bluuebunny/crossref_metadata_2025 huggingface dataset:
Ouabain-resistant hyperpolarization induced by insulin in aggregates of embryonic heart cells.
Authors: Lantz R C , Elsas L J , DeHaan R L
Published: May 2006

Spheroidal aggregates formed from trypsin-dissociated 14-day embryonic chicken hearts after 48 hr of rotation on a gyratory shaker. Intracellularly recorded resting membrane potentials of aggregates bathed in 1.3 mM K+ balanced salt solution had a mean (+/- SD) of 64 +/- 4 mV. After a stable potential was achieved, addition of 1-100 nM sodium bovine insulin caused a slow hyperpolarization of up to 19 mV after 4-5 min, followed, in some cases, by a further, more rapid, shift to a potential near EK. Equivalent hyperpolarizations were observed when insulin was added in the presence of 10 mM ouabain, indicating that enhanced Na+,K+ pump activity was not responsible for the change in membrane potential. The concentration of insulin that produced half-maximal hyperpolarization (2 nM) corresponded to the association constant of a high-affinity insulin receptor, suggesting that binding to this class of receptors led to the change in membrane potential. Steady-state current-voltage curves from current clamp experiments suggested that insulin produced an increase in slope conductance at potentials near rest by inducing an outward current with an apparent potential negative to -90 mV.
Identification of an N2 line protein of striated muscle.
Authors: Wang K , Williamson C L
Published: May 2006

Three unusually large myofibrillar proteins (Mr, > 500,000) are present in major amounts in the striated muscle of various species. the molecular properties and localization of two of these proteins (Mr, approximately 1 X 10(6)), named "titin," have been reported recently [Wang, K., McClure, J. & Tu, A.(1979) Proc. Natl. Acad. Sci. USA 76, 3698-3702]. We have now purified the third protein (Mr, approximately 5-6 X 10(5)), designated "band 3," from rabbit psoas myofibrils. Band 3 is distinct chemically and immunochemically from titin and myosin heavy chain. Immunofluorescent localization studies of band 3 in glycerinated rabbit psoas myofibrils indicated a biphasic distribution. In sarcomeres 1.8-3.7 micrometer long, band 3 is localized predominately on the N2 line in the phase-contrast lucent region of the I band. Quantitative analysis of light micrographs confirmed the unique behavior of N2 lines described in early electron microscopic studies. As the sarcomeres lengthen, the N2 line moves away from both the Z line and the M line, maintaining the same proportional distance. In very short sarcomeres (1.5-1.8 micrometer), band 3 is found mainly in the A band, suggesting that either the N2 line moves into the A band or that band 3 has dissociated from the N2 line which remains near the Z line. We conclude that band 3 is an N2 line component of rabbit skeletal myofibrils.
Attenuation and processing of RNA from the rplJL--rpoBC transcription unit of Escherichia coli.
Authors: Barry G , Squires C , Squires C L
Published: May 2006

Attenuation and processing of the mRNA from the ribosomal protein-RNA polymerase operon rplJL--rpoBC have been demonstrated by the analysis of nuclease SI-resistant RNA . DNA hybrids. These hybrids were formed between RNA produced in vivo and specific DNA restriction fragments which span the rplL--rpoB intercistronic region. The 3' end of the predominant attenuated RNA lies 69 nucleotides beyond the end of the rplL gene following sequence features that are similar to those of other known attenuators. The nonattenuated transcript is normally cleaved in the intercistronic region. However, in an RNase III mutant strain, the hybrids corresponding to the cleaved nonattenuated mRNAs disappear and the expected full-sized hybrid is seen. We have localized the cleavage to an area of possible secondary structure in the transcript approximately 200 nucleotides beyond the end of the rplL gene. This demontrates RNase III processing of Escherichia coli mRNA. The methods used in this study permit the examination of specific ends of large and complex polycistronic mRNAs. Such experiments should help in understanding how posttranscriptional events influence gene expression.
Embryonic-fetal erythroid characteristics of a human leukemic cell line.
Authors: Benz E J , Murnane M J , Tonkonow B L , Berman B W , Mazur E M , Cavallesco C , Jenko T , Snyder E L , Forget B G , Hoffman R
Published: May 2006

We have studied a number of cell surface, enzyme, and protein markers in the human leukemic K562 cell line. We have confirmed previous observations that these cells accumulate human embryonic hemoglobins after exposure to hemin. In addition, our results demonstrated that these cells possess in the "ininduced" state i surface antigen, lactate dehydrogenase isoenzymes characteristic of embryonic or fetal erythroid cells, fetal and embryonic globin chains, and globin mRNAs. The levels if i antigen, embryonic globin chains, and embryonic globin mRNA increased substantially after exposure of the cells to hemin in suspension culture. In contrast, K562 cells lacked several surface, enzymatic, and functional properties typical of granulocytes, lymphocytes, monocytes, or adult erythroblasts, including HLA surface antigens, surface immunoglobulins, sheep erythrocyte rosetting, phagocytosis, terminal deoxynucleotidyl transferase, carbonic anhydrase, ABO and Rh blood groups, and adult hemoglobins. The K562 cell line therefore exhibits phenotypic properties of embryonic erythroid progenitor cells and a quantitative increase in the expression of some of these properties can be achieved by exposure of the cells to hemin.
Gene deletions in alpha thalassemia prove that the 5' zeta locus is functional.
Authors: Pressley L , Higgs D R , Clegg J B , Weatherall D J
Published: May 2006

The deletions in the zeta-alpha globin gene cluster in two infants with the hemoglobin Bart's hydrops fetalis syndrome (homozygous alpha thalassemia 1) have been mapped by restriction endonuclease analysis using a zeta-specific probe. DNA from a Thai infant lacked the psi alpha 1 gene and both alpha genes, but the zeta genes were present. A Greek infant's DNA had also lost the 3' zeta 1 gene. Because zeta globin was synthesized in the infant's cord blood, this indicates that the 5' zeta 2 gene recently identified by Lauer et al. [Lauer, J., Shen, C. J. & Maniatis, T. (1980) Cell, in press] must be functional.
Measurement of changes in high-energy phosphates in the cardiac cycle using gated 31P nuclear magnetic renonance.
Authors: Fossel E T , Morgan H E , Ingwall J S
Published: May 2006

Levels of the high-energy phosphate-containing compounds, ATP and creatine phosphate, and of inorganic phosphate (Pi) were measured as a function of position in the cardiac cycle. Measurements were made on isolated, perfused, working rat hearts through the use of gated 31P nuclear magnetic resonance spectroscopy. Levels of ATP and creatine phosphate were found to vary during the cardiac cycle and were maximal at minimal aortic pressure and minimal at maximal aortic pressure. Pi varied inversely with the high-energy phosphates.
Identification of mineralocorticoid target sites in the isolated rabbit cortical nephron
Authors: Marver Diana , Schwartz Michael Jay
Published: May 2006

Previous evidence suggests that the activity of the mitochondrial enzyme citrate synthase [citrate oxaloacetate-lyase (
pro
-3
S
-CH
2
COO → acetyl-CoA), EC 4.1.3.7] is increased in target tissues upon acute administration of aldosterone. Therefore, an ultramicro assay was established to determine citrate synthase levels in isolated rabbit nephron segments as a means of localizing mineralocorticoid-responsive sites within the renal cortex. The relative citrate synthase activities in normal rabbit segments (per kg of dry tissue) correlated with the metabolic activity of the segments. The order was: distal convoluted tubule > proximal convoluted tubule > cortical thick ascending limb of Henle > cortical collecting duct > pars recta. When these segments were isolated from adrenalectomized rabbits, only the citrate synthase activity in the cortical collecting duct was significantly decreased compared to normal values (3.2 mol of citrate/kg dry wt per hr compared to 7.1;
P
< 0.001). Furthermore, enzyme activities in segments isolated from adrenalectomized rabbits 90 min after intravenous injection of aldosterone (10 μg/kg) were unchanged from normal or adrenalectomized rabbit tubule values for all segments except the cortical collecting duct. In this segment, aldosterone significantly increased citrate synthase activity compared to adrenalectomized rabbit values (8.1 mol/kg per hr compared to 3.2;
P
< 0.001), in contrast to the effect of dexamethasone at 10 μg/kg (4.4 mol/kg per hr compared to 3.2;
P
, NS). Spirolactone SC 26304 administered 30 min prior to injection of aldosterone inhibited the increase in collecting duct citrate synthase activity seen with aldosterone alone (3.4 mol/kg per hr compared to 8.1;
P
< 0.001). These findings suggest that the collecting duct is the primary target for aldosterone in the renal cortex.
Macular corneal dystrophy: failure to synthesize a mature keratan sulfate proteoglycan.
Authors: Hassell J R , Newsome D A , Krachmer J H , Rodrigues M M
Published: May 2006

Corneal specimens obtained during surgery from patients with macular corneal dystrophy and obtained at autopsy from control eyes were incubated in a medium containing radioactive precursors of glycoproteins and proteoglycans. Biosynthetically radiolabeled material was extracted and characterized by using molecular sieve chromatography and specific enzymes. Cells in control corneas synthesized both a chondroitin sulfate proteoglycan and a keratan sulfate proteoglycan similar to those present in monkey and bovine corneas. Cells in macular corneas synthesized a normal chondroitin sulfate proteoglycan but did not synthesize either keratan sulfate or a mature keratan sulfate proteoglycan. Instead, macular corneas synthesized a glycoprotein with unusually large oligosaccharide side chains. This glycoprotein was not detected in normal corneas and is slightly smaller than normal keratan sulfate proteoglycan. The failure to synthesize a mature keratan sulfate proteoglycan may produce corneal opacity and result in blindness. Because of evidence indicating that the corneal keratan sulfate proteoglycan is normally synthesized through a glycoprotein intermediate [Hart, G. W. & Lennarz, W. (1978) J. Biol. Chem. 253-5795-5801], macular corneal dystrophy may be a defect in glycoprotein processing.
The protein encoded by the transforming gene of avian sarcoma virus (pp60src) and a homologous protein in normal cells (pp60proto-src) are associated with the plasma membrane.
Authors: Courtneidge S A , Levinson A D , Bishop J M
Published: May 2006

Oncogenesis by avian sarcoma virus is attributable to a single viral gene (src) which encodes a phosphoprotein (pp60src) with the enzymatic activity of a protein kinase. A closely related protein, pp60proto-src, occurs in uninfected cells from a wide variety of vertebrate species and is presumed to be the product of a cellular gene that served as progenitor for src. We explored the location of these proteins within the cell by using immunoprecipitation to analyze subcellular fractions prepared from avian sarcoma virus-transformed rat and chicken cells and from uninfected rat cells. We found that both pp60src and pp60proto-src were associated with the plasma membrane as active protein kinases and could be recovered efficiently only by disrupting the membranes with nonionic detergent. Our findings, in conjunction with those of other investigators, indicate that both proteins are embedded in the membrane by means of a hydrophobic domain(s); available evidence indicates that pp60src is not exposed on the surface of the cell but is accessible at the cytoplasmic aspect of the plasma membrane. These conclusions lend credence to two current speculations. First, pp60src and pp60proto-src may have similar or even identical functions. Second, neoplastic transformation may originate from derangements in the plasma membrane or its affiliated structures.
Replicator regions of the yeast mitochondrial DNA responsible for suppressiveness.
Authors: Blanc H , Dujon B
Published: May 2006

Hypersuppressiveness is a heritable property of some rho- mutants (called HS) that, in crosses to rho+, give rise to about 100% rho- cells. The mtDNAs of all HS rho- mutants reveal a common organization: they all share a homologous region of about 300 base pairs (called rep) and the fragments retained are always short (ca. 1% of the wild-type genome) and tandemly repeated. Using one HS rho- mutant as an example, we show that, after crosses with rho+ strains, the mitochondrial genome of the progeny is indistinguishable from that of the HS parent. This suggests that HS mtDNA molecules have a decisive selective advantage for replication during the transient heteroplasmic stage that follows zygote formation, the rep regions playing a role in the control of replication initiation of the mtDNA molecules. The complete nucleotide sequence of one HS rho- mutant and its localization in the oli1-rib3 segment of the rho+ mitochondrial genome are presented. Comparison of the nucleotide sequences of the rep regions of two different HS rho- mutants reveals that several rep sequences must exist in the wild-type genome, probably as a result of duplications of an originally unique ancestor.
In vitro aggregation of the gene-sized DNA molecules of the ciliate Stylonychia mytilus.
Authors: Lipps H J
Published: May 2006

Macronuclear DNA of hypotrichous ciliates exists in the form of gene-sized DNA molecules. It can be resolved on agarose gels into a continuum of sizes upon which is imposed a set of characteristic DNA bands. Most or all of the DNA molecules carry identical terminal inverted repeat sequences. By incubating macronuclear DNA under increasingly stronger ionic conditions, high molecular weight DNA aggregates and ring-like DNA structures are formed. Experimental evidence is presented that this aggregation is not due to the presence of identical single-stranded DNA ends on each macronuclear DNA fragment, and an alternative model for DNA aggregation is discussed.
Monoclonal antibodies against trophectoderm-specific markers during mouse blastocyst formation.
Authors: Brûlet P , Babinet C , Kemler R , Jacob F
Published: May 2006

Two-dimensional gel electrophoresis has allowed the detection of proteins characteristic of inner cell mass and trophectoderm in mouse blastocyst. Certain of the proteins characterizing trophectoderm copurify with intermediate filaments from trophectoderm and a trophoblastoma cell line. A monoclonal antibody prepared against proteins of these intermediate filaments labels a filament network in trophectoderm but not in inner cell mass cells.
Scale-invariance in reaction-diffusion models of spatial pattern formation.
Authors: Othmer H G , Pate E
Published: May 2006

We propose a reaction-diffusion model of spatial pattern formation whose solutions can exhibit scale-invariance over any desired range for suitable choices of parameters in the model. The model does not invoke preset polarity or any other ad hoc distinction between cells and provides a solution to the French flag problem without sources at the boundary. Furthermore, patterns other than the polar pattern that usually arises first in a growing one-dimensional system described by Turing's model can be obtained. Evidence is given that suggests that the model may apply in the slug stage of Dictyostelium discoideum.
Ontogenetic patterns of thyrotropin-releasing hormone-like material in rat hypothalamus, pancreas, and retina: selective effect of light deprivation.
Authors: Martino E , Seo H , Lernmark A , Refetoff S
Published: May 2006

Recent observations have shown the presence of thyrotropin-releasing hormone-like material (TRH-LM) in rat pancreatic islets and in retina. Its immunological and biological properties are identical to those of synthetic thyrotropin-releasing hormone (thyroliberin). This communication deals with the ontogenesis of TRH-LM in rat pancreas and retina as compared to that of rat hypothalamus. Effects of sex and exposure to constant dark were also studied. Results show that asynchronous changes in the concentration of TRH-LM occur during the postnatal maturation of these tissues, presumably mediated by organ-specific control mechanisms--e.g., light affects only the accumulation of TRH-LM in the retina. TRH-LM may act as neurotransmitter in the regulation of pancreatic islet cell function and in the development of photo-reception in the retina. Increases in hypothalamic TRH-LM seem to parallel the development of the pituitary-thyroid secretory activity, but the function of extrahypothalamic TRH-LM remains speculative.
Antibody-directed cytotoxic agents: use of monoclonal antibody to direct the action of toxin A chains to colorectal carcinoma cells.
Authors: Gilliland D G , Steplewski Z , Collier R J , Mitchell K F , Chang T H , Koprowski H
Published: May 2006

We have constructed cell-specific cytotoxic agens by covalently coupling the A chain from diphtheria toxin or ricin toxin to monoclonal antibody directed against a colorectal carcinoma tumor-associated antigen. Antibody 1083-17-1A was modified by attachment of 3-(2-pyridyldithio)propionyl or cystaminyl groups and then treated with reduced A chain to give disulfide-linked conjugates that retained the original binding specificity of the antibody moiety. the conjugates showed cytotoxic activity for colorectal carcinoma cells in culture, but were not toxic in the same concentration range for a variety of cell lines that lacked the antigen. Under defined conditions virtually 100% of antigen-bearing cultured cells were killed, whereas cells that lacked the antigen were not affected. Conjugates containing toxin A chains coupled to monoclonal antibodies may be useful in studying functions of various cell surface components and, possibly, as tumor-specific therapeutic agents.
Partial purification and characterization of a macromolecule which enhances fluoride activation of adenylate cyclase.
Authors: Rasenick M M , Bitensky M W
Published: May 2006

Fluoride activation of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] is significantly enhanced (2 to 5 times) by a protein factor isolated from rat brain. The fluoride-dependent adenylate cyclase stimulator (FCS) is nondialyzable, trypsin-labile, and stable at 90 degrees C for 10 min. FCS stimulates adenylate cyclase activity only in the presence of NaF (2-25 mM) and this effect is independent of added GTP, 5'-guanylylimidodiphosphate, or calcium. FCS has been purified roughly 3000-fold from a 12,000 X g supernatant fraction of rat brain homogenate. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis and sucrose density gradient sedimentation suggest that FCS is a monomer with an apparent Mr of 59,000. Isoelectric focusing indicates FCS has a pI of 8.9. FCS from rat brain stimulates fluoride-activated adenylate cyclase from a variety of cell types, and FCS can also be isolated from rat liver. The effects of FCS are not reversed by washing membranes when the membranes and FCS are preincubated with NaF. The Km of adenylate cyclase for ATP and the fluoride concentration causing half-maximal activation are unchanged by FCS; however, FCS increases the Vmax by 2.5-fold. FCS may act to increase the catalytic efficiency of fluoride-activated complexes of the GTP-binding unit with adenylate cyclase or to enhance the formation of additional active complexes.
Chemical probes for higher-order structure in RNA.
Authors: Peattie D A , Gilbert W
Published: May 2006

Three chemical reactions can probe the secondary and tertiary interactions of RNA molecules in solution. Dimethyl sulfate monitors the N-7 of guanosines and senses tertiary interactions there, diethyl pyrocarbonate detects stacking of adenosines, and an alternate dimethyl sulfate reaction examines the N-3 of cytidines and thus probes base pairing. The reactions work between 0 degrees C and 90 degrees C and at pH 4.5--8.5 in a variety of buffers. As an example we follow the progressive denaturation of yeast tRNAPhe terminally labeled with 32P as the tertiary and secondary structures sequentially melt out. A single autoradiograph of a terminally labeled molecule locates regions of higher-order structure and identifies the bases involved.
Microtubule-associated proteins: a monoclonal antibody to MAP2 binds to differentiated neurons.
Authors: Izant J G , McIntosh J R
Published: May 2006

Hybridomas that secret IgG reacting specifically with the brain microtubule-associated protein MAP2 have been prepared with speen cells from BALB/c mice hyperimmunized with high molecular weight neurotubule-associated proteins. Immunofluorecence microscopy using dual fluorochrome labeling of tubulin and MAP2 antigens revealed identical patterns of interphase fiber networks in cells from explants of newborn mouse brain. The anti-MAP2 antibody did not stain primary mouse kidney cells or CHO, 3T3, HeLa, or PtK1 cell lines. Immunoprecipitation and antibody gel staining techniques failed to demonstrate any crossreacting antigen in these cells. MAP2 antigen was not seen in association with the mitotic spindle in any of the cells examined. Radioimmunoassay showed species crossreactivity of the anti-MAP2 antibody with mammalian but not avian neural cell extracts. Glial cells and some neuroblastoma cell lines did not appear to contain MAP2. However, in the B104 rat neuroblastoma cell line the MAP2 antigen appeared to be associated with the cytoskeleton concomitant with differentiation induced by dibutyryl cyclic AMP. In disagreement with most previously published reports, our data suggest that MAP2 is found only in differentiated neuronal cells and raises the possibility that MAP2 is involved in neuronal differentiation or neuron-specific processes.
Sertoli cells contain a mitogenic polypeptide.
Authors: Feig L A , Bellvé A R , Erickson N H , Klagsbrun M
Published: May 2006

The seminiferous epithelium of the mouse testis contains a potent mitogen that induces DNA synthesis and cell division in cutures of confluent, quiescent BALB/c 3T3 cells. Homogenates of adult mouse seminiferous tubules, when added to 3T3 cells for 48 hr, produce a dose-dependent increase in DNA synthesis with half-maximal activity at 400 micrograms of protein per ml. Maximal stimulation causes a 70- to 100-fold increase in [methyl-3H]thymidine incorporation into DNA, a labeling of > 99% of 3T3 cell nuclei, and a doubling in cell number. The level of mitogenic activity in seminiferous tubules isolated from newborn mice is five times greater than that in tubules of adult mice. Furthermore, prepuberal Sertoli cells contain substantially greater levels of mitogenic activity than any other cell type purified from the seminiferous epithelium. A homogenate of prepuberal Sertoli cells induces half-maximal DNA synthesis at 20 micrograms of protein per ml. The mitogenic factor is sensitive to proteases and to heating at 100 degrees C for 2 min but not to dithiothreitol. Gel-filtration chromatography reveals that the factor is a polypeptide with a Mr of approximately 15,500. These observations provide evidence for the presence of a growth factor in the mammalian testis.
A cell surface molecule involved in aggregation of embryonic liver cells.
Authors: Bertolotti R , Rutishauser U , Edelman G M
Published: May 2006

Aggragation of chicken enbryo hepatocytes can be inhibited by Fab' fragments of antibodies prepared against the cells. An aqueous extract of liver cell membranes contained antigens that neutralized the adhesion-blocking properties of the Fab' fragments. This neutralization activity was associated with a polypeptide of Mr68,000 in NaDodSO4; the polypeptide was distinct from serum albumin. Specific antibodies prepared against the 80-fold purified active fraction inhibited liver cell adhesion and immunoprecipitated the 68,000 Mr polypeptide from active fractions as well as from a detergent extract of liver cell membranes. In hepatocyte cultures, Fab' fragments of antibodies against the liver molecule prevented both colony formation and appearance of histotypic patterns. Liver cell adhesion was compared at the cellular and molecular levels to that of embryonic neural retina cells. Antibodies against the cell adhesion molecule from neural tissue inhibited retinal but not liver cell aggregation; conversely, antibodies against the liver polypeptide inhibited liver but not retinal cell aggregation. By means of antibody absorption and immunoprecipitation, it was confirmed that the two cell adhesion molecules are antigenically unrelated.
Indiscriminate recombination in simian virus 40-infected monkey cells.
Authors: Winocour E , Keshet I
Published: May 2006

DNA transfection of African green monkey BSC-1 cells with simian virus 40 (SV40) DNA and bacterial virus phi X174 replicative form DNA ("cotransfection") yielded stocks containing SV40/phi X174 recombinant virus, which was detected by an infectious-center in situ plaque hybridization procedure and which was sensitive to anti-SV40 antiserum. The recombinant virus replicated during serial passage. Restriction endonuclease cleavage of the SV40/phi X174 DNA indicated that several different types of recombinant DNA structures had arisen. Similar SV40 DNA cotransfection experiments with polyoma virus DNA, bacterial plasmid (pBR322) DNA, and a plasmid-cloned segment of the mouse genome (coding for intracisternal type A particles) yielded stocks that generated recombinant plaques as judged by in situ plaque hybridization with the appropriate labeled probe. It appears, therefore, that an active indiscriminate recombination process, incapable of distinguishing between diverse DNAs of prokaryotic and eukaryotic origin, occurs in SV40-infected monkey cells.
Somatic rearrangements forming active immunoglobulin mu genes in B and T lymphoid cell lines.
Authors: Cory S , Adams J M , Kemp D J
Published: May 2006

We have cloned an active gene for an immunoglobulin mu heavy (H) chain, bearing the variable (VH), joining (JH), and constant (C mu) sequences expressed in the IgM-secreting mouse plasmacytoma HPC-76. The mu gene was formed by somatic recombination between a VH gene and one of several JH genes, which are located about 7.7 kilobase pairs from the C mu gene in embryo DNA. The JH-C mu intervening sequence has suffered a deletion of about 2.7 kilobase pairs in HPC-76. Because the delection encompasses sequences required to switch an expressed VH-JH gene from C mu to another CH gene, it may represent a mechanism for "freezing" a lymphocyte clone at the stage of IgM expression. For the second (inactive) C mu allele in HPC-76, the entire joining and switch regions have been deleted; functional inactivation of one allele may thus represent one mechanism by which a lymphocyte clone restricts expression to a single allele (allelic exclusion). Probes generated from the cloned mu gene allowed examination of the JH locus in B, Abelson "pre-B," and T lymphoma cell lines and a myeloid line, all of which cotain RNA species bearing C mu sequences. The B and pre-B lines exhibited recombination within both alleles of the JH locus, suggesting that both alleles may be expressed in some cells. The absence of the JH gene 5' to the recombination sites favors a deletion mechanism for VH-JH joining. Recombination within the JH locus was also detected in two out of four T lymphoma lines, but not in the myeloid line. This indicates that the mechanism by which B cells generate immunoglobulin diversity is operational in some T cells. Lines that synthesize mu RNA without JH rearrangement may have activated the C mu gene directly or have undergone recombination at a more distant locus.
Further evidence for peptidergic transmission in sympathetic ganglia.
Authors: Jan Y N , Jan L Y , Kuffler S W
Published: May 2006

We previously proposed that, in sympathetic ganglia of the bullfrog, a peptide which resembles luteinizing hormone-releasing factor (LH-RF, luliberin) functions as the transmitter for the late slow excitatory postsynaptic potential (epsp), a signal that may last 5-10 min. To test this hypothesis further, we have compared the physiological andpharmacological effects of LH-RF with those of the natural transmitter and have found a close parallel. (i) LH-RF, when ejected with a brief pulse of pressure through a micropipette near a ganglion cell, produces a depolarizing response lasting for minutes. (ii) The LH-RF-induced response is associated with changes in input resistance similar to thoe during a late slow epsp. (iii) The amplitudes of the LH-RF-induced response and the late slow epsp vary in parallel as the membrane potential is shifted over a wide range. (iv) Both responses increase the excitability of ganglion cells. (v) The two responses interact with the cholinergic epsps in a parallel manner: they cause diminution of the slow epsp but not of the fast epsp. (vi) Both responses are blocked by an analog of LH-RF that antagonizes the effects of LH-RF in the rat.
13
C and
199
Hg nuclear magnetic resonance spectroscopic study of alkenemercurinium ions: Effect of methyl substituents on
199
Hg chemical shifts
Authors: Olah George A. , Garcia-Luna Armando
Published: May 2006

The long-lived ethylene, cyclohexene, and norbornenemercurinium ions prepared in superacidic, low-nucleophilic media have been studied by
13
C and
199
Hg NMR spectroscopy. The norbornenemercurinium ion shows temperature-dependent
13
C and
199
Hg NMR spectra, consistent with equilibration via rapid hydride and Wagner-Meerwin shifts. The
199
Hg NMR shifts of a series of alkylmercury bromides were also obtained in order to elucidate the effect of methyl substituents on
199
Hg NMR chemical shifts.
Nucleosomal packaging of the thymidine kinase gene of herpes simplex virus transferred into mouse cells: an actively expressed single-copy gene.
Authors: Camerini-Otero R D , Zasloff M A
Published: May 2006

We have studied the nucleoprotein structure of the herpes thymidine kinase gene introduced into mouse Ltk-aprt- cells by means of DNA-mediated gene transfer. Using the technique of Southern blotting, we examined staphylococcal digests of the nuclei from the relatively stable transformants that contain one or less integrated copies of the thymidine kinase gene per haploid genome. Out experiments show that, under selection for the active expression of this gene, it is packaged in nucleosomes with a repeat length identical to the average for the host mouse sequences.
Crystallization of the middle part of the mitochondrial electron transfer chain: cytochrome bc1-cytochrome c complex.
Authors: Ozawa T , Tanaka M , Shimomura Y
Published: May 2006

Complex III (cytochrome bc1 particle; ubiquinol:ferricytochrome c oxidoreductase, EC 1.10.22) was purified from beef heart mitochondria by affinity chromatography. Phospholipids were depleted by washing the particle with detergent while it still was on the affinity column. The particle first was mixed with an excess of cytochrome c in 1.5% cholate (wt/vol); slow removal of the detergent from the mixture was achieved by dialysis. Freezing of the mixture resulted in crystallization of the cytochrome bc1 particle in the form of a 1:2 complex with cytochrome c. The chemical composition and spectrophotometric properties of the crystal are described. The same crystallization maneuver used in the case of cytochrome oxidase has been demonstrated to be effective in crystallizing the middle part of the mitochondrial electron-transfer chain.
Monoclonal antibodies to human estrogen receptor.
Authors: Greene G L , Nolan C , Engler J P , Jensen E V
Published: May 2006

Extranuclear estrogen receptor protein (estrophilin) of MCF-7 human breast cancer cells was purified by passage of the cytosol fraction of a cell homogenate through an affinity column of estradiol linked to Sepharose by a substituted di-n-propyl sulfide bridge in the 17 alpha position. Elution with 50 micro M [3H]estradiol in 10% (vol/vol) dimethyl formamide/0.5 M sodium thiocyanate gave 40% recovery of [3H]estradiol-estrophilin showing 14% of the specific radioactivity expected for the pure complex. Serum from a Lewis rat immunized with this partially purified estradiol-receptor complex contained antiestrophilin antibodies that reacted not only with nuclear and extranuclear estradiol-receptor complexes from MCF-7 cells but also with estrophilin from rat, calf, and monkey uterus, hen oviduct, and human breast cancers. Splenic lymphocytes from the immunized rat were fused with cells of two different mouse myeloma lines (P3-X63-Ag8 and Sp2/0-Ag14) to yield hybridoma cultures, 2% of which produced antibodies to estrophilin. After cloning by limiting dilution, three hybridoma lines secreting antiestrophilin were expanded in suspension culture and as ascites tumors in athymic mice to provide substantial quantities of monoclonal antibodies that recognize mammalian but not avian estrophilin and that show different degrees of reactivity with receptor from nonprimate sources. By growing the clone from Sp2/0 in the presence of [35S]methionine, radiolabeled monoclonal IgG has been prepared. These monoclonal antibodies should prove useful in the study of estrogen receptors of human reproductive tissues, in particular for the radioimmunochemical assay and immunocytochemical localization of receptors in breast cancers.
Pyrroline-5-carboxylate synthase activity in mammalian cells.
Authors: Smith R J , Downing S J , Phang J M , Lodato R F , Aoki T T
Published: May 2006

Although glutamic acid is known to be a precursor for proline biosynthesis, the enzymatic conversion of glutamic acid to pyrroline-5-carboxylic acid, the immediate precursor of proline, has not been demonstrated in cell-free systems. By providing appropriate concentrations of ATP and NADPH and blocking further metabolism of pyrroline-5-carboxylic acid, we have developed a method for measuring the formation of pyrroline-5-carboxylic acid from glutamic acid in homogenates of mammalian cells. We have designated this activity pyrroline-5-carboxylate synthase. To confirm that our assay is a valid measure of the initial step in proline biosynthesis from glutamic acid, we have compared two mutant lines of Chinese hamster ovary cell. Proline prototrophic cells, which can synthesize proline from glutamic acid, have easily measurable pyrroline-5-carboxylate synthase activity (5.97 nmol of pyrroline-5-carboxylic acid per hr per mg of homogenate protein). In contrast, proline auxotrophic cells, which are unable to synthesize proline from glutamic acid, have no detectable pyrroline-5-carboxylate synthase activity.
Effect of diethyldithiocarbamate rescue on tumor response to cis-platinum in a rat model.
Authors: Borch R F , Katz J C , Lieder P H , Pleasants M E
Published: May 2006

The nephrotoxic effects of cis-dichlorodiamineplatinum(II) (NSC-119875) (DDP) in female F344 rats were effectively inhibited by administration of sodium diethyldithiocarbamate (DDTC) in doses of 750 mg/kg intraperitoneally or 100 mg/kg intravenously 2 hr after administration of DDP. Rats were inoculated with mammary tumor 13762 and treated after 10 days with DDP (2.0 or 8.0 mg/kg) with or without DDTC rescue (750 mg/kg intraperitoneally or 100 mg/kg intravenously). Initial reductions in tumor size were identical with or without rescue in all experiments. High-dose intraperitoneal rescue, however, resulted in earlier relapse and more rapid progressions at both DDP doses than was observed in the absence of rescue. Low-dose intravenous rescue led to a tumor response identical to that observed without rescue. Urinary excretion of free DDTC was increased by prior administration of acetazolamide; however, this combination was more toxic to rats after DDP administration than was DDTC alone. Intravenous administration of DDTC appeared to be the most effective route for delivery of this ligand to the kidney. These results support our earlier mechanistic hypothesis and demonstrate the feasibility of inhibition of cis-platinum toxicity by DDTC without inhibition of the antitumor effect.
Protection of cultured renal tubular epithelial cells from anoxic cell swelling and cell death.
Authors: Kreisberg J I , Mills J W , Jarrell J A , Rabito C A , Leaf A
Published: May 2006

In order to study the relationship between cell swelling and cell death due to ischemia, we have developed an in vitro model by using primary cultures of renal tubular epithelial cells. With this model, we have studied two components of ischemia--namely, anoxia along with substrate deprivation. After 2 hr of anoxia in the absence of substrate, the cultured cells swelled and blebbed. Cells similarly treated in the presence of 8% polyethylene glycol, an oncotic agent, did not swell and bleb, and when cells were counted 18 hr later, similar numbers of cells were seen as in the untreated cultures. However, tubule cells exposed to anoxia without 8% polyethylene glycol had 50% fewer cells 18 hr later. Therefore, if cell swelling is prevented during 2 hr of anoxia, cell viability is improved.
Beta limits for torsatrons
Authors: Bauer F. , Betancourt O. , Garabedian P. , Shohet J. L.
Published: May 2006

An ideal magnetohydrodynamic equilibrium and stability code is used to study ballooning modes in torsatrons. The most dangerous modes turn out to be those with low poloidal and toroidal wave numbers. Beta limits for equilibrium and stability are determined for an [unk] = 2 ultimate torsatron with large [unk] = 1 and [unk] = 3 sidebands.
Sites of termination of in vitro DNA synthesis on ultraviolet- and N-acetylaminofluorene-treated phi X174 templates by prokaryotic and eukaryotic DNA polymerases.
Authors: Moore P D , Bose K K , Rabkin S D , Strauss B S
Published: May 2006

In vitro DNA synthesis on a phi X174 template primed with a restriction fragment and catalyzed by the Escherichia coli DNA polymerase I large (Klenow) fragment (pol I) terminates at the nucleotide preceding a site that has been altered by ultraviolet irradiation or treatment with N-acetylaminofluorene. Termination on ultraviolet-irradiated templates is similar when synthesis is catalyzed by E. coli DNA polymerase III holoenzyme (pol III), phage T4 DNA polymerase, a polymerase alpha from human lymphoma cells, or avian myeloblastosis virus reverse transcriptase. 3' leads to 5' exonuclease activity cannot be detected in the reverse transcriptase and DNA polymerase alpha preparations. On N-acetylaminofluorene templates, pol I, pol III, and T4 polymerase reactions terminate immediately preceding the lesion, whereas reverse transcriptase-catalyzed reactions and, at some positions in the sequence, polymerase alpha-catalyzed reactions terminate at the site of the lesion. Substitution of Mn2+ for Mg2+ changes the pattern of pol I-catalyzed termination sites. The data suggest that termination is a complicated process that does not depend exclusively on the 3' leads to 5' exonuclease activity associated with many polymerases.
Apolipoprotein B is structurally and metabolically heterogeneous in the rat.
Authors: Elovson J , Huang Y O , Baker N , Kannan R
Published: May 2006

Electrophoresis of rat apolipoprotein B (apoB) on 5% polyacrylamide gels in the presence of NaDodSO4 separates three major components: PI, which comigrates with human low density lipoprotein (LDL) apoB; PII, a slightly faster-moving satellite band; and PIII, which migrates somewhat more slowly than myosin heavy chain. The proportion of PIII decreases with increasing density of the parent rat lipoprotein, from 90% an 70%, respectively, in chylomicrons and very low density lipoproteins (VLDL), to 7% in the major LDL2 (density 1.038-1.063 g/ml) fraction. A major component that comigrates with rat PIII is a marker for human chylomicron apoB, being absent from human VLDL, intermediate density lipoprotein (IDL), and LDL. Preliminary immunological and peptide mapping data show that rat apoB PI and PIII are closely related structurally, with the latter possibly being a large fragment of the former. Both peptides are synthesized in rat liver and found in Golgi secretory vesicles. Kinetic tracer experiments show that rat PI and PIII are present on separate VLDL particles, both of which are extensively removed from the circulation at the remnant stage, and that the declining PIII-to-PI/II ratios in IDL and LDL may be attributed to the more rapid turnover of PIII-containing lipoproteins at all levels, particularly within the LDL density range.
Diphtheria toxin forms transmembrane channels in planar lipid bilayers.
Authors: Donovan J J , Simon M I , Draper R K , Montal M
Published: May 2006

When exposed to a lipid bilayer, diphtheria toxin binds to it and forms transmembrane, voltage-dependent, anion-selective channels. The mean (+/- SD) conductance of these channels in asolectin membranes is 6.2 +/- 0.7 pmho (pS) in 0.2 M NaCl and 20 +/- 2 pmho in 1.0 M NaCl. The rate of channel formation depends on the pH in the toxin-containing compartment; it is very low at pH greater than 5.0 and increases abruptly as the pH decreases from 4.9 to 4.0. Binding of toxin to the membrane is also pH dependent, being unmeasurable at pH 7 and increasing monotonically with decreasing pH. The rate of channel formation depends upon membrane potential as well; channels form only at negative potentials. These channels are permanent in the time scale of the experiments (about 1 hr). The membrane conductance caused by the channels is also voltage dependent, being constant at positive potentials and decreasing at negative potentials. Hence, the current-voltage curve is linear at positive potentials and sublinear at negative potentials. The conditions necessary for insertion of toxin into the bilayer and formation of channels are similar to those that prevail inside the lysosome. Thus, these results lend credence to the idea that toxin enters the cytoplasm from the lysosomal compartment.
Secretory granules of an anterior pituitary cell line, AtT-20, contain only mature forms of corticotropin and beta-lipotropin.
Authors: Gumbiner B , Kelly R B
Published: May 2006

The pituitary cell line, AtT-20, synthesizes the precursor to corticotropin (adrenocorticotropic hormone; ACTH) and beta-endorphin and correctly glycosylates and cleaves it to make the mature forms of the hormones before they are secreted. This cell line was used to study the intracellular transport, packaging, and secretion of these hormones. Secretory granules from the cells were isolated by homogenization and differential centrifugation and isopycnic sedimentation on a 2H2O-Ficoll gradient to give a preparation having a specific activity of 90micrograms ACTH per mg of protein, which is 30- to 90-fold greater than that of whole cells. The granules have density characteristics and a sedimentation coefficient that are appropriate for spheres of 1000 A radius. They contain all of the fragments of the initial ACTH/endorphin precursor but almost undetectable amounts of the intact precursor. The fragments constitute about 50% of the protein in the secretory granule fraction and, from density measurements, we estimate that they are present in approximately 60,000 copies per vesicle. The cell line secretory granules appear, therefore, to be similar to mature secretory granules in normal differentiated tissues. ACTH first appears in the secretory granule at 30-45 min after synthesis. Cleavage of the precursor to mature ACTH occurs at about the same time in the whole cell. Therefore, proteolysis of the prohormone to ACTH and to beta-lipotropin is a metabolic event that can be correlated with the packaging of the hormone into a mature secretory granule. Cleavage of beta-lipotropin to beta-endorphin occurs later, probably in the secretory granule.
Sequence arrangement of a highly methylated satellite DNA of a plant,
Scilla
: A tandemly repeated inverted repeat
Authors: Deumling Barbara
Published: May 2006

G+C-rich satellite DNA, representing about 19% of total nuclear DNA, was isolated from various tissues of the monocotyledonous plant,
Scilla siberica
, by using Ag
+
-Cs
2
SO
4
gradient techniques. This satellite DNA had an unusually high melting point and a high methylcytosine (m
5
C) content (≈25% of total bases; m
5
C/cytosine ratio ≈1.5) and was localized, by
in situ
hybridization, in the heterochromatin regions of the chromosomes. Digestion with restriction endonuclease Hae III yielded a series of fragments ranging from 35 to several hundred nucleotide pairs. The major fragments, I-IV (35, 50, 59, and 69, nucleotide pairs, respectively), were isolated, and their nucleotide sequences were determined. The dominant fragment I was a highly symmetrical molecule, with a basically palindromic arrangement. This sequence represented the basic unit of
Scilla
satellite DNA and was tandemly repeated many times, with some base substitutions and multiple successive insertions of the tetranucleotide G-T-C-C. The dinucleotide CpG was the commonest nearest-neighbor sequence. Thin layer chromatography, DNA sequence analysis, and gas chromatography combined with mass spectrometry showed the high m
5
C content (m
5
C/Cyt = 2.2 and 2.8, respectively, for fragments II and III). Identical cleavage fragments were found in satellite DNAs from two other species of this genus (
S. amoena
and
S. ingridae
), which suggests that this constitutively methylated sequence is evolutionarily stable. The sequence arrangement of this plant satellite DNA is compared with those reported for several animal satellite DNAs.
Distinct calcium-independent and calcium-dependent adhesion systems of chicken embryo cells.
Authors: Brackenbury R , Rutishauser U , Edelman G M
Published: May 2006

Three criteria have been used to distinguish among different systems of embryonic cell adhesion: dependence on Ca2+, involvement of particular cell-surface molecules, and binding specificity. The characterization of the adhesion with respect to cell-surface molecules was carried out by using specific antibodies against the neural and liver cell adhesion molecules (N-CAM and L-CAM) and antibodies raised against retinal cells prepared by limited trypsinization in the presence of Ca2+ (called "T/Ca cells"). Aggregation of cells prepared from retina or brain without Ca2+ did not require Ca2+ and was inhibited by anti-(N-CAM) antibodies but not by anti-(L-CAM) or anti-T/Ca cell antibodies. In contrast, cells obtained from the same tissues in the presence of Ca2+ did require Ca2+ to aggregate. This aggregation was inhibited by anti-T/Ca cell antibodies but not by anti-(N-CAM) or anti-(L-CAM) antibodies. Hepatocyte aggregation also required Ca2+ and was inhibited only by anti-(L-CAM) antibodies. These results define three antigenically distinct cell adhesion systems in the embryo and raise the possibility that additional systems will be found. The neural Ca2+-independent system displayed a limited tissue specificity, mediating binding to neural but not liver cells. In contrast, the Ca2+-dependent systems of both neural and liver cells caused binding to all cell types tested. The Ca2+-dependent system was most active in retinal cells from 6-7 day embryos, whereas the Ca2+-independent system was most active at later times during development. In addition, treatments that inhibited the Ca2+-independent or Ca2+-dependent systems had very different effects on the fasciculation of neurites from dorsal root ganglia. All of the results suggest that Ca2+-independent and Ca2+-dependent adhesion systems play different functional roles during embryogenesis.
Rescue of noncultivatable human rotavirus by gene reassortment during mixed infection with ts mutants of a cultivatable bovine rotavirus.
Authors: Greenberg H B , Kalica A R , Wyatt R G , Jones R W , Kapikian A Z , Chanock R M
Published: May 2006

Fastidious human rotaviruses that did not undergo productive infection in tissue culture were rescued by genetic reassortment during mixed infection with a temperature-sensitive (ts) mutant of a cultivatable bovine rotavirus. In this manner, the genes of the fastidious rotavirus that restricted growth in vitro were replaced by the corresponding genes from a tissue culture-adapted rotavirus. We recovered genetically reassorted viruses that grew to high titer and were neutralized specifically by hyperimmune guinea pig type 1 or type 2 human rotavirus antiserum. Preliminary RNA analysis of these clones disclosed that they were indeed viruses with reassorted genes.