bluuebunny/crossref_metadata_2025 huggingface dataset:
Imaging of calcium and aluminum in neurofibrillary tangle-bearing neurons in parkinsonism-dementia of Guam.
Authors: Garruto R M , Fukatsu R , Yanagihara R , Gajdusek D C , Hook G , Fiori C E
Published: May 2006

We report the distribution and imaging of calcium and aluminum in neurofibrillary tangle (NFT)-bearing neurons within Sommer's sector of the hippocampus in Guamanian patients with parkinsonism-dementia, using a method of computer-controlled electron beam x-ray micro-analysis and wavelength dispersive spectrometry. Calcium and aluminum were distributed in cell bodies and axonal processes of NFT-bearing neurons. The elemental images show that both calcium and aluminum deposits occur within the same NFT-bearing hippocampal neuron in this dementing disease, suggesting that these elements are involved in NFT formation. No prominent concentrations of calcium and aluminum were imaged in non-NFT-containing regions within the pyramidal cell layer of the parkinsonism-dementia cases or in the control cases. These findings support the hypothesis that secondary hyperparathyroidism resulting from low environmental calcium and magnesium in the high-incidence focus of amyotrophic lateral sclerosis and parkinsonism-dementia on Guam had led to abnormal deposition of calcium and aluminum in the central nervous system.
Gastrin and cholecystokinin in pituitary neurons.
Authors: Rehfeld J F , Hansen H F , Larsson L I , Stengaard-Pedersen K , Thorn N A
Published: May 2006

Gastrins occur in the hypothalamo-hypophyseal neurons of all mammalian species examined. In addition, human, bovine, and murine hypothalamo-hypophyseal neurons contain the homologous cholecystokinins (CCKs). CCK also occurs in neurons innervating bovine melanotrophs. Although the concentration of gastrin is of the same magnitude (15-30 pmol/g) in all neural lobes, the concentration of CCK varies from undetectable in pig and cat to 1 nmol/g in the cow. The constant occurrence of neurohypophyseal gastrin suggests a role different from that of the species-dependent CCK.
Purification of cowpea mosaic virus RNA replication complex: Identification of a virus-encoded 110,000-dalton polypeptide responsible for RNA chain elongation
Authors: Dorssers Lambert , van der Krol Sander , van der Meer Jos , van Kammen Albert , Zabel Pim
Published: May 2006

An endogenous cowpea mosaic virus (CPMV) RNA-protein complex (CPMV replication complex) capable of elongating
in vitro
preexisting nascent chains to full-length viral RNAs has been solubilized from the membrane fraction of CPMV-infected cowpea leaves using Triton X-100 and purified by Sepharose 2B chromatography and glycerol gradient centrifugation in the presence of Triton X-100. Analysis of the polypeptide composition of the complex by NaDod-SO
4
/PAGE and silver staining revealed major polypeptides with molecular masses of 110, 68, and 57 kilodaltons (kDa), among which the 110-kDa polypeptide was consistently found to cosediment precisely with the RNA polymerase activity. Using antisera to specific viral proteins, we found the 110-kDa polypeptide to be the only known viral polypeptide associated with the RNA replication complex, the 68- and 57-kDa polypeptides being most probably host-specific. The host-encoded 130-kDa monomeric RNA-dependent RNA polymerase, which is known to be stimulated in CPMV-infected cowpea leaves, did not copurify with the virus-specific RNA polymerase complex. Our results dispute the hypothesis that plant viral RNA replication may be mediated by the RNA-dependent RNA polymerase of uninfected plants. We tentatively conclude that the 110-kDa polypeptide encoded by the bottom component RNA of CPMV constitutes the core of the CPMV RNA replication complex.
Association of phorbol ester-induced hyperphosphorylation and reversible regulation of transferrin membrane receptors in HL60 cells.
Authors: May W S , Jacobs S , Cuatrecasas P
Published: May 2006

Phorbol diesters are tumor-promoting agents that cause differentiation of HL60 human leukemic cells and concomitantly alter surface transferrin-receptor expression [Rovera, G., Ferreo, D., Pagliardi, G. L., Vartikar, J., Pessano, S., Bottero, L., Abraham, S. & Lebman, D. (1982) Ann. N.Y. Acad. Sci. 397, 211-220]. Transferrin-receptor regulation is shown here to result from a rapid and reversible internalization process that is temporally associated with reversible increased phosphorylation (hyperphosphorylation) of the transferrin receptor. Such a reversible mechanism involving regulation of these surface proteins could result in the rapid generation of an early signal for HL60 cellular differentiation.
Functional and inducible expression of a transfected murine class II major histocompatibility complex gene.
Authors: Folsom V , Gold D P , White J , Marrack P , Kappler J , Tonegawa S
Published: May 2006

Using the spheroplast fusion technique, we have introduced the cloned E beta b gene into two d haplotype cell lines, the B lymphoma line A20-2J and the macrophage tumor line P388D1. Analysis with a monoclonal antibody indicates that the product of the transfected E beta b gene associates with the endogenous E alpha chain to form an E alpha dE beta b complex. While expression of E alpha dE beta b is constitutive in A20-2J cells transfected with the E beta b gene, surface expression of E alpha dE beta b is detected in transfected macrophage cells only after treatment of cells with culture supernatants from concanavalin A (Con A)-stimulated T cells. Transfected B lymphoma cells and transfected Con A supernatant-treated macrophage cells have acquired the ability to present antigen to E alpha dE beta b-restricted T-cell hybridomas. The observed inducible expression of the transfected gene in the macrophage host indicates that sequences responsible for regulated expression of the E beta b gene may be associated with the transfected gene. In combination with directed mutagenesis, the system described here provides a means to study (i) E beta b sequences that are important in determining the restriction specificity of the E molecule and (ii) sequences associated with the E beta gene that may be important in the regulation of E beta chain expression.
The presence of cysteine in the cytoplasmic domain of the vesicular stomatitis virus glycoprotein is required for palmitate addition.
Authors: Rose J K , Adams G A , Gallione C J
Published: May 2006

The transmembrane glycoprotein (G protein) of vesicular stomatitis virus (VSV) is known to contain 1-2 mol of covalently linked fatty acid (palmitate) per mol of protein. G protein is oriented in cellular membranes such that the carboxyl-terminal 29 amino acids protrude into the cytoplasm. We have obtained expression in eukaryotic cells of mutagenized cDNA clones that encode VSV G proteins lacking portions of this cytoplasmic domain. Labeling of these truncated proteins with [3H]palmitate indicated that the palmitate might be linked to an amino acid residue within the first 14 residues on the carboxyl-terminal side of the transmembrane domain. Using oligonucleotide directed mutagenesis, we changed the single codon specifying cysteine in this domain to a codon specifying serine. Expression of this mutant gene results in synthesis of a G protein lacking palmitate. We suggest that linkage of palmitate to G protein is through the cysteine in the cytoplasmic domain and that such a linkage may occur in many viral and cellular glycoproteins. The G protein lacking palmitate is glycosylated and is transported normally to the cell surface.
Identification of glial filament protein and vimentin in the same intermediate filament system in human glioma cells.
Authors: Wang E , Cairncross J G , Liem R K
Published: May 2006

We have used a human glioma cell line (U-251MG) to study the expression and cytoplasmic organization of vimentin (decamin) and the glial filament protein (GFP). Four clones of the parental U-251 cultures were isolated and found to express GFP from 1-2% to 99% of the cells in the population. Double immunofluorescence microscopy with antibodies to vimentin and GFP has shown that, in all four clonal cell lines, vimentin-containing filaments are expressed in most cells as an organized network and, in GFP-positive cells, GFP and vimentin are associated with the same filament network. Immunoelectron microscopy with specific antibodies labeled with colloidal gold particles of various sizes shows that GFP and vimentin are localized in the same filaments. These findings confirm in vitro studies of the copolymerization of subunits of different biochemical nature into the same intermediate filament and suggest the in vivo probability of the coassembly of GFP and vimentin from a possible soluble pool of monomers.
Structure, expression, and mutation of the hypoxanthine phosphoribosyltransferase gene.
Authors: Melton D W , Konecki D S , Brennand J , Caskey C T
Published: May 2006

The wild-type mouse hypoxanthine phosphoribosyltransferase (HPRT; IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) gene has been isolated from genomic libraries and its structure has been determined. This X chromosome-linked gene is greater than 33 kilobases long and is split into nine exons. All the exon sequences have been determined, and a single-base substitution in the HPRT cDNA coding sequence from a mouse neuroblastoma cell line that overproduces a mutant HPRT protein has been identified. The 5' end of the gene has been defined, both by nuclease S1 protection and primer extension studies and by a functional assay in which an HPRT minigene, capable of expression in cultured cells, was created by ligating the 5' end of the gene onto wild-type human HPRT cDNA. Sequences normally associated with eukaryotic promoters are not present in the immediate 5'-flanking region of the HPRT gene, which is instead highly G+C rich. This observation is discussed in relation to the possible link between DNA methylation and X-chromosome inactivation.
Thymus-derived lymphocytes and their interactions with macrophages are required for the production of osteoclast-activating factor in the mouse.
Authors: Horowitz M , Vignery A , Gershon R K , Baron R
Published: May 2006

A bone-resorbing factor, comparable to the osteoclast-activating factor (OAF) produced from peripheral blood leukocytes, is shown to be produced by murine spleen cells activated with the T-cell mitogen Con A. Murine OAF is demonstrated here as being a product of the interaction between thymus-derived T lymphocytes and macrophages. Activation of T cells in the presence of macrophages with Con A yields culture supernatants with OAF activity. This OAF activity is not dialyzable and is not extracted by lipid solvents. Purified B cells in the presence or absence of macrophages and cocultured with Con A or activated with the B-cell-specific mitogen lipopolysaccharide yield culture supernatants with no detectable OAF activity. Similarly, macrophages cocultured with Con A or activated with lipopolysaccharide fail to yield culture supernatants with bone resorbing activity. These types of immune cell interactions are similar to that required for the production of lymphokines. These data support the hypothesis that one aspect of regulation of bone remodeling is through cells of the immune system.
Leukotrienes stimulate insulin release from the rat pancreas.
Authors: Pek S B , Walsh M F
Published: May 2006

We and others had observed that drugs that inhibit the lipoxygenase enzymes inhibit insulin release. The aim of the study was to search for evidence that, among the products of lipoxygenase-activated metabolism of arachidonic acid, leukotrienes (LTs) promote insulin release. Synthetic LTB4, LTC4, LTD4, and LTE4 were administered over 1-min periods to the isolated, perfused rat pancreas in the presence of 5.6 mM glucose. Perfusion effluent levels of insulin and glucagon were measured by radioimmunoassay. LTB4 and LTC4 and, to a lesser extent, LTE4 and LTD4 stimulated insulin release in a dose-related manner, in the concentration range of 10(-11) to 10(-7) M. Only 10(-7) M LTC4 stimulated glucagon release. We conclude that (i) among the arachidonic acid metabolites, LTs are involved in the modulation of secretion of pancreatic islet hormones and (ii) LTs preferentially promote insulin release.
Isolation and characterization of a stage-specific transforming gene, Tlym-I, from T-cell lymphomas.
Authors: Lane M A , Sainten A , Doherty K M , Cooper G M
Published: May 2006

A cellular transforming gene detected by transfection of mouse T-cell lymphoma DNA has been isolated by molecular cloning. This gene (designated Tlym-I) is homologous to a small conserved family of sequences present in normal mouse and human DNAs but is not related to any of the previously described viral or cellular transforming genes.
Chemically defined media for cultivation of
Naegleria gruberi
Authors: Fulton Chandler , Webster Cecelia , Wu Jennifer S.
Published: May 2006

Cultivation of amebae of the axenic strain of
Naegleria gruberi
, NEG-M, was achieved in media consisting entirely of chemically defined components. A complete medium that contains 31 components allows growth with yields up to 5 × 10
6
amebae per ml. A minimal medium gives lower yields but defines 22 components that are essential for continuous cultivation: 11 amino acids, 6 vitamins, hematin, guanosine, D-glucose, Mg
2+
, and inorganic phosphate. These media allow precise studies of the metabolism and differentiation of this unusual eukaryote.
The 2p breakpoint of a 2;8 translocation in Burkitt lymphoma interrupts the V kappa locus.
Authors: Emanuel B S , Selden J R , Chaganti R S , Jhanwar S , Nowell P C , Croce C M
Published: May 2006

The majority of chromosomal rearrangements observed in Burkitt lymphomas involve a translocation between 8q and 14q, while the remaining minority carry variant translocations between chromosome 8 and either 2 or 22. We have studied the JI Burkitt lymphoma cell line carrying the variant 2;8 chromosome translocation using a combination of high-resolution and molecular cytogenetic techniques. We have determined that the chromosome 2 breakpoint of the 2;8 translocation in these cells is in the distal portion of 2p11.2. In situ hybridization of a DNA probe for kappa light chain variable (V kappa) region demonstrated that this 2p11.2 breakpoint is within the V kappa region. There was significant hybridization of the probe to both the 2p- and 8q+ chromosomes, with 23% of all grains considered to be specific for V kappa located over the middle one-third of the long arm of the 8q+ chromosome. Thus, there is translocation of the entire kappa constant (C kappa) region and a portion of the region carrying V kappa genes from 2p to a region 3' of the c-myc oncogene on the involved chromosome 8, resulting in transcriptional activation of the c-myc that is quite distant from the 5' end of the C kappa gene. These results provide direct evidence for translocation-related rearrangement of the kappa immunoglobulin gene cluster in this Burkitt lymphoma and for the assignment of the V kappa locus to 2p11.2.
Development of a human T-cell hybridoma secreting separate B-cell growth and differentiation factors.
Authors: Butler J L , Falkoff R J , Fauci A S
Published: May 2006

A cloned human T-cell hybridoma (7D5) secreting B-cell growth factor (BCGF) and B-cell differentiation factor (BCDF) was established. Supernatant from this hybrid was capable of maintaining proliferation in anti-IgM-activated normal human B cells. In addition, the hybridoma supernatant induced differentiation and antibody secretion in Staphylococcus aureus Cowan I-stimulated B cells. No interleukin 2 was present in supernatant from this hybridoma. Molecular size of the hybridoma-derived BCGF and BCDF was determined by gel filtration chromatography. BCGF activity was present in the 20-kDa fractions, and BCDF activity eluted in the 30- to 35-kDa fractions. The isoelectric points of the factors, determined by chromatofocusing, were 6.6 for BCGF and 5.9 for BCDF. Finally, absorption experiments were performed using specific target cells. Phytohemagglutinin-stimulated T-cell blasts did not remove either BCGF or BCDF activity. Anti-IgM-activated B cells absorbed BCGF but not BCDF. In contrast, CESS cells removed BCDF but not BCGF. Thus, a human T-cell hybridoma secreting two distinct B-cell lymphokines was developed. Further immunochemical and functional studies of these immunoregulatory molecules should greatly enhance our understanding of the regulation of human B-cell function in normal and disease states.
Single-cell studies on hapten-specific B cells: response to T-cell-dependent antigens.
Authors: Hebbard G S , Pike B L , Nossal G J
Published: May 2006

The effectiveness of the hapten-gelatin antigen-affinity fractionation technique for selection of hapten-specific B cells activatable by "T-cell-dependent" (TD) stimuli was assessed. Normal adult murine spleen cells were fractionated on fluorescein (Flu)-gelatin layers and the adherent cells were cultured singly or in small numbers with various sources of syngeneic keyhole limpet hemocyanin (KLH)-primed T lymphocytes. Conditions were defined under which the addition of Flu-KLH caused optimal clonal proliferation and differentiation of B cells into anti-Flu directly hemolytic plaque-forming cells (pfc). It was found that 3-5% of the Flu-specific B cells could be activated, versus 1 in 5000 unfractionated spleen cells. The mean enrichment factor for fractionation was 179, almost identical to that seen when the stimulus is "T-cell-independent" (TI), showing that the method is capable of isolating B cells responsive to antigenic stimuli requiring specific T-cell help. Efforts were made to determine whether TD B cells constituted a separate population from TI B cells by determining clone frequencies using Flu-KLH, the TI antigen Flu-polymerized flagellin (Flu-POL), or a mixture of both for stimulation. With Flu-POL alone and with the mixed stimulus 2-3 times more pfc clones were produced than with Flu-KLH, yet evidence for separate B-cell subsets was not obtained because of strong "bystander" stimulation due to the presence of the carrier-primed T cells in a confined volume of 10 microliters.
Primary structural evidence that the H-2Dq region encodes at least three distinct gene products: Dq, Lq, and Rq.
Authors: Lillehoj E P , Hansen T H , Sachs D H , Coligan J E
Published: May 2006

Class I gene products D, L, and R encoded by the D region of the H-2q haplotype were isolated by sequential immunoprecipitation from Nonidet P-40 extracts of B10.AKM murine spleen cells. Primary structural comparisons of these molecules were made by tryptic peptide map analyses and radiochemical amino acid sequence determinations. Partial NH2-terminal radiosequence analyses of the peptides generated by CNBr cleavage of these three molecules defined the Rq molecule as a gene product that is distinct from the Dq and Lq molecules. Comparative tryptic peptide map analyses of [3H]arginine-, [3H]lysine-, or [3H]mannose-labeled molecules were performed to determine if the Dq and Lq molecules could be distinguished. The two molecules were identical in their [3H]lysine-labeled peptides (17 in common) and [3H]mannose-labeled glycopeptides but could be differentiated by their [3H]arginine-labeled peptides (18 of 21 in common). These results suggest an amino acid sequence homology of 99% or greater between the Dq and Lq molecules. These data therefore provide structural evidence for three D region gene products and suggest that their antigenic differences are not due to post-translational glycosylation differences. Thus, the D region of the H-2q haplotype may provide an example of a relatively recent class I gene duplication, indicating that these genes are in a dynamic state of evolution through expansion or contraction of the gene pool.
Detection of cerebral lactate in vivo during hypoxemia by 1H NMR at relatively low field strengths (1.9 T).
Authors: Behar K L , Rothman D L , Shulman R G , Petroff O A , Prichard J W
Published: May 2006

1H NMR was used to monitor lactate production and clearance during hypoxemia and recovery in the rabbit brain at the relatively low magnetic-field strength of 1.89 T. An array of conventional physiological variables were recorded simultaneously with spectrum acquisition, including the electroencephalogram and electrocardiogram. The sensitivity and spectral resolution achieved at this field strength should be applicable to studies of human brain pathophysiology in the large-bore magnets now available.
Cultures of shiverer mutant cerebellum injected with normal oligodendrocytes make both normal and shiverer myelin.
Authors: Billings-Gagliardi S , Hall A L , Stanhope G B , Altschuler R J , Sidman R L , Wolf M K
Published: May 2006

Earlier reports suggested that injecting normal optic nerve into organotypic cerebellar cultures from three of the central nervous system hypomyelinated mutant mice resulted in striking local increases in myelination of mutant axons. It has been questioned whether this myelin was produced by the introduced normal glia because there was no "marker" by which the genotype of an individual myelin sheath could be rigorously determined. The present study shows that the myelin sheaths of the central nervous system hypomyelinated mutant shiverer (shi/shi) can be distinguished in vitro from genetically normal myelin by their immunocytochemical reactions and ultrastructure. When shi/shi cultures are injected with normal optic nerve, they produce two kinds of myelin as distinguished by these techniques: shi/shi myelin throughout and, in addition, ultrastructurally and immunocytochemically normal myelin near the optic nerve. These results suggest that the primary defect of the shi/shi mutation involves the oligodendrocyte and support the earlier conclusion that in all of the glial injection experiments to date the genotype of the oligodendrocyte determines the phenotype of the myelin produced.
Phosphatidylserine functions as the major precursor of phosphatidylethanolamine in cultured BHK-21 cells.
Authors: Voelker D R
Published: May 2006

Pulse-chase experiments with [3H]serine provide evidence that significant amounts of phosphatidylserine turn over to form phosphatidylethanolamine in mammalian cells in tissue culture. Phospholipase C hydrolysis of [3H]phosphatidylethanolamine synthesized from [3H]serine by baby hamster kidney (BHK-21) cells demonstrates that nearly all of the radiolabel remains in the ethanolamine moiety. Uniform labeling experiments with [3H]serine further demonstrate that the distribution of radiolabel in phosphatidylserine and phosphatidylethanolamine is nearly identical to the mass ratio of these lipids. Physiological concentrations of ethanolamine (20 microM) have only a marginal effect upon the ability of cells in culture to incorporate radiolabeled serine into either phosphatidylserine or phosphatidylethanolamine. These data provide compelling evidence that phosphatidylethanolamine synthesis via phosphatidylserine and phosphatidylserine decarboxylase contributes significantly to membrane biogenesis in mammalian cells.
Nondisulfide polymerization of gamma- and beta-crystallins in the human lens.
Authors: Roy D , Dillon J , Wada E , Chaney W , Spector A
Published: May 2006

The water-soluble 43,000-dalton fraction (WS43) of the human lens has been shown to be heterogeneous. It appears to contain, in addition to actin, components related to the crystallins. Immunoblot reactions indicate that this polypeptide fraction is composed of dimers containing beta- and gamma-crystallin components. It has been estimated that 10-30% of this fraction arises by dimerization of gamma-crystallin. A possible route for the formation of the 43,000-dalton fraction is suggested by the observation that photolysis of gamma-crystallin with light greater than 295 nm leads to polymer formation, including the 43,000-dalton fraction. The polymerization products react with anti-WS43. The results suggest that photochemical reactions may lead to the accumulation of polymers of some of the crystallins with aging of the human lens. Similar covalently linked polypeptides have previously been shown to be present in the high molecular weight aggregates associated with cataract formation.
Formation of a transformed follicle is necessary but not sufficient for development of an avian leukosis virus-induced lymphoma.
Authors: Baba T W , Humphries E H
Published: May 2006

Avian leukosis virus (ALV) infection of susceptible chickens induces bursal lymphomas after a latent period of several months. The clonal development of these B-cell tumors is believed to be a multistep process. Histopathological changes, referred to as transformed follicles, occur within the target organ soon after virus infection and may represent a proximal stage of lymphomagenesis. To establish further the significance of this lesion and its relationship to the subsequent development of lymphomas, we have compared the incidence of transformed follicles observed in animals susceptible or resistant to ALV-induced tumor development. During the 8 weeks following ALV infection, transformed follicles were detected in 82% of the susceptible animals and in 11% of the resistant animals. These results indicate that the incidence of transformed follicles in these animals correlates with their susceptibility to lymphoma development. Furthermore, each transformed follicle does not develop into a tumor. These observations suggest that the formation of a transformed follicle is necessary but not sufficient for lymphoma development.
Statistical test of linearity of photoreceptor transduction process: Limulus passes, others fail.
Authors: Grzywacz N M , Hillman P
Published: May 2006

We present the results of a theoretical analysis of a completely general linear chain model for transduction in photoreceptors from which we have derived a statistical test for the intrinsic linearity of the single-photon transduction process. By linearity we mean comprising first-order chemical reactions only. We show results of our own measurements in Limulus ventral photoreceptors that pass this linearity test, suggesting that the single-photon transduction in Limulus may be a simple chain of first-order biochemical reactions (plus possible diffusional processes). However, we also demonstrate that published data show the existence of strong nonlinearities in the single-photon responses of toad and perhaps also of locust. Such nonlinearities are not difficult to construct from existing biochemical notions (feedback, cooperativity), but all but one [Kramer, L. (1975) Biophys. Struct. Mech. 1,239-257] of the published analytical models of the single-photon process have been linear. The test we have used is the distribution of "areas" (time integrals of conductance changes) of single-photon responses or "bumps." Reasonable molecular linear chain models do not allow distributions very sharply peaked at non-zero values. Such peaked distributions are seen in toad and locust but not in Limulus.
Sequence context effects in DNA replication blocks induced by aflatoxin B1.
Authors: Refolo L M , Conley M P , Sambamurti K , Jacobsen J S , Humayun M Z
Published: May 2006

The genotoxic effects of the potent mutagenic carcinogen aflatoxin B1 (AFB1) are believed to be mediated by its reaction with the N-7 atom of guanine residues in DNA. We have analyzed the effect of AFB1-induced chemical modification on the template function of single-stranded DNA in vitro. The experimental strategy involves the elongation of a primer on a modified template by Escherichia coli DNA polymerase I (large fragment) and analysis of the products by high-resolution gel electrophoresis. Our data show that (i) AFB1 induces specific replication blocks one nucleotide 3' to the sites of occurrence of guanine residues on template DNA; (ii) AFB1-induced replication blocks occur predominantly at sequences capable of participation in intrastrand base pairing; (iii) within the intrastrand base-paired regions there are strong sequence context effects, in accordance with the previously described [Muench, K. F., Misra, R. P. & Humayun, M. Z. (1983) Proc. Natl. Acad. Sci. USA 80, 6-10] specificity "rules" that apply to the reaction of AFB1 with guanine residues in double-stranded DNA; (iv) there is evidence that the (7-guanyl)-AFB1 adducts as well as secondary derivatives such as the formamidopyrimidine-AFB1 act as replication blocks. In summary, these data suggest that previously observed inhibition of DNA replication and transcription by AFB1 is directly attributable to (7-guanyl)-AFB1 adducts or their secondary reaction products.
1H homonuclear editing of rat brain using semiselective pulses.
Authors: Hetherington H P , Avison M J , Shulman R G
Published: May 2006

We have used a semiselective Hahn spin-echo sequence of the form (1331)-tau-(2662)-tau-AQ, delivered by a surface coil to obtain high-resolution 1H NMR spectra from the brains of intact dead rats. This sequence gave suppression of the tissue water resonance by a factor of 80,000 when tau = 68 ms. Delivery of a frequency-selective Dante pulse train to the alpha-CH resonance of lactate at 4.11 ppm, simultaneously with the 2662 refocusing pulse, altered the j-modulation in the spin-coupled beta-CH3 protons. Subtraction of this spectrum from one in which the Dante was ineffective gave an edited spectrum containing only the beta-CH3 resonance of lactate at 1.31 ppm. When the position of the Dante was shifted to 3.78 ppm to selectively invert the alpha-CH protons of alanine, an edited spectrum of alanine was obtained.
Beta-endorphin-(1-27) is a naturally occurring antagonist to etorphine-induced analgesia.
Authors: Nicolas P , Li C H
Published: May 2006

The potent opioid peptide beta-endorphin is found in the brain and pituitary with two related fragments, beta-endorphin-(1-27) and beta-endorphin-(1-26). The fragments retain substantial opioid-receptor binding activity but are virtually inactive analgesically. beta-Endorphin-(1-27) inhibits beta-endorphin-induced and etorphine-induced analgesia when coinjected intracerebroventricularly into mice. Antagonism by competition at the same site(s) is suggested from parallel shifts of the dose-response curves of etorphine or beta-endorphin in the presence of beta-endorphin-(1-27). Its potency is 4-5 times greater than that of the opiate antagonist naloxone. beta-Endorphin-(1-26) does not antagonize the antinociceptive action of etorphine or beta-endorphin in doses up to 500 pmol per animal.
T-cell receptor gene rearrangements as markers of lineage and clonality in T-cell neoplasms.
Authors: Flug F , Pelicci P G , Bonetti F , Knowles D M , Dalla-Favera R
Published: May 2006

Ig gene rearrangements represent markers of lineage, clonality, and differentiation of B cells, allowing a molecular diagnosis and immunogenotypic classification of B-cell neoplasms. We sought to apply a similar approach to the study of T-cell populations by analyzing rearrangements of the T-cell receptor beta-chain (T beta) gene. Our analysis, by Southern blotting hybridization using T beta-specific probes of DNAs from polyclonal T cells and from 12 T-cell tumors, indicates that T beta gene rearrangement patterns can be used as markers of (i) lineage, allowing the identification of polyclonal T-cell populations, and (ii) clonality, allowing the detection of monoclonal T-cell tumors. In addition, our data indicate that T beta gene rearrangements represent early and general markers of T-cell differentiation since they are detectable in histologically different tumors at all stages of T-cell development. The ability to determine lineage, clonality, and stage of differentiation has significant implications for future experimental and clinical studies on normal and neoplastic T cells.
The 200- and 150-kDa neurofilament proteins react with IgG autoantibodies from patients with kuru, Creutzfeldt-Jakob disease, and other neurologic diseases.
Authors: Toh B H , Gibbs C J , Gajdusek D C , Goudsmit J , Dahl D
Published: May 2006

Sera from 65 patients with spongiform virus encephalopathies (29 with kuru, 36 with Creutzfeldt-Jakob disease), 79 with other neurologic diseases, and 65 control subjects were examined for reactivity in immunoblots of preparations of myelinated axons and neurofilaments from mouse brain. The sera reacted most frequently with the 200-kDa and 150-kDa neurofilament proteins and less frequently with the 70-kDa neurofilament protein and a 62-kDa neurofilament-associated protein. The sera reacted with the same proteins as those which reacted with rabbit and mouse polyclonal antibodies and mouse monoclonal antibody to neurofilament proteins. Serum reactions were also seen with Trixon X-100 extracts of chimpanzee brain and bovine spinal cord but not with Triton extracts of liver, kidney, and muscle.
Expression of human choriogonadotropin in monkey cells using a single simian virus 40 vector.
Authors: Reddy V B , Beck A K , Garramone A J , Vellucci V , Lustbader J , Bernstine E G
Published: May 2006

We have inserted the cDNAs coding for both polypeptide subunits, alpha and beta, of human choriogonadotropin (hCG) into a single simian virus 40 expression vector in such a way that they replace the viral VP2 and VP1 coding sequences, respectively. Monkey cells infected with this virus and the appropriate helper virus produce dimeric hCG. hCG produced in this system was shown to chromatograph identically to standard hCG preparations on gel filtration and to be biologically active in the mouse uterine weight assay.
An electrophoretic karyotype for yeast.
Authors: Carle G F , Olson M V
Published: May 2006

The chromosomal DNA molecules of a standard laboratory strain of Saccharomyces cerevisiae have been separated into 12 well-resolved bands by orthogonal-field-alternation gel electrophoresis. DNA X DNA hybridization probes derived from cloned genes have been used to correlate this banding pattern with yeast's genetically defined chromosomes. The 12 bands are shown to represent 9 singlets and 3 comigrating doublets, thereby accounting for 15 chromosomes that were identified as I-XI and XIII-XVI. Because the three comigrating doublets could be readily resolved in certain laboratory yeast strains that contain chromosome-length polymorphisms relative to our standard strain, all 15 of these chromosomes could be displayed as a single band in at least one of four strains that were studied. A 16th chromosome (number XII), which is known to contain the genes for rRNA, does not reproducibly enter the gels. By making use of the band identifications, the previously unmapped fragment F8 was assigned to chromosome XIII. With the possible exception of chromosomes that differ greatly in size or electrophoretic behavior from all the known chromosomes, the results appear to define a complete "electrophoretic karyotype" for yeast.
Specific 5' and 3' regions of the mu-chain gene are undermethylated at distinct stages of B-cell differentiation.
Authors: Blackman M A , Koshland M E
Published: May 2006

The mu-chain gene is expressed differently in successive stages of B-lymphocyte development. The heavy chain product appears as a cytoplasmic constituent in pre-B-cells, as part of the IgM receptor in maturing B cells, and as a component in the pentamer IgM antibody synthesized and secreted by the antigen-stimulated cell. We have used the methylation of CpG sequences as an assay system to define the chromatin changes associated with different expression of the mu-chain. The methylation status of eight index sites was followed by restriction enzyme analysis of murine cell lines representing the major stages in the developmental pathway. The analyses showed that a single Msp I/Hpa II site 5' to the immunoglobulin enhancer becomes undermethylated with the onset of mu-chain gene transcription. Four midgene Msp I/Hpa II sites exhibit a progressive loss of methyl groups unrelated to changes in mu-chain gene expression, whereas a Msp I/Hpa II site and two Hha I sites surrounding the exon encoding the carboxyl terminus of the secreted form of mu chain (mus) become undermethylated during the transition to IgM secretion. These results indicate that structural changes in local regions of the mu-chain gene correlate with specific developmental events.
Use of monoclonal antibodies to a human cytotoxin for its isolation and for examining the self-induction of resistance to this protein.
Authors: Hahn T , Toker L , Budilovsky S , Aderka D , Eshhar Z , Wallach D
Published: May 2006

Crude preparations of cytotoxins (CTXs) produced by human peripheral blood mononuclear cells exert a marked cytotoxic effect when applied to cells in the presence of cycloheximide but in its absence can induce resistance to cytotoxicity. To examine the relationship between these cytotoxic and protective activities, we attempted to fully dissociate the CTX from the other proteins secreted by mononuclear cells. Mice injected with preparations of the cytokines secreted by peripheral blood mononuclear cells developed significant titers of serum antibodies to CTX(s). Splenocytes of such immunized mice were fused with NSO myeloma cells; a few among the resulting hybridoma cells secreted CTX-binding antibodies. Immunoadsorbents constructed with a monoclonal antibody produced by one of these hybridomas were used to purify to homogeneity a CTX (Mr approximately 17,500) from crude preparations of cytokines, by a single adsorption and elution cycle. Purified CTX was cytotoxic in the presence of cycloheximide but in its absence induced resistance to cytotoxicity; this resistance was manifested by decreased vulnerability to CTX in a subsequent incubation in the presence of cycloheximide. We conclude that CTX itself can induce certain changes in cells, which are reflected in resistance to its own cytotoxic effect.
Frequency of molecular alterations affecting ras protooncogenes in human urinary tract tumors.
Authors: Fujita J , Srivastava S K , Kraus M H , Rhim J S , Tronick S R , Aaronson S A
Published: May 2006

Members of the ras gene family are activated as oncogenes in many different human cancers. To systematically determine the frequency at which such genes might be involved in the neoplastic process affecting a specific target tissue, urothelial cells, we surveyed a large series of urinary tract tumors for ras oncogenes by DNA transfection and by molecular genetic analysis. Harvey (Ha)-ras oncogenes were detected in 2 of 38 tumors by transfection, molecularly cloned in biologically active form, and shown to contain single base changes at codon 61 leading to substitutions of arginine and leucine, respectively, for glutamine at this position. One additional Ha-ras oncogene was identified in a bladder carcinoma by restriction polymorphisms at codon 12. In one of 21 tumors, we observed a 40-fold amplification of the Kirsten (Ki)-ras gene. No amplification of other ras genes was detected in any of the tumors analyzed. Our findings strengthen the conclusion that codons 12 and 61 are the major "hot spots" of ras oncogene activation and suggest that quantitative alterations in expression due to gene amplification may provide an alternative mechanism for ras gene activation in primary human tumors.
The 200- and 150-kDa neurofilament proteins react with IgG autoantibodies from chimpanzees with kuru or Creutzfeldt-Jakob disease; a 62-kDa neurofilament-associated protein reacts with sera from sheep with natural scrapie.
Authors: Toh B H , Gibbs C J , Gajdusek D C , Tuthill D D , Dahl D
Published: May 2006

Sera from 46 chimpanzees with spongiform encephalopathy (18 kuru, 28 Creutzfeldt-Jakob disease) and sera from 12 sheep with natural scrapie were tested for reactivity with immunoblots of neurofilament preparations obtained from mouse brain. The sera from the chimpanzees reacted mainly with the 200- and 150-kDa proteins of the neurofilament triplet and less frequently with the 70-kDa component of the triplet and with a 62-kDa neurofilament-associated protein. In contrast, the sera of sheep with natural scrapie reacted exclusively against the 62-kDa protein. The specificity of the reactions was established by comparison of sera reactivities with those of rabbit and mouse polyclonal antibodies and mouse monoclonal antibody to neurofilament proteins.
Evidence from two transformed cell lines that the phosphorylations of peptide tyrosine and phosphatidylinositol are catalyzed by different proteins.
Authors: MacDonald M L , Kuenzel E A , Glomset J A , Krebs E G
Published: May 2006

Two transformed rodent cell lines (RS-1 and LSTRA) were studied in vitro to determine if their major protein tyrosine kinases catalyzed the phosphorylation of phosphatidylinositol (PtdIns), phosphatidylinositol 4-phosphate (PtdIns4P), or diacylglycerol. RS-1 cells, transformed by Rous sarcoma virus, contain high levels of pp60src; LSTRA cells, transformed by Moloney murine leukemia virus, contain a tyrosine kinase (pp56) that is the product of an unknown cellular gene. Rates of phosphorylation of peptide tyrosine were elevated more than 20-fold in RS-1 and LSTRA particulate fractions compared to fractions from suitable control cells (N2 and YAC-1), but there was not a proportional increase in rates of phosphorylation of PtdIns, PtdIns4P, or diacylglycerol. Heat (34 degrees C) completely inactivated the LSTRA tyrosine kinase, while it enhanced the phosphorylation of PtdIns and PtdIns4P and had no effect on the phosphorylation of diacylglycerol. PtdIns4P inhibited the phosphorylation of PtdIns but had no effect on tyrosine kinase activity. An antibody, raised against a peptide with a sequence homologous to the autophosphorylation site of pp60src, immunoprecipitated tyrosine kinase activity from RS-1 and LSTRA extracts but had no effect on PtdIns kinase or PtdIns4P kinase activity. These results provide evidence that the phosphorylations of tyrosine and PtdIns are catalyzed by different proteins. An additional observation was that a monoclonal antibody that binds to pp60src and pp56 removed PtdIns kinase as well as tyrosine kinase activity from RS-1 and LSTRA particulate extracts. This antibody also removed PtdIns kinase from N2 and YAC-1 extracts, in which tyrosine kinase activity was low or undetectable. Thus, the anti-pp60src monoclonal antibody may recognize the PtdIns kinase in addition to pp60src and pp56.
Torsional stress induces an S1 nuclease-hypersensitive site within the promoter of the Xenopus laevis oocyte-type 5S RNA gene.
Authors: Reynolds W F , Gottesfeld J M
Published: May 2006

The internal promoter of the Xenopus laevis oocyte-type 5S RNA gene is preferentially cleaved by S1 and Bal-31 nucleases in plasmid DNA. S1 nuclease sensitivity is largely dependent on supercoiling; however, Bal-31 cleaves within the 5S RNA gene in linear as well as in supercoiled DNA. The S1 nuclease-hypersensitive site is centered at position +48-52 of the gene at the 5' boundary of the promoter. A DNAase I-hypersensitive site is induced at this position upon binding of the transcription factor, TFIIIA, specific for the 5S RNA gene. The somatic-type 5S RNA gene promoter is not preferentially cleaved by S1 nuclease or Bal-31 nuclease in supercoiled DNA, nor does TFIIIA induce a DNase I site at position +50. This differential promoter response may be related to a 4-fold difference in TFIIIA affinity between the oocyte and somatic 5S RNA genes.
Platelet-derived growth factor mimics phorbol diester action on epidermal growth factor receptor phosphorylation at threonine-654.
Authors: Davis R J , Czech M P
Published: May 2006

Addition of platelet-derived growth factor (PDGF) to quiescent WI-38 human fetal lung fibroblasts mimics the effect of tumor-promoting phorbol diesters to inhibit the high-affinity binding of 125I-labeled epidermal growth factor (125I-EGF). PDGF, like phorbol diesters, was found to increase the phosphorylation state of EGF receptors immunoprecipitated from intact fibroblasts that were labeled to equilibrium with [32P]phosphate. Phosphoamino acid analysis of the EGF receptors indicated that both PDGF and phorbol diesters increased the level of [32P]phosphoserine and [32P]phosphothreonine. Phosphopeptide mapping of the EGF receptor demonstrated that PDGF increased the phosphorylation of several sites and induced the phosphorylation of a site that was not observed to be phosphorylated on EGF receptors isolated from control cells. This latter phosphorylation site on the EGF receptor was identified as threonine-654, previously shown to be phosphorylated in response to phorbol diesters in intact cells or by purified protein kinase C in vitro. Further, it was observed that PDGF mimicked the action of phorbol diesters to inhibit the EGF-dependent tyrosine phosphorylation of the EGF receptor in [32P]phosphate-labeled fibroblasts. These results are consistent with the hypothesis that increases in diacylglycerol and Ca2+ levels caused by addition of PDGF to fibroblasts activate protein kinase C and that this kinase, at least in part, mediates the effect of PDGF on the phosphorylation of the EGF receptor. The data further suggest that protein kinase C may play an important role in the regulation of cellular metabolism and proliferation by PDGF.
Bacillus subtilis sigma factor sigma 29 is the product of the sporulation-essential gene spoIIG.
Authors: Trempy J E , Bonamy C , Szulmajster J , Haldenwang W G
Published: May 2006

Evidence is presented that the sporulation-essential locus spoIIG codes for both sigma 29 and a structurally related protein, P31. This demonstrates that at least one specific Bacillus subtilis RNA polymerase binding protein provides a critical function in endospore formation. spoIIG-specific RNA is present in B. subtilis cultures that are synthesizing P31 and sigma 29 and is absent in those that are not. A monoclonal antibody specific for an antigenic determinant on P31/sigma 29 detected crossreacting proteins (P25/P21) but not P31 or sigma 29 in a Spo- B. subtilis strain with a mutation at the spoIIG locus (spoIIG41). The appearance of P25 and P21 occurs in this mutant at a time when P31 and sigma 29 would normally appear and suggests that they are homologous proteins. Transformation of the spoIIG41 strain with plasmid DNA carrying the structural gene for spoIIG complements the Spo- phenotype and results in the synthesis of P31, sigma 29, P25, and P21 at the appropriate times during sporulation. In Escherichia coli, the cloned spoIIG sequence encoded a protein that reacted with the anti-P31/sigma 29 monoclonal antibody and had the electrophoretic mobility of authentic P31.
Regulation of c-myc mRNA levels in normal human lymphocytes by modulators of cell proliferation.
Authors: Reed J C , Nowell P C , Hoover R G
Published: May 2006

Increased expression of the cellular oncogene c-myc has recently been demonstrated in some types of proliferating non-neoplastic cells, including lectin mitogen-stimulated lymphocytes, suggesting a role for this protooncogene in the regulation of growth of normal cells. Here we report the effects of several modulators of lymphocyte proliferation on the steady-state levels of c-myc mRNA in human peripheral blood mononuclear cells (PBMC). Stimulating PBMC with lectin mitogen phytohemagglutinin (PHA), phorbol ester phorbol 12-myristate 13-acetate, calcium ionophore ionomycin, or monoclonal antibody OKT3 (anti-antigen receptor complex) produced marked increases in c-myc mRNA levels within 3 hr. Recombinant interleukin 2 (rIL-2; Cetus) had little effect on c-myc expression but, in combination with PHA, it augmented levels of c-myc transcripts measured at 24 hr but not at 3 hr. Adding various inhibitors of lymphocyte proliferation to PHA-stimulated cultures revealed that cyclosporin A, dexamethasone, and OKT11A antibody (anti-sheep erythrocyte receptor) diminished levels of c-myc mRNA measured at 3 hr and 24 hr, whereas anti-Tac (anti-IL-2-receptor) inhibited at 24 hr but not at 3 hr. Thus, cyclosporin A, dexamethasone, and OKT11A interfere with early events of T-cell activation, whereas anti-Tac acts later. Hydroxyurea and 42/6 antibody (anti-transferrin receptor), which impair the G1----S transition in cycling cells, failed to inhibit c-myc expression and instead delayed the decrease in c-myc mRNA levels that normally occurs with the onset of DNA synthesis. These data indicate that c-myc is regulated (in normal lymphocytes) at several points in the cell cycle.