bluuebunny/crossref_metadata_2025 huggingface dataset:
CD4 and CD8 molecules can physically associate with the same T-cell receptor.
Authors: Gallagher P F , Fazekas de St Groth B , Miller J F
Published: May 2006

The expression of the cell-surface glycoproteins CD4 and CD8 on functionally mature T cells is usually mutually exclusive and correlates with class II and class I major histocompatibility complex (MHC) restriction, respectively. CD4 and CD8 function by binding to class II and class I MHC molecules on the antigen-presenting cell (APC), thereby increasing the adhesion between the T cell and the APC. From antibody-blocking studies and from cocapping and comodulation experiments, CD4 and CD8 come into close physical contact with their appropriately restricted T-cell receptor (TCR) at the time of antigen recognition. By the use of affinity chromatography followed by two-dimensional diagonal gel electrophoresis, we have identified a Mr 43,000 disulfide-bonded heterodimer copurifying with CD4. This protein was identified as the TCR by its removal after preclearing with the anti-TCR antibody F23.1 and by its generation after protease digestion of the same peptides as the TCR from this clone. When CD4 and CD8 were similarly isolated from an unusual CD4+ CD8+ class II-restricted T-cell clone, the TCR was identified as associating with either accessory molecule in the absence of activation. Therefore, CD4 and CD8 do not distinguish between class I- and class II-restricted TCRs in their ability to form membrane complexes, indicating a need for both the TCR and its associated accessory molecule to recognize the same individual MHC molecule on the APC to optimize TCR triggering.
Positive inotropic effects of the endogenous Na+/K(+)-transporting ATPase inhibitor from the hypothalamus.
Authors: Hallaq H A , Haupert G T
Published: May 2006

Bovine hypothalamus contains a nonpeptidic substance that inhibits purified Na+/K(+)-transporting ATPase [ATP phosphohydrolase (Na+/K(+)-transporting), EC 3.6.1.37] reversibly with high affinity by a mechanism similar to, but not identical to, that of the cardiac glycosides. It possesses some of the characteristics ascribed to a putative endogenous "digitalis-like" compound that has been implicated in the control of renal sodium excretion and the pathogenesis of essential hypertension in man. To determine whether this hypothalamic Na+/K(+)-transporting ATPase inhibitor might have physiologic properties in cardiac tissues, its effects on Na+ pump inhibition, accumulation of cytosolic free calcium, and contractile response were studied in cultured, spontaneously contracting neonatal rat cardiocytes. The hypothalamic factor potently inhibited the Na+ pump in these cells, increased myoplasmic free calcium in a dose-dependent manner, and reversibly enhanced myocyte contractility by up to 40%, comparable in degree to maximal positive inotropic effects caused by the cardiac glycoside ouabain. Comparative studies further indicate that cardiotoxic effects of ouabain in the myocytes may be more complex than simple progressive elevation of intracellular free calcium concentration because at a free calcium concentration in excess of that produced by a toxic dose of ouabain, no toxicity with the hypothalamic Na+/K(+)-transporting ATPase inhibitor occurred.
Increased tumor necrosis factor alpha mRNA after cellular exposure to ionizing radiation.
Authors: Hallahan D E , Spriggs D R , Beckett M A , Kufe D W , Weichselbaum R R
Published: May 2006

We report that tumor necrosis factor alpha (TNF-alpha) mRNA is increased after treatment with x-rays in certain human sarcoma cells. An increase in TNF-alpha mRNA is accompanied by the increased production of TNF-alpha protein. TNF-alpha enhances radiation lethality in both TNF-alpha-producing and -nonproducing tumor cells. These data suggest that, in addition to the direct cytotoxic effects of x-rays, production of TNF-alpha may add to radiation lethality through autocrine and paracrine mechanisms. Combinations of TNF-alpha and therapeutic radiation may be useful in clinical cancer therapy.
Cloning of ligand-specific cell lines via gene transfer: identification of a D2 dopamine receptor subtype.
Authors: Todd R D , Khurana T S , Sajovic P , Stone K R , O'Malley K L
Published: May 2006

Using rat genomic DNA, we have established a transfected mouse fibroblast cell line that expresses a spiperone binding site with the pharmacological characteristics of a D2 dopamine receptor. The expressed D2 receptors are the product of a gene that is distinct from that reported by Bunzow et al. [Bunzow, J. R., Van Tol, H. H. M., Granoly, D. K., Albert, P., Salon, J., Christie, M., Machida, C. A., Neve, K. A. & Civelli, O. (1988) Nature (London) 336, 783-787]. Flow cytometry with the Ca2+-sensitive dye indo-1 demonstrated that activation of the expressed D2 sites resulted in increases in intracellular calcium that were dependent on the influx of external Ca2+. These general cloning procedures should be applicable to the production of cell lines expressing a variety of genes for which only functional assays are available.
Crystallographic refinement of interleukin 1 beta at 2.0 A resolution.
Authors: Priestle J P , Schär H P , Grütter M G
Published: May 2006

The structure of human recombinant interleukin 1 beta (IL-1 beta) has been refined by a restrained least-squares method to a crystallographic R factor of 17.2% to 2.0 A resolution. One-hundred sixty-eight solvent molecules have been located, and isotropic temperature factors for each atom have been refined. The overall structure is composed of 12 beta-strands that can best be described as forming the four triangular faces of a tetrahedron with hydrogen bonding resembling normal antiparallel beta-sheets only at the vertices. The interior of this tetrahedron is filled by hydrophobic side chains. Analysis of sequence alignments with IL-1 beta from other mammalian species shows the interior to be very well conserved with the exterior residues markedly less so. There does not appear to be a clustering of invariant amino acid side chains on the surface of the molecule, suggesting an area of interaction with the IL-1 receptor. Comparison of the IL-1 beta structure with IL-1 alpha sequences indicates that IL-1 alpha probably has a similar overall folding as IL-1 beta but binds to the receptor in a different fashion. The three-dimensional structure of the IL-1 beta is analyzed in light of what has been suggested by previously published work on mutants and fragments of the molecule.
Sequence-specific recognition and cleavage of duplex DNA via triple-helix formation by oligonucleotides covalently linked to a phenanthroline-copper chelate.
Authors: François J C , Saison-Behmoaras T , Barbier C , Chassignol M , Thuong N T , Hélène C
Published: May 2006

Homopyrimidine oligodeoxynucleotides recognize the major groove of the DNA double helix at homopurine.homopyrimidine sequences by forming local triple helices. Phenanthroline was covalently attached to the 5' end of an 11-mer homopyrimidine oligonucleotide of sequence d(TTTCCTCCTCT). Simian virus 40 DNA, which contains a single target site for this oligonucleotide, was used as a substrate for the phenanthroline-oligonucleotide conjugate. In the presence of copper ions and a reducing agent, a single specific double-strand cleavage site was observed at 20 degrees C by agarose gel electrophoresis. The efficiency of double-strand cleavage was greater than 70% at 20 degrees C and pH 7.4. Secondary cleavage sites were observed when binding of the oligonucleotide to mismatched sequences was allowed to take place at low temperature. The exact location of the cleavage sites was determined by polyacrylamide gel electrophoresis of denatured fragments by using both simian virus 40 DNA and a synthetic DNA fragment containing the target sequence. The asymmetric distribution of the cleavage sites on the two strands revealed that the cleavage reaction took place in the minor groove even though the phenanthroline linker was located in the major groove. Linkers of different lengths were used to tether phenanthroline to the oligonucleotide and their relative efficacies of DNA cleavage were compared. Based on these comparative studies and on model building, it is proposed that the phenanthroline ring carried by the oligonucleotide intercalates from the major groove and that copper chelation locks the complex in place from within the minor groove where the cleavage reaction occurs.
Isolation of a cDNA clone of the 14-kDa subunit of the signal recognition particle by cross-hybridization of differently primed polymerase chain reactions.
Authors: Strub K , Walter P
Published: May 2006

Using an enhancement of the polymerase chain reaction (PCR) technique, we have isolated a complementary DNA encoding SRP14 (14-kDa subunit), one of six proteins contained in the signal recognition particle (SRP). Several pools of degenerate oligonucleotides encoding different peptide sequences of SRP14 were used to generate amplified DNA by the PCR. A cross-hybridization procedure was developed to identify the authentic SRP14 cDNA clone among the amplified DNA products obtained by PCR. The basis of this approach is the assumption that a partial cDNA of SRP14 should be the only DNA product common to two amplification reactions primed with different degenerate oligonucleotide mixtures. The partial canine cDNA of SRP14 identified by this procedure served as a probe to isolate a complete cDNA clone of SRP14 from a mouse embryonic cDNA library in lambda phage gt10.
Three-dimensional ordered distribution of crystals in turkey tendon collagen fibers.
Authors: Traub W , Arad T , Weiner S
Published: May 2006

The organization of apatite crystals and collagen fibrils in mineralized turkey tendon has been studied by electron microscopy and electron diffraction. To minimize artifactual distortions the tissue was examined, for the first time, as isolated fibrils in an aqueous environment of vitreous ice, as well as in conventionally prepared sections. The electron micrographs show that the plate-shaped apatite crystals are arranged in parallel arrays across the collagen fibrils. This provides direct evidence for highly asymmetric assembly in collagen fibrils, and, indeed, the fibrils were observed to be elongated rather than round in cross-section. There is, furthermore, a pronounced tendency for the layers of crystals to be coherently aligned in adjacent fibrils. These observations may also be important for understanding the mechanical behavior of bone at the molecular level, as such extended, aligned aggregates of flat crystals could develop into natural fracture planes in mature bone.
The yeast cell fusion protein FUS1 is O-glycosylated and spans the plasma membrane.
Authors: Trueheart J , Fink G R
Published: May 2006

Previous work has shown that efficient cell fusion during conjugation in Saccharomyces cerevisiae requires a pheromone-induced surface protein encoded by FUS1. We show that the FUS1 protein migrates on SDS/polyacrylamide gels with an apparent molecular mass of 80 kDa, although the mass is predicted to be 58 kDa from the gene coding capacity. This discrepancy results from the presence of O-linked mannose oligosaccharides attached to the clustered serines and threonines at the amino terminus of the protein. The addition of mannose is completely abolished in the early secretory mutant sec53, attenuated in the late-endoplasmic reticulum-blocked sec18, and unaffected in sec7, which is blocked late in the Golgi phase of secretion. Membrane fractionation and protease protection experiments indicate that FUS1 spans the plasma membrane, with its glycosylated amino terminus projecting into the periplasmic space.
Phosphatidylserine translocation to the mitochondrion is an ATP-dependent process in permeabilized animal cells.
Authors: Voelker D R
Published: May 2006

Chinese hamster ovary (CHO-K1) cells were pulse labeled with [3H]serine, and the synthesis of phosphatidyl[3H]ethanolamine from phosphatidyl[3H]serine during the subsequent chase was used as a measure of lipid translocation to the mitochondria. When the CHO-K1 cells were pulse labeled and subsequently permeabilized with 50 micrograms of saponin per ml, there was no significant turnover of nascent phosphatidyl[3H]serine to form phosphatidyl[3H]ethanolamine during an ensuing chase. Saponin treatment rendered greater than 99% of the cells permeable as judged by trypan blue exclusion and depleted them of 85% of their complement of cytosolic proteins as determined by residual lactic acid dehydrogenase activity. Supplementation of the permeabilized cells with 2 mM ATP resulted in significant phosphatidyl[3H]ethanolamine synthesis (83% of that found in intact cells) from phosphatidyl[3H]serine during a subsequent 2-hr chase. Phosphatidyl[3H]ethanolamine synthesis essentially ceased after 2 hr in the permeabilized cells. The translocation-dependent synthesis of phosphatidyl[3H]ethanolamine was a saturable process with respect to ATP concentration in permeabilized cells. The conversion of phosphatidyl[3H]serine to phosphatidyl[3H]ethanolamine did not occur in saponin-treated cultures supplemented with 2 mM AMP, 2 mM 5'-adenylyl imidodiphosphate, or apyrase (2.5 units/ml) plus 2 mM ATP. ATP was the most effective nucleotide, but the addition of GTP, CTP, UTP, and ADP also supported the translocation-dependent synthesis of phosphatidyl[3H]ethanolamine albeit to a lesser extent. These data provide evidence that the interorganelle translocation of phosphatidylserine requires ATP and is largely independent of soluble cytosolic proteins.
Ancient roots for polymorphism at the HLA-DQ alpha locus in primates.
Authors: Gyllensten U B , Erlich H A
Published: May 2006

The genes encoding the human histocompatibility antigens (HLA) exhibit a remarkable degree of polymorphism as revealed by immunologic and molecular analyses. This extensive sequence polymorphism either may have been generated during the lifetime of the human species or could have arisen before speciation and been maintained in the contemporary human population by selection or, possibly, by genetic drift. These two hypotheses were examined using the polymerase chain reaction method to amplify polymorphic sequences from the DQ alpha locus, as well as the DX alpha locus, an homologous but nonexpressed locus, in a series of primates that diverged at known times. In general, the amino acid sequence of a specific human DQ alpha allelic type is more closely related to its chimpanzee or gorilla counterpart than to other human DQ alpha alleles. Phylogenetic analysis of the silent nucleotide position changes shows that the similarity of allelic types between species is due to common ancestry rather than convergent evolution. Thus, most of the polymorphism at the DQ alpha locus in the human species was already present at least 5 million years ago in the ancestral species that gave rise to the chimpanzee, gorilla, and human lineages. However, one of the DQ alpha alleles may have arisen after speciation by recombination between two ancestral alleles.
Nonanesthetic alcohols dissolve in synaptic membranes without perturbing their lipids.
Authors: Miller K W , Firestone L L , Alifimoff J K , Streicher P
Published: May 2006

While many theories of general anesthesia postulate a lipid site of action, there has been no adequate explanation for the lack of anesthetic potency of the highly hydrophobic primary alkanols with more than 12 carbons (the cut-off). Some work suggests that these nonanesthetic alcohols do not dissolve in membranes. Other work contradicts this and suggests that an anesthetic site on a protein provides a better explanation. Here we show that both the anesthetic dodecanol and the nonanesthetic tetradecanol are taken up equally well into the tissues of animals and into isolated postsynaptic membranes. When a group of Rana pipiens tadpoles were treated with dodecanol, half were anesthetized by 4.7 microM (free aqueos concentration), and the corresponding concentration in the tissues was found to be 0.4 mmol per kg wet weight. Prolonged exposure (92 hr) to tetradecanol produced even higher tissue concentrations (0.7 mmol per kg wet weight), yet no anesthetic effects were observed. Furthermore, general anesthetics are thought to act on postsynaptic membranes but both alkanols partitioned into postsynaptic membranes from Torpedo electroplaques. The spin label, 12-doxyl stearate, was incorporated into these membranes. The lipid order parameter it reported was decreased by the anesthetic alcohols (octanol, decanol, and dodecanol), whereas the nonanesthetic alcohols either did not change it significantly (tetradecanol) or actually increased it (hexadecanol and octadecanol). Thus, although lipid solubility is unable to account for the pharmacology of the cut-off in potency of the long-chain alcohols, lipid perturbations provide an accurate description.
Growth hormone stimulates c-fos gene expression by means of protein kinase C without increasing inositol lipid turnover.
Authors: Doglio A , Dani C , Grimaldi P , Ailhaud G
Published: May 2006

Growth hormone (GH) is required for the terminal differentiation of preadipose Ob1771 cells that have entered the differentiation program as evidenced by the expression of early marker genes (pOb24 and lipoprotein lipase). Induction of c-fos mRNA within 15 min and induction of insulin-like growth factor I mRNA within a few hours take place in response to GH. The role of GH is mediated, at least in part, by means of the activation of protein kinase C, as shown by the inhibition of epidermal growth factor binding and by the expression of the c-fos gene, and is thus analogous to the action of prostaglandin F2 alpha and 4 beta-phorbol-12,13-didecanoate in this respect. However, in contrast to that of the c-fos gene, the regulation of insulin-like growth factor I gene expression by GH is not mediated by means of the activation of protein kinase C, and, in line with this, prostaglandin F2 alpha and 4 beta-phorbol-12,13-didecanoate were ineffective. GH and prostaglandin F2 alpha were able to stimulate the formation of diacyglycerol within a few seconds, but GH did not elicit an accumulation of inositol phosphates, in contrast to that generated by prostaglandin F2 alpha. We conclude that the transduction signal of GH action in c-fos mRNA induction is the formation of diacylglycerol and that the mechanism whereby GH can activate protein kinase C is associated with a phospholipase C-mediated hydrolysis of glycerophospholipids other than inositol phospholipids.
Low density of sodium channels supports action potential conduction in axons of neonatal rat optic nerve.
Authors: Waxman S G , Black J A , Kocsis J D , Ritchie J M
Published: May 2006

The density of sodium channels in premyelinated axons was estimated from measurements of the binding of [3H]saxitoxin to neonatal rat optic nerve. The maximum saturable binding capacity of the nerve was 16.2 +/- 1.2 fmol/mg of wet weight, with an equilibrium dissociation constant of 0.88 +/- 0.18 nM (mean +/- SEM). These values correspond to a high-affinity saxitoxin-binding site density of approximately 2/microns 2 within premyelinated axon membrane. Action potential propagation in neonatal rat optic nerve is completely blocked by 5 nM saxitoxin, indicating that action potential electrogenesis is mediated by channels that correspond to high-affinity saxitoxin-binding sites. These results demonstrate that action potential conduction is supported by a low density of sodium channels in this system. Since the internodal axon membrane of myelinated fibers may contain a low density of sodium channels, it is possible that restoration of conduction in some demyelinated fibers may not require additional sodium channel incorporation into the demyelinated axon membrane.
Principles determining the structure of high-pressure forms of metals: The structures of cesium(IV) and cesium(V)
Authors: Pauling Linus
Published: May 2006

Consideration of the relation between bond length and bond number and the average atomic volume for different ways of packing atoms leads to the conclusion that the average ligancy of atoms in a metal should increase when a phase change occurs on increasing the pressure. Minimum volume for each value of the ligancy results from triangular coordination polyhedra (with triangular faces), such as the icosahedron and the Friauf polyhedron. Electron transfer may permit atoms of an element to assume different ligancies. Application of these principles to Cs(IV) and Cs(V), which were previously assigned structures with ligancy 8 and 6, respectively, has led to the assignment to Cs(IV) of a primitive cubic unit cell with
a
= 16.11 Å and with about 122 atoms in the cube and to Cs(V) of a primitive cubic unit cell resembling that of Mg
32
(Al,Zn)
49
, with
a
= 16.97 Å and with 162 atoms in the cube.
Natural-abundance 13C NMR study of glycogen repletion in human liver and muscle.
Authors: Jue T , Rothman D L , Tavitian B A , Shulman R G
Published: May 2006

Optimizing the surface-coil design and spectral-acquisition parameters has lead to the observation of the 13C NMR natural abundance glycogen signal in man at 2.1 T. Both the human muscle and hepatic glycogen signals can be detected definitively with a time resolution of approximately equal to 13 min. A 1H/13C concentric surface coil was used. The 1H outer coil was 11 cm in diameter; the 13C inner coil was 8 cm in diameter. The coils were tuned to 89.3 MHz and 22.4 MHz, respectively. The 1H coil was used for optimizing field homogeneity (shimming) the magnet and for single-frequency decoupling of the C1 glycogen signal. Total power deposition from both the transmitter pulse and the continuous wave decoupling did not exceed the Food and Drug Administration guideline of 8 W/kg of tissue. Experiments were done for which healthy subjects returned to the magnets at different times for 13C NMR measurement. The spectral difference between experiments was within the noise in the C1 glycogen region. Because of the spectral reproducibility and the signal sensitivity, hepatic glycogen repletion can be followed. Four hours postprandial, hepatic glycogen increases by 3.8 times from the basal fasted state. The hepatic glycogen data correspond directly to previous biopsy results and support the use of 13C NMR as a noninvasive probe of human metabolism.
Tissue-specific and differentiation-specific expression of a human K14 keratin gene in transgenic mice.
Authors: Vassar R , Rosenberg M , Ross S , Tyner A , Fuchs E
Published: May 2006

A construct containing approximately 2500 base pairs (bp) of 5' upstream and approximately 700 bp of 3' downstream sequence was used to drive the expression of an intronless human K14 gene in vitro and in vivo. To track the expression of the gene, a small sequence encoding the antigenic portion of neuropeptide substance P was inserted in frame 5' to the TGA translation stop codon of the gene. Surprisingly, this gene was expressed promiscuously in a wide variety of cultured cells transiently transfected with the construct. In contrast, when introduced into the germ line of transgenic mice, the construct was expressed in a fashion analogous to the endogenous K14 gene--namely, in the basal layer of stratified squamous epithelia. Our results suggest that some regulatory mechanism is overridden as a consequence of transient transfection but that sequences that can control proper K14 expression are present in the construct. The appropriate tissue-specific and differentiation-specific expression of K14.P in transgenic mice is an important first step in characterizing a promoter that could be employed to drive the foreign expression of drug-related genes in the epidermis of skin grafts.
Evidence for the progressive and adaptive nature of spontaneous transformation in the NIH 3T3 cell line.
Authors: Rubin H , Xu K
Published: May 2006

The NIH 3T3 mouse cell line is widely used as a recipient of DNA from tumors to demonstrate the presence of transforming oncogenes. We show that these cells produce transformed foci spontaneously if kept in the confluent state for more than 10 days. The formation of foci depends on the type and concentration of bovine serum used in the medium and passage history of the cells. Cells maintained in continuous exponential multiplication in the subconfluent state by transfer every 2-3 days in medium with 10% calf serum failed to develop the capacity to produce foci in 2% calf serum, but those transferred the same way in 2% calf serum or in 10% fetal bovine serum, which is a less potent growth stimulant, did develop that capacity to an increasing degree over time. The number of transformed cells increased sharply with the time that a culture remained in the confluent state. There are several morphological types and degrees of transformation, which indicates that the underlying changes are varied and the process is progressive. The results also suggest that transformation occurs in a fraction of an entire cell population that is undergoing a physiological adaptation to moderate constraints on its growth.
Membrane binding sites for plasma lipoproteins on endosomes from rat liver.
Authors: Jaeckle S , Brady S E , Havel R J
Published: May 2006

Highly purified endosomal membranes from rat liver, enriched in receptors for a number of macromolecules taken up into hepatocytes via the coated pit/endosome/lysosome pathway [including the receptor for low density lipoproteins (LDL)], were used to characterize binding sites for lipoproteins containing apolipoprotein E. In endosomal membranes from livers of estradiol-treated rats, in which LDL receptors are induced manyfold, two high-affinity binding sites were found for two apolipoprotein E-rich lipoproteins: very low density beta-lipoproteins (beta-VLDL) from cholesterol-fed rabbits and rat chylomicron remnants. One of these sites, binding to which is inhibited by 30 mM EDTA, appears identical to the LDL receptor by ligand and immunoblotting and other characteristics. The other site, highly resistant to EDTA, does not bind LDL. Binding to the EDTA-resistant site, however, is readily inhibited by heparin (as is the LDL receptor) and also by antisera prepared against rat or bovine LDL receptor. The distribution of the EDTA-resistant site among early endosomes, late endosomes, and endosome-derived receptor-recycling membranes is similar to that of the LDL receptor and other recycling receptors. The LDL receptor was present in endosomal membranes from livers of untreated rats at about 10% of the level found in membranes from estradiol-treated rats, but the EDTA-resistant site was barely detectable. No saturable binding of beta-VLDL that could not be inhibited by antisera to the LDL receptor could be detected in endosomal membranes from livers of either untreated or estradiol-treated rats. The EDTA-resistant site may be a modified form of the LDL receptor that recognizes apolipoprotein E but not the B apolipoprotein of LDL. Alternatively, it may be a distinct receptor sharing immunological determinants with the LDL receptor, specialized for the endocytosis of certain lipoproteins containing apolipoprotein E, including chylomicron remnants.
CD4+ lymphocyte function with early human immunodeficiency virus infection.
Authors: Gurley R J , Ikeuchi K , Byrn R A , Anderson K , Groopman J E
Published: May 2006

The pathogenesis of cellular immune deficiency following human immunodeficiency virus (HIV) infection could result from quantitative and/or qualitative dysfunction of the CD4+ lymphocyte population. To better characterize the T-cell response to soluble antigen with HIV infection, we have isolated peripheral blood lymphocytes and purified populations of CD4+ lymphocytes from healthy HIV antibody-positive subjects, patients with acquired immunodeficiency syndrome (AIDS)-related complex (ARC), and healthy HIV antibody-negative controls. T-lymphocyte function was determined by proliferative response to lectin (phytohemagglutinin), phorbol 12-myristate 13-acetate (PMA), calcium ionophore, purified recombinant HIV envelope gp120, tetanus toxoid antigen, and tetanus toxoid antigen in the presence of recombinant gp120 or purified recombinant soluble CD4. PBLs and CD4+ lymphocytes from asymptomatic HIV-infected subjects responded equally well to lectin, PMA, and/or calcium ionophore and to tetanus toxoid as cells from uninfected control subjects. The cells that proliferated in response to a soluble antigenic stimulus did not respond to gp120. Cells from subjects with ARC had a selective antigen recognition defect independent of the number of CD4+ lymphocytes. Recombinant gp120 inhibited CD4+ lymphocyte proliferation to antigenic stimulus by 30-40%. Recombinant soluble CD4, a proposed therapeutic for HIV, had no effect on T-cell response to antigen. A selective antigen recognition response was not compromised early in HIV infection but was compromised in subjects with ARC. Inhibition of proliferation to tetanus toxoid by gp120 suggests that HIV may affect major histocompatibility complex II restricted antigen recognition independent of CD4+ cell loss.
Influenza viruses induce autoantibodies to a brain-specific 37-kDa protein in rabbit.
Authors: Laing P , Knight J G , Hill J M , Harris A G , Oxford J S , Webster R G , Markwell M A , Paul S M , Pert C B
Published: May 2006

Immunization of rabbits with certain H1N1 influenza viruses, including the neurotropic strains NWS/33 and WSN/33 and the New Jersey/76 strain, resulted in the production of autoantibodies to a brain-specific protein of 37 kDa that is present in various species, including humans. Autoantibodies were produced to brain only; various other tissues tested were negative. These antibodies were not elicited by other influenza A or B viruses, including closely related recombinant strains, but were elicited by the isolated hemagglutinin of A/Bellamy/42 strain and by formaldehyde-fixed WSN virus--demonstrating that infection was not essential for the induction of autoantibodies. In histological studies, reaction with anti-viral antisera was specific to gray matter and was confined to sera that recognized the 37-kDa protein. Antibody binding was prominent in regions comprised of neuronal cell bodies in cellular layers of the dentate gyrus, hippocampus, cerebral cortex, and cerebellum and was undetectable in myelin-rich regions, such as the corpus callosum. The 37-kDa protein, therefore, appears to be a neuronal antigen. Antibodies directed against this protein may be involved in the pathogenesis of one or more of the neuropsychiatric disorders that occur after infection with influenza.
Chromosomal translocation in a human leukemic stem-cell line disrupts the T-cell antigen receptor delta-chain diversity region and results in a previously unreported fusion transcript.
Authors: Begley C G , Aplan P D , Davey M P , Nakahara K , Tchorz K , Kurtzberg J , Hershfield M S , Haynes B F , Cohen D I , Waldmann T A
Published: May 2006

We have studied a leukemic stem-cell line, DU.528, that is able to differentiate into myeloid and lymphoid cells. The leukemic cells have a translocation between chromosomes 1 and 14, t(1;14)(p33;q11), which we have molecularly cloned and sequenced. Initial screening used joining (J)-segment probes from the T-cell receptor (TCR) alpha- and delta-chain loci. In apparent concert with the translocation, a deletion has occurred between delta-chain diversity (D)-region genes D delta 1 and D delta 2. D delta 2 was observed on derivative chromosome 1 [der(1)] and D delta 1 on der(14) with a deletion of the intervening 10 kilobases of germ-line DNA. The nature of the D delta 1-D delta 2 deletional event implicates a lymphoid recombinase in the mechanism of the translocation. As a consequence of the translocation, an unusual fusion transcript was generated. Probes from chromosome 1 detected a previously unreported transcript in RNA from both the cell line and the patient. A chromosome 14 probe identified the same transcript, thus confirming a fusion transcript derived from both chromosomes 1 and 14. This translocation may identify a gene for which we propose the name SCL (stem-cell leukemia) that is important for hemopoietic development and oncogenesis and that has been disrupted or altered in this stem-cell line.
Characterization of high-affinity receptors for interleukin 5 on interleukin 5-dependent cell lines.
Authors: Mita S , Tominaga A , Hitoshi Y , Sakamoto K , Honjo T , Akagi M , Kikuchi Y , Yamaguchi N , Takatsu K
Published: May 2006

Interleukin 5 (IL-5) is a glycosylated polypeptide that acts as a key factor for B-cell growth and differentiation. We demonstrated previously that there are two classes (high and low affinity) of IL-5 receptors on murine chronic B-cell leukemic cells (BCL1-B20). Treatment of surface-bound radiolabeled IL-5 with bivalent crosslinkers identified a polypeptide of Mr 92,500. In this study, we analyzed characteristics of high-affinity IL-5 receptors on IL-5-dependent early B-cell lines (T-88 and T88-M), mouse myeloma cells (MOPC-104E), and BCL1-B20 cells. All cell lines had two classes of IL-5 binding sites, but T88-M cells bore the highest number of high-affinity receptors. The number of high-affinity IL-5 receptors on BCL1-B20 cells could be up-regulated 3-fold by lipopolysaccharide and down-regulated by IL-5. Disuccinimidyl tartarate crosslinking of 35S-labeled IL-5 to the receptors on the T88-M and lipopolysaccharide-stimulated BCL1-B20 cells revealed two major 35S-labeled components of Mr 92,500 and Mr 160,000, even when the binding of 35S-labeled IL-5 was carried out under high-affinity conditions (100 pM 35S-labeled IL-5). The Mr 92,500 component, but not the Mr 160,000 component, was detected in the lysates of MOPC-104E and T-88 cells, both of which bore a large number of low-affinity receptors and a limited number of high-affinity receptors. The results suggest that the Mr 92,500 component represents the complex of IL-5 with the low-affinity Mr 46,500 receptor, whereas the high-affinity receptor consists of the Mr 46,500 peptide and an additional peptide.
Tumor necrosis factor alpha induces expression of human immunodeficiency virus in a chronically infected T-cell clone.
Authors: Folks T M , Clouse K A , Justement J , Rabson A , Duh E , Kehrl J H , Fauci A S
Published: May 2006

Tumor necrosis factor alpha (TNF-alpha), also known as cachectin, was demonstrated to induce the expression of human immunodeficiency virus (HIV) in a chronically infected T-cell clone (ACH-2). Concentrations of recombinant TNF-alpha as low as 50 pg/ml induced a significant increase over background of HIV expression in the ACH-2 cells as determined by supernatant reverse transcriptase activity. The HIV-inducing effects of TNF-alpha could not be explained by toxic effects on the cells. In addition, both the uninfected parental cell line (A3.01) and the infected ACH-2 cells were shown to have high-affinity receptors for TNF-alpha. Transient-transfection experiments demonstrated that the inductive effects of TNF-alpha were due to specific activation of the HIV long terminal repeat. These studies provide evidence that TNF-alpha may play a role in the mechanisms of pathogenesis of HIV infection.
c-myc gene amplification during hepatocarcinogenesis by a choline-devoid diet.
Authors: Chandar N , Lombardi B , Locker J
Published: May 2006

Liver tumors arise in rats fed a choline-devoid diet without added carcinogens. We found amplification of the c-myc gene in 13/13 of these tumors. The amplification ranged from 2- to 70-fold and was accompanied by an increase in c-myc gene expression. Amplification of c-myc was larger in tumors of rats fed a choline-devoid diet followed by a choline-supplemented diet than in tumors from animals fed a choline-devoid diet exclusively. In the former animals, low levels of c-myc gene amplification were also detected in nontumorous regions of tumor-bearing livers. The choline-devoid diet provides an in vivo experimental model for the induction of gene amplification in the rat liver. In this setting, amplification of the c-myc gene may be an early and critical event in carcinogenesis.
Variant translocation of the bcl-2 gene to immunoglobulin lambda light chain gene in chronic lymphocytic leukemia.
Authors: Adachi M , Cossman J , Longo D , Croce C M , Tsujimoto Y
Published: May 2006

The bcl-2 gene has been identified as a gene directly involved in the consistent chromosome translocation t(14;18), which is found in approximately 90% of human follicular lymphoma cases, and is a prime candidate for the oncogene playing a crucial role in follicular lymphomagenesis. In this paper, we describe a case of chronic lymphocytic leukemia showing the juxtaposition of the bcl-2 gene on chromosome 18 to immunoglobulin lambda light chain (Ig lambda) gene on chromosome 22 in a head-to-head configuration. Sequencing analysis of the joining site of the bcl-2 gene and Ig lambda gene has shown that the breakpoint is within the 5' flanking region of the bcl-2 gene and about 2.2 kilobases 5' to the joining segment of Ig lambda locus in a germ-line configuration. The extranucleotide, commonly appearing at the joining site of the t(14;18) translocation involving the IgH locus, is absent from the joining site of bcl-2 and Ig lambda. The lack of extranucleotide suggests that the juxtaposition of the bcl-2 and Ig lambda genes occurred during physiological rearrangement of the Ig lambda gene since it has been shown that the rearrangement of the Ig lambda locus is not accompanied by extranucleotides.
Mitotic recombination of chromosome 17 in astrocytomas.
Authors: James C D , Carlbom E , Nordenskjold M , Collins V P , Cavenee W K
Published: May 2006

Allelic combinations at seven loci on human chromosome 17 defined by restriction fragment length polymorphisms were determined in tumor and normal tissues from 35 patients with gliomas. Loss of constitutional heterozygosity at one or more of these loci was observed in 8 of the 24 tumors displaying astrocytic differentiation and in the single primitive neuroectodermal tumor examined. The astrocytomas showing these losses included examples of each adult malignancy grade of the disease, including glioblastoma (malignancy grade IV), and seven of them demonstrated concurrent maintenance of heterozygosity for at least one chromosome 17 locus. Determination of allele dosage together with the genotypic data indicated that the tumor chromosomes 17 were derived by mitotic recombination in 7 of the 9 cases with shared homozygosity of the region 17p11.2-pter in all cases. In contrast, tumors of oligodendrocytic, ependymal, or mixed cellular differentiation did not exhibit loss of alleles at any of the loci examined. These data suggest that the somatic attainment of homozygosity for loci on chromosome 17p is frequently associated with the oncogenesis of central nervous system tumors, particularly those showing solely astrocytic differentiation, and that mitotic recombination mapping is a useful approach towards the subregional localization of a locus whose rearrangement is involved in this disease.
Detection of sequences homologous to human retroviral DNA in multiple sclerosis by gene amplification.
Authors: Greenberg S J , Ehrlich G D , Abbott M A , Hurwitz B J , Waldmann T A , Poiesz B J
Published: May 2006

Twenty-one patients with multiple sclerosis, chronic progressive type, were examined for DNA sequences homologous to a human retrovirus. Genomic DNA from peripheral blood mononuclear cells was analyzed for the presence of homologous sequences to the human T-cell leukemia/lymphoma virus type I (HTLV-I) long terminal repeat, 3' gag, pol, and env domains by the enzymatic in vitro gene amplification technique, polymerase chain reaction. Positive identification of homologous pol sequences was made in the amplified DNA from six of these patients (29%). Three of these six patients (14%) also tested positive for the env region, but not for the other regions tested. In contrast, none of the samples from 35 normal individuals studied was positive when amplified and tested with the same primers and probes. Comparison of patterns obtained from controls and from patients with adult T-cell leukemia or tropical spastic paraparesis suggests that the DNA sequences identified are exogenous to the human genome and may correspond to a human retroviral species. The data support the detection of a human retroviral agent in some patients with multiple sclerosis.
Isolation and identification of a diuretic hormone from the tobacco hornworm,
Manduca sexta
Authors: Kataoka Hiroshi , Troetschler Ruth G. , Li Jorge P. , Kramer Steven J. , Carney Robert L. , Schooley David A.
Published: May 2006

A diuretic hormone (DH) has been isolated from pharate adult heads of
Manduca sexta
by a nine-step purification procedure. The primary structure of the amino-terminal 40 residues was determined by sequence analysis of intact DH. The structure of an amidated carboxyl-terminal tryptic hexapeptide was characterized by sequence analysis of the peptide, and this hexapeptide was later compared by reversed-phase liquid chromatography with two synthetic hexapeptides with the free acid or amide at the carboxyl terminus. The complete structure of
M. sexta
DH was established as a 41-residue peptide without disulfide bonds: H-Arg-Met-Pro-Ser-Leu-Ser-Ile-Asp-Leu-Pro-Met-Ser-Val-Leu-Arg-Gln-Lys-Leu-Ser -Leu-Glu-Lys-Glu-Arg-Lys-Val-His-Ala-Leu-Arg-Ala-Ala-Ala-Asn-Arg-Asn-Phe-Leu- Asn-Asp-Ile-NH
2
.
M. sexta
DH was synthesized and shown to have chromatographic and biological properties identical with those of the native material. Synthetic DH stimulated fluid excretion
in vivo
upon injection into larval
M. sexta
and newly emerged adult
Pieris rapae. M. sexta
DH has considerable sequence homology with corticotropin-releasing factor, urotensin I, and sauvagine.
A hydrogenase-linked gene in Methanobacterium thermoautotrophicum strain delta H encodes a polyferredoxin.
Authors: Reeve J N , Beckler G S , Cram D S , Hamilton P T , Brown J W , Krzycki J A , Kolodziej A F , Alex L , Orme-Johnson W H , Walsh C T
Published: May 2006

The genes mvhDGA, which encode the subunit polypeptides of the methyl viologen-reducing hydrogenase in Methanobacterium thermoautotrophicum strain delta H, have been cloned and sequenced. These genes, together with a fourth open reading frame designated mvhB, are tightly linked and appear to form an operon that is transcribed starting 42 base pairs upstream of mvhD. The organization and sequences of the mvhG and mvhA genes indicate a common evolutionary ancestry with genes encoding the small and large subunits of hydrogenases in eubacterial species. The product of the mvhB gene is predicted to contain six tandomly repeated bacterial-ferredoxin-like domains and, therefore, is predicted to be a polyferredoxin that could contain as many as 48 iron atoms in 12 Fe4S4 clusters.
Regulation of the gastrin promoter by epidermal growth factor and neuropeptides.
Authors: Godley J M , Brand S J
Published: May 2006

The regulation of gastrin gene transcription was studied in GH4 pituitary cells transfected with constructs comprised of the first exon of the human gastrin gene and various lengths of 5' regulatory sequences ligated upstream of the reporter gene chloramphenicol acetyltransferase. Gastrin reporter gene activity in GH4 cells was equal to the activity of a reporter gene transcribed from the endogenously expressed growth hormone promoter. The effect of a variety of peptides on gastrin gene transcription including epidermal growth factor (normally present in the gastric lumen), gastrin-releasing peptide, vasoactive intestinal peptide, and somatostatin (present in gastric nerves) was assessed. Epidermal growth factor increased the rate of gastrin transcription almost 3-fold, whereas thyrotropin-releasing hormone and vasoactive intestinal peptide increased gastrin transcription 2- and 1.5-fold, respectively. Gastrin-releasing peptide, a peptide that strongly stimulates gastrin release, weakly increased gastrin transcription (1.3-fold). Somatostatin inhibited the increase in gastrin transcription induced by epidermal growth factor, thyrotropin-releasing hormone, and vasoactive intestinal peptide. Constructs containing various lengths of 5' regulatory sequences defined a response element -40 to -82 base pairs (bp) 5' to the transcription initiation site. This 40-bp sequence contains Sp1 and AP2 binding sites, which suggests that epidermal growth factor and thyrotropin-releasing hormone stimulate gastrin gene transcription through transcription factors that bind to Sp1 and/or AP2 motifs.
Molecular cloning and sequencing of cDNAs encoding the entire rat fatty acid synthase.
Authors: Amy C M , Witkowski A , Naggert J , Williams B , Randhawa Z , Smith S
Published: May 2006

Overlapping cloned cDNAs representing the entire sequence of the rat fatty acid synthase mRNA have been isolated from a cDNA library and sequenced. Authenticity of the cDNA clones was supported by hybridization to fatty acid synthase mRNA and by amino-terminal sequencing of 39 fatty acid synthase CNBr fragments. The full-length fatty acid synthase mRNA is 9156 nucleotides long and includes an 84-nucleotide 5' noncoding region, a 7515-nucleotide coding sequence, and a 1537-nucleotide 3' noncoding region; a second mRNA species containing a shortened 3' noncoding sequence is also transcribed in the rat. The encoded fatty acid synthase subunit contains 2505 amino acids and has a molecular weight of 272,340. Active sites and substrate binding sites were located within the sequence, thus establishing the order of domains on the multifunctional animal fatty acid synthase as condensing enzyme-transferase-dehydrase-enoyl reductase-ketoreductase-acyl carrier protein-thioesterase.
A ras-related protein is phosphorylated and translocated by agonists that increase cAMP levels in human platelets.
Authors: Lapetina E G , Lacal J C , Reep B R , Molina y Vedia L
Published: May 2006

The antigenicity of platelet proteins was assayed against various monoclonal antibodies (mAbs) that recognize specific epitopes of the ras-encoded p21 protein. mAb M90, which detects the region of p21 protein within amino acids 107-130 and inhibits its GTP-binding activity, strongly reacted with a 22-kDa protein present in the particulate fraction of human platelets. Other mAbs against ras-encoded proteins, including Y13-259, which efficiently detects ras proteins from a variety of organisms, did not recognize the platelet 22-kDa protein. Transfer of the platelet 22-kDa protein to nitrocellulose paper showed that the protein binds [alpha-32P]GTP. Moreover, preincubation of the transferred protein with mAb M90 drastically reduced its GTP-binding activity. Treatment of platelets with iloprost, a prostacyclin analog, caused (i) a time-dependent increase of a 24-kDa protein that is recognized by mAb M90 in particulate and cytosolic fractions and (ii) the gradual decrease of the 22-kDa protein from the particulate fraction. When platelets were labeled with 32P and then treated with iloprost, the 24-kDa protein was found to be phosphorylated. The 32P-labeled 24-kDa protein was specifically immunoprecipitated by mAb M90. These results suggest that appearance of the 24-kDa protein results from phosphorylation of the 22-kDa protein, which shifts its mobility to a higher molecular mass area.