. However, most carcinoma cell lines examined do not express gro mRNA, suggesting a third function of gro as a negative growth regulator in epithelial cells.
Rapid separation and purification of oligonucleotides by high-performance capillary gel electrophoresis.
Authors: Cohen A S , Najarian D R , Paulus A , Guttman A , Smith J A , Karger B L
Published: May 2006

Picomole amounts of oligodeoxynucleotides [polydeoxyadenylic acids, (dA)40-60] were baseline resolved and analyzed in less than 8 min by high-performance capillary electrophoresis with polyacrylamide gels. In addition, fast analysis of a crude 70-mer oligodeoxynucleotide and a slab gel-purified 99-mer oligodeoxynucleotide was accomplished, demonstrating the ability of high-performance capillary electrophoresis to characterize rapidly synthesized oligonucleotides. Besides analytical separations, 800 ng of a primer (20-mer) was isolated in less than 20 min. The purified species was collected in water and subsequently used as a probe in a standard dot-blot analysis. The use of high-performance capillary electrophoresis for the analysis and purification of a variety of biopolymers is simple, rapid, and has the potential for automation.
In vivo stability of ester- and ether-linked phospholipid-containing liposomes as measured by perturbed angular correlation spectroscopy.
Authors: Derksen J T , Baldeschwieler J D , Scherphof G L
Published: May 2006

To evaluate liposome formulations for use as intracellular sustained-release drug depots, we have compared the uptake and degradation in rat liver and spleen of liposomes of various compositions, containing as their bulk phospholipid an ether-linked phospholipid or one of several ester-linked phospholipids, by perturbed angular correlation spectroscopy. Multilamellar and small unilamellar vesicles (MLVs and SUVs), composed of egg phosphatidylcholine, sphingomyelin, distearoyl phosphatidylcholine (DSPC), dipalmitoyl phosphatidylcholine (DPPC) or its analog dihexadecylglycerophosphorylcholine (DHPC), and cholesterol plus phosphatidylserine, and containing 111In complexed to nitrilotriacetic acid, were injected intravenously in rats. Recovery of 111In-labeled liposomes in blood, liver, and spleen was assessed at specific time points after injection and the percentage of liposomes still intact in liver and spleen was determined by measurement of the time-integrated angular perturbation factor [G22(infinity)] of the 111In label. We found that MLVs but not SUVs, having DHPC as their bulk phospholipid, showed an increased resistance against lysosomal degradation as compared to other phospholipid-containing liposomes. The use of diacyl phospholipids with a high gel/liquid-crystalline phase-transition temperature, such as DPPC and DSPC, also retarded degradation of MLV, but not of SUV in the dose range tested, while the rate of uptake of these liposomes by the liver was lower.
Patterns of gene expression and sites of latency in human nerve ganglia are different for varicella-zoster and herpes simplex viruses.
Authors: Croen K D , Ostrove J M , Dragovic L J , Straus S E
Published: May 2006

The cellular localization and viral transcription patterns of acute and latent varicella-zoster virus (VZV) infections of human sensory nerve ganglia were studied by in situ hybridization and compared with those of latent herpes simplex virus (HSV) infection. Trigeminal and dorsal root ganglia obtained at autopsy were hybridized with 35S-labeled single-stranded RNA probes homologous to VZV or HSV fragments. We have reported that HSV persists in human sensory neurons and expresses only one family of transcripts that overlap extensively with, but are opposite in polarity to, the mRNA encoding the immediate early protein termed infected cell protein 0 (ICP0). In the present study we find that latent VZV infection involves nonneuronal cells, and multiple, but not all, VZV genes are transcribed. In contrast, during varicella both neuronal and nonneuronal cells are infected, with all regions of the VZV genome analyzed being expressed. Thus, the patterns of gene expression and cellular locations of VZV and HSV infections of human ganglia differ. The differences may underlie clinical features that distinguish these infections.
Localization of mRNA for low density lipoprotein receptor and a cholesterol synthetic enzyme in rabbit nervous system by in situ hybridization.
Authors: Swanson L W , Simmons D M , Hofmann S L , Goldstein J L , Brown M S
Published: May 2006

The low density lipoprotein receptor and one of its ligands, apoprotein E, are known to be synthesized in the central nervous system. In the current study, we used in situ hybridization to localize the receptor mRNA in selected neurons and glia throughout the nervous system of 9-day-old rabbits. Particularly high levels were found in sensory ganglia, sensory nuclei, and motor-related nuclei. The same regions contained high levels of mRNA for 3-hydroxy-3-methylglutaryl-coenzyme A synthase, a regulated enzyme in cholesterol biosynthesis. The distribution of low density lipoprotein receptor mRNA was similar in mature and immature rabbits. The data suggest that certain cells in the nervous system have high requirements for cholesterol, which they satisfy through cholesterol synthesis and through receptor-mediated uptake of cholesterol-carrying lipoproteins. The latter originate in astrocytes which synthesize and secrete apoprotein E. These data suggest that the nervous system of mammals contains an active system for continuous redistribution and recycling of cholesterol that is physically distinct from the lipoprotein transport system in plasma.
Gin-mediated DNA inversion: product structure and the mechanism of strand exchange.
Authors: Kanaar R , van de Putte P , Cozzarelli N R
Published: May 2006

Inversion of the G loop of bacteriophage Mu requires the phage-encoded Gin protein and a host factor. The topological changes in a supercoiled DNA substrate generated by the two purified proteins were analyzed. More than 99% of the inversion products were unknotted rings. This result excludes synapsis by way of a random collision of recombination sites, because the resulting entrapped supercoils would be converted into knots by recombination. Instead, the recombination sites must come together in the synaptic complex in an ordered fashion with a fixed number of supercoils between the sites. The linking number of the substrate DNA increases by four during recombination. Thus, in three successive rounds of inversion, the change in linking number was +4, +8, and +12, respectively. These results lead to a quantitative model for the mechanism of Gin recombination that includes the distribution of supercoils in the synaptic complex, their alteration by strand exchange, and specific roles for the two proteins needed for recombination.
Several distinct "CCAAT" box binding proteins coexist in eukaryotic cells.
Authors: Raymondjean M , Cereghini S , Yaniv M
Published: May 2006

Even though a "CCAAT" box element is localized approximately equal to 80 base pairs upstream of the transcription initiation site in many eukaryotic promoters, it is not recognized by a single ubiquitous transcription factor. We show here that such a sequence present in the rat albumin promoter binds a protein distinct from the CCAAT-binding transcription factor CTF/NFI or from the CCAAT-binding protein (CBP) previously described. The protein binding to the albumin CCAAT sequence (ACF or albumin CCAAT factor) is not exclusive to liver, since we found a protein with identical properties in spleen.
Tumorigenic human squamous lung cancer cells have defined cell surface carbohydrates that are absent from nontumorigenic cells.
Authors: Pettijohn D E , Pfenninger O , Brown J , Duke R , Olsson L
Published: May 2006

Cells of cloned lines derived from human squamous lung carcinomas spontaneously become heterogeneous with respect to several tumor-associated cell surface carbohydrates such as the sialosyl-Lea oligosaccharide antigen or the recently described oligosaccharide recognized by monoclonal antibody 43-9F. Subclones derived from these cultures are initially homogeneous with respect to the presence or absence of a specific cell surface carbohydrate but gradually revert back to a heterogeneous population. Cells of homogeneous subclones having both the sialosyl-LEa and 43-9F cell surface antigens and other subclones lacking them were injected subcutaneously in nude mice. All clones expressing these tumor-associated cell surface carbohydrates were found to be highly tumorigenic, whereas those lacking them were nontumorigenic or, at most, weakly tumorigenic. Clones having the tumor-associated cell surface carbohydrates were more resistant to cytotoxic attack by purified mouse natural killer cells than those clones lacking these carbohydrates, suggesting that the tumorigenicity of the former clones may be influenced by immunoprotective effects of these novel carbohydrates.
Oligodendrocyte substratum adhesion modulates expression of adenylate cyclase-linked receptors.
Authors: Vartanian T , Sprinkle T J , Dawson G , Szuchet S
Published: May 2006

The molecular mechanisms of myelin formation/reformation in the central nervous system are unknown. In previous work we have demonstrated that mature oligodendrocytes (OLG) respond to a signal(s), elicited by their adhesion to a substratum, by turning on a myelinogenic metabolism. Events occurring within 24 hr of adhesion include generation of diacylglycerol, activation of protein kinase C, phosphorylation of myelin basic protein, and enhanced synthesis of myelin lipids and proteins. To elucidate the mechanism(s) of signal transduction, we have investigated whether OLG-substratum interaction influences the level of basal cAMP and the expression of receptors coupled to adenylate cyclase. By using ovine brain OLG we have found that adhesion to a polylysine-coated surface for 24 hr increased the basal level of cAMP 2-fold and altered the expression (assessed by cAMP production) of receptors coupled to adenylate cyclase. Isoproterenol (beta-adrenergic agonist) augmented cAMP from 4 to 26 pmol/mg of protein in adhering OLG but had no such effect in nonattached OLG. Adhesion of OLG was accompanied by rapid synthesis of ethanolamine plasmalogen, a class of lipids believed to be associated with beta-adrenergic receptors. Nonattached OLG responded to prostaglandin E1 with only a 3-fold stimulation in their cAMP content; in attached OLG, 6-fold stimulation was observed. In contrast, vasoactive intestinal polypeptide elicited a 3-fold increase in cAMP in nonattached OLG but, following 24 hr of attachment, OLG did not respond to vasoactive intestinal polypeptide. The increase of cellular cAMP levels was accompanied by a 2.5-fold gain in protein kinase A. OLG-substratum adhesion resulted also in phosphorylation of the OLG/myelin protein, 2',3'-cyclic nucleotide 2'-phosphodiesterase, which proved to be a substrate for cAMP and phospholipid-, Ca2+-dependent protein kinases. These findings, in conjunction with our earlier work, implicate cAMP and diacylglycerol in signaling myelinogenesis; they suggest that phosphorylation/dephosphorylation of myelin basic protein and 2',3'-cyclic nucleotide 2'-phosphodiesterase may be key processes in the cascade of events that are initiated by adhesion of OLG to a polylysine surface (possibly acting as a surrogate for axons) and culminate in the reformation of myelin.
Regulation of the ecdysteroid titer of Manduca sexta: reappraisal of the role of the prothoracic glands.
Authors: Warren J T , Sakurai S , Rountree D B , Gilbert L I , Lee S S , Nakanishi K
Published: May 2006

It is generally accepted that the prothoracic glands of insects produce ecdysone, which is converted by a 20-monooxygenase in peripheral tissues to the major molting hormone, 20-hydroxyecdysone. Incubation in vitro of the prothoracic glands of larval or pupal Manduca sexta in the presence of a hemolymph protein fraction (HPF) increased the ecdysteroid content of the medium almost 8-fold. A comparable increase was noted when HPF was added to medium preconditioned with prothoracic glands but from which the glands had been removed. We used a differential RIA to show that a major product of the prothoracic glands in vitro cross-reacts with antiserum (20-hydroxyecdysone-2-succinylthyroglobulin amide; H-2) that retains affinity to ecdysteroids having a modified A ring. However, this product did not bind to antiserum (ecdysone-22-succinylthyroglobulin amide; H-22) that has affinity mainly for ecdysteroids modified at the side chain. We employed radiolabeled precursor studies with prothoracic glands in vitro and a combination of analytical techniques (NMR, CD, MS) to demonstrate that the major ecdysteroid release from the glands is a mixture of 2-dehydroecdysone and 3-dehydroecdysone (1:2), which is rapidly reduced to ecdysone in the presence of HPF. We postulate that the active component of HPF is 3 beta 3 beta (2 beta)-formin-3(2)-ketoecdysteroid reductase. These results may explain several anomalous observations pertaining to the molting of insect fragments in the absence of prothoracic glands and suggest a complex system for the control of insect molting and metamorphosis.
Quasi-random graphs
Authors: Chung F. R. K. , Graham R. L. , Wilson R. M.
Published: May 2006

We introduce a large equivalence class of graph properties, all of which are shared by so-called random graphs. Unlike random graphs, however, it is often relatively easy to verify that a particular family of graphs possesses some property in this class.
Isolation of a second yeast Saccharomyces cerevisiae gene (GPA2) coding for guanine nucleotide-binding regulatory protein: studies on its structure and possible functions.
Authors: Nakafuku M , Obara T , Kaibuchi K , Miyajima I , Miyajima A , Itoh H , Nakamura S , Arai K , Matsumoto K , Kaziro Y
Published: May 2006

In a previous paper, we demonstrated that a gene coding for a protein homologous to the alpha subunit of mammalian guanine nucleotide-binding regulatory (G) proteins occurs in Saccharomyces cerevisiae. The gene, designated GPA1, encodes a protein (GP1 alpha) of 472 amino acids with a calculated Mr of 54,075. Here we report the isolation of another G-protein-homologous gene, GPA2, which encodes an amino acid sequence of 449 amino acid residues with a Mr of 50,516. The predicted primary structure of the GPA2-encoded protein (GP2 alpha) is homologous to mammalian G proteins [inhibitory and stimulatory G proteins (Gi and Gs, respectively), a G protein of unknown function (Go), and transducins (Gt)] as well as yeast GP1 alpha. When aligned with the alpha subunit of Gi (Gi alpha) to obtain maximal homology, GP2 alpha was found to contain a stretch of 83 additional amino acid residues near the NH2 terminus. The gene was mapped in chromosome V, close to the centromere. Haploid cells carrying a disrupted GPA2 gene are viable. Cells carrying a high copy number of plasmid GPA2 (YEpGPA2) had markedly elevated levels of cAMP and could suppress a temperature-sensitive mutation of RAS2. These results suggest that GPA2 may be involved in the regulation of cAMP levels in S. cerevisiae.
A 40-base-pair sequence in the 3' end of the beta-actin gene regulates beta-actin mRNA transcription during myogenesis.
Authors: DePonti-Zilli L , Seiler-Tuyns A , Paterson B M
Published: May 2006

In an earlier report, evidence was presented that the down-regulation of beta-actin mRNA during myogenesis was controlled by a region 3' to the promoter of the gene. In this paper we report the location of this regulatory sequence, determined by deletion analysis and the use of chimeric genes, transfected stably into the mouse myogenic cell line C2C12. The domain responsible for the reduction in beta-actin mRNA levels is at most 40 base pairs long and is located just 5' to the canonical polyadenylylation signal in the gene. Placement of this sequence in the corresponding 3' position both in the alpha-cardiac-actin gene and in the neomycin-resistance gene in pSV2-neo confers the beta-actin mRNA regulatory pattern when these constructs are stably introduced into C2C12 cells. Nuclear run-on experiments indicate that transcriptional control can account for the decrease observed in beta-actin mRNA levels during myogenesis for both the endogenous as well as the transfected beta-actin gene constructs. This 3' transcriptional control sequence is conserved in all of the vertebrate beta-actin genes sequenced and is not similar to any of the 3' processing-adenylylation or termination sequences described previously. This mode of gene regulation may reflect a more general mechanism involved in the process of gene suppression during development.
Ligand activation of overexpressed epidermal growth factor receptors transforms NIH 3T3 mouse fibroblasts.
Authors: Riedel H , Massoglia S , Schlessinger J , Ullrich A
Published: May 2006

The cell surface receptor for the mitogenic peptide epidermal growth factor (EGF) is involved in control of normal cell growth and may play a role in the genesis of human neoplasia such as squamous carcinoma and glioblastoma. Soft-agar growth and focus-formation experiments with NIH 3T3 mouse fibroblasts transfected with an expression plasmid demonstrated the ligand-dependent transforming potential of the human EGF receptor without structural alterations. Activation of overexpressed normal receptor alone appears to be sufficient for transformation of NIH 3T3 cells in vitro.
Expression and developmental control of platelet-derived growth factor A-chain and B-chain/Sis genes in rat aortic smooth muscle cells.
Authors: Majesky M W , Benditt E P , Schwartz S M
Published: May 2006

Cultured arterial smooth muscle cells (SMC) can produce platelet-derived growth factor (PDGF)-like molecules. This property raises the possibility that SMC-derived PDGFs function as autocrine/paracrine regulators in the formation and maintenance of the artery wall. In this study we have asked if levels of mRNAs directing synthesis of PDGF are modulated in aortic SMC during postnatal development. We report here that genes encoding PDGF A- and B-chain precursors are expressed at similar low levels in intact aortas from newborn and adult rats. Marked differences in regulation of transcript abundance of these genes were revealed when aortic SMC were grown in cell culture. PDGF B-chain transcripts accumulated in passaged newborn rat SMC but not adult rat SMC, whereas PDGF A-chain RNA was found in comparable amounts in SMC from both age groups. Similarly, SMC from newborn rats secreted at least 60-fold more PDGF-like activity into conditioned medium than did adult rat SMC. PDGF B-chain transcripts in newborn rat aortic SMC are short-lived and increased 5-fold by 3 hr after treatment with cycloheximide. In contrast, PDGF A-chain transcripts are more stable, and their constitutive levels were generally unaffected by cycloheximide. These results show that PDGF A- and B-chain genes are transcribed in the normal rat aorta and provide evidence for age-related change in the control of PDGF B-chain gene expression in aortic SMC. Independent regulation of transcript levels in cultured SMC leaves open the possibility that PDGFs of different composition (AA, AB, BB) play different roles in normal function of the artery wall.
Transforming growth factor beta mRNA increases during liver regeneration: a possible paracrine mechanism of growth regulation.
Authors: Braun L , Mead J E , Panzica M , Mikumo R , Bell G I , Fausto N
Published: May 2006

Transforming growth factor beta (TGF-beta) is a growth factor with multiple biological properties including stimulation and inhibition of cell proliferation. To determine whether TGF-beta is involved in hepatocyte growth responses in vivo, we measured the levels of TGF-beta mRNA in normal liver and during liver regeneration after partial hepatectomy in rats. TGF-beta mRNA increases in the regenerating liver and reaches a peak (about 8 times higher than basal levels) after the major wave of hepatocyte cell division and mitosis have taken place and after the peak expression of the ras protooncogenes. Although hepatocytes from normal and regenerating liver respond to TGF-beta, they do not synthesize TGF-beta mRNA. Instead, the message is present in liver nonparenchymal cells and is particularly abundant in cell fractions enriched for endothelial cells. TGF-beta inhibits epidermal growth factor-induced DNA synthesis in vitro in hepatocytes from normal or regenerating liver, although the dose-response curves vary according to the culture medium used. We conclude that TGF-beta may function as the effector of an inhibitory paracrine loop that is activated during liver regeneration, perhaps to prevent uncontrolled hepatocyte proliferation.
Merosin, a protein specific for basement membranes of Schwann cells, striated muscle, and trophoblast, is expressed late in nerve and muscle development.
Authors: Leivo I , Engvall E
Published: May 2006

We have identified a tissue-specific basement membrane-associated protein by using monoclonal antibodies prepared against a protein fraction of human placenta. In immunofluorescence, the monoclonal antibodies stained basement membranes of Schwann cells, striated muscle, and trophoblast, whereas no reaction was seen with any other basement membrane or tissue structure. In antibody-affinity chromatography of proteolytic digests of human placenta, a 65-kDa polypeptide was bound by these monoclonal antibodies. Rabbit antisera and monoclonal antibodies raised against the isolated 65-kDa polypeptide stained human and monkey tissues identically to the original monoclonal antibodies and reacted with an 80-kDa polypeptide in tissue extracts prepared without proteolysis. The 65-kDa and 80-kDa polypeptides were shown to be immunologically distinct from laminin, type IV collagen, fibronectin, and major serum proteins. They presumably represent a novel basement membrane-associated protein, which we have named merosin. No merosin immunoreactivity could be detected in cultures of any of 28 established cell lines. In developing mouse tissues, merosin staining first appeared at the newborn stage. The restricted tissue distribution and late developmental appearance of merosin suggest that the protein has a tissue-specific function associated with a high level of differentiation.
Phylogeographic population structure of Red-winged Blackbirds assessed by mitochondrial DNA
Authors: Ball R. Martin , Freeman Scott , James Frances C. , Bermingham Eldredge , Avise John C.
Published: May 2006

A continent-wide survey of restriction-site variation in mitochondrial DNA (mtDNA) of the Red-winged Blackbird (
Agelaius phoeniceus
) was conducted to assess the magnitude of phylogeographic population structure in an avian species. A total of 34 mtDNA genotypes was observed among the 127 specimens assayed by 18 restriction endonucleases. Nonetheless, population differentiation was minor, as indicated by (
i
) small genetic distances in terms of base substitutions per nucleotide site between mtDNA genotypes (maximum
P
≈ 0.008) and by (
ii
) the widespread geographic distributions of particular mtDNA clones and phylogenetic arrays of clones. Extensive morphological differentiation among redwing populations apparently has occurred in the context of relatively little phylogenetic separation. A comparison between mtDNA data sets for Red-winged Blackbirds and deermice (
Peromyscus maniculatus
) also sampled from across North America shows that intraspecific population structures of these two species differ dramatically. The lower phylogeographic differentiation in redwings is probably due to historically higher levels of gene flow.
Secretion from chromaffin cells is controlled by chromogranin A-derived peptides.
Authors: Simon J P , Bader M F , Aunis D
Published: May 2006

Chromogranin A (CGA) is the major protein of the secretory granule from chromaffin cells and also is found in a variety of endocrine cells. Although the sequence of this acidic glycoprotein has been elucidated recently, its biological function is unknown. Here we have purified CGA from chromaffin granules; the final preparation contained the 74-kDa native CGA together with two degradation products--three bands near 60 kDa and a single band of 43 kDa. This preparation was found to inhibit (a maximum inhibition of 60% at 1 microM) the nicotine-induced, but not the high K+-evoked, catecholamine secretion from bovine chromaffin cells maintained in primary culture. Spontaneous release was also affected in the nanomolar CGA protein concentration range. The observation that the inhibitory effect is strictly dependent on a preincubation step together with the modification of the CGA protein profile during this preincubation step suggests that the degradation peptide(s) rather than the 74-kDa native CGA--the approximately equal to 60-kDa bands or the 43-kDa singlet band--is actually involved in secretory cell activity. This was demonstrated by using trypsin-generated peptides that were inhibitory without the preincubation period. The finding that unprocessed CGA is not active on chromaffin cell secretion suggests that this molecule is a precursor of a peptide(s) that is able to regulate catecholamine secretion. Thus, the present data suggest that a CGA-derived peptide(s) could exert a feedback control on chromaffin cell secretory activity--a mechanism that might be of importance during stress situations.
RecA protein-dependent cleavage of UmuD protein and SOS mutagenesis.
Authors: Shinagawa H , Iwasaki H , Kato T , Nakata A
Published: May 2006

Induction of the Escherichia coli SOS system increases the ability of the cell to perform DNA repair and mutagenesis. Products of the recA and umuD,C genes are required for mutagenesis induced by radiation and many chemicals. Transcription of the SOS genes including recA and umuD,C is repressed by a repressor, LexA protein, and is derepressed by the proteolytic cleavage of LexA facilitated by RecA protein that had been activated by inducing signals produced in the cell by agents that damage DNA. An activated form of RecA protein, RecA, seems to have roles in SOS mutagenesis other than its known role as an antirepressor. Derepression of the genes involved in SOS mutagenesis such as recA and umuD,C in defective chromosomal lexA(Def) mutants does not increase the ability of the cell to perform mutagenesis. Activation of RecA protein is essential to this ability. RecA facilitates the proteolytic cleavage of several repressors such as lambda, P22, and 434 phage repressors and LexA, and UmuD protein contains a sequence homologous to the regions surrounding the cleavage sites of these repressors; therefore, we examined the possibility that UmuD protein is cleaved by RecA. We found evidence that the intact UmuD protein was cleaved after mutagenic treatment and that the cleavage was dependent on RecA. The results suggested that UmuD protein may be proteolytically processed by RecA, and that processed UmuD may be the active form of the protein participating in mutagenesis.