bluuebunny/crossref_metadata_2025 huggingface dataset:
Safe and efficient generation of recombinant retroviruses with amphotropic and ecotropic host ranges.
Authors: Danos O , Mulligan R C
Published: May 2006

We have constructed a set of packaging cell lines useful for the generation of helper-free recombinant retroviruses with amphotropic and ecotropic host ranges. To eliminate the problems of transfer of packaging functions and helper virus formation encountered with the previously available packaging systems, two mutant Moloney murine leukemia virus-derived proviral genomes carrying complementary mutations in the gag-pol or env regions were sequentially introduced into NIH 3T3 cells by cotransformation. Both genomes contained a deletion of the psi sequence necessary for the efficient encapsidation of retroviral genomes into virus particles and additional alterations at the 3' end of the provirus. We show that the resulting packaging cell lines psi CRIP and psi CRE can be used to isolate clones that stably produce high titers (10(6) colony-forming units/ml) of recombinant retroviruses with amphotropic and ecotropic host ranges, respectively. More importantly, we demonstrate that viral producers derived from the packaging cell lines do not transfer the packaging functions, or yield helper virus, even under conditions where existing packaging cell lines can be shown to yield transfer of packaging functions and/or helper virus. These properties of the psi CRIP and psi CRE packaging lines make them particularly valuable reagents for in vivo gene transfer studies aimed at cell lineage analysis and the development of human gene replacement therapies.
Chromosomal localization of the human diazepam binding inhibitor gene.
Authors: DeBernardi M A , Crowe R R , Mocchetti I , Shows T B , Eddy R L , Costa E
Published: May 2006

We have used in situ chromosome hybridization and human-mouse somatic cell hybrids to map the gene(s) for human diazepam binding inhibitor (DBI), an endogenous putative modulator of the gamma-aminobutyric acid receptor acting at the allosteric regulatory center of this receptor that includes the benzodiazepine recognition site. In 784 chromosome spreads hybridized with human DBI cDNA, the distribution of 1476 labeled sites revealed a significant clustering of autoradiographic grains (11.3% of total label) on the long arm of chromosome 2 (2q). Furthermore, 63.5% of the grains found on 2q were located on 2q12-21, suggesting regional mapping of DBI gene(s) to this segment. Secondary hybridization signals were frequently observed on other chromosomes and they were statistically significant mainly for chromosomes 5, 6, 11, and 14. In addition, DNA from 32 human-mouse cell hybrids was digested with BamHI and probed with human DBI cDNA. A 3.5-kilobase band, which probably represents the human DBI gene, was assigned to chromosome 2. Four higher molecular weight bands, also detected in BamHI digests, could not be unequivocally assigned. A chromosome 2 location was excluded for the 27-, 13-, and 10-kilobase bands. These results assign a human DBI gene to chromosome 2 (2q12-21) and indicate that three of the four homologous sequences detected by the human DBI probe are located on three other chromosomes.
Properties of a mutant recA-encoded protein reveal a possible role for Escherichia coli recF-encoded protein in genetic recombination.
Authors: Madiraju M V , Templin A , Clark A J
Published: May 2006

A mutation partially suppressing the UV sensitivity caused by recF143 in a uvrA6 background was located at codon 37 of recA where GTG (valine) became ATG (methionine). This mutation, originally named srf-803, was renamed recA803. Little if any suppression of the recF143 defect in UV induction of a lexA regulon promoter was detected. This led to the hypothesis that a defect in recombination repair of UV damage was suppressed by recA803. The mutant RecA protein (RecA803) was purified and compared with wild-type protein (RecA+) as a catalyst of formation of joint molecules. Under suboptimal conditions, RecA803 produces both a higher rate of formation and a higher yield of joint molecules. The suboptimal conditions tested included addition of single-stranded DNA binding protein to single-stranded DNA prior to addition of RecA. We hypothesize that the ability of RecA803 to overcome interference by single-stranded DNA binding protein is the property that allows recA803 to suppress partially the deficiency in repair caused by recF mutations in the uvrA6 background. Implications of this hypothesis for the function of RecF protein in recombination are discussed.
Relaxation of muscle fibers with adenosine 5'-[gamma-thio]triphosphate (ATP[gamma S]) and by laser photolysis of caged ATP[gamma S]: evidence for Ca2+-dependent affinity of rapidly detaching zero-force cross-bridges.
Authors: Dantzig J A , Walker J W , Trentham D R , Goldman Y E
Published: May 2006

The relationship between the mechanical and biochemical states of the muscle cross-bridge cycle and the control of contraction were investigated by using the nucleotide analogs adenosine 5'-[gamma-thio]triphosphate (ATP[gamma S]) and caged ATP[gamma S] [the O-1(2-nitrophenyl)ethyl P3-ester of ATP[gamma S]]. ATP[gamma S] interacts with actomyosin in a manner similar to ATP but is hydrolyzed (by a factor of 500) more slowly. Generation of ATP[gamma S] by photolysis of caged ATP[gamma S] within a permeabilized fiber in rigor in the absence of Ca2+ relaxed tension and stiffness as occurs with ATP. The transient rise in tension prior to final relaxation observed with photolysis of caged ATP was absent with caged ATP[gamma S]. This result suggests that following detachment of a cross-bridge, ATP is normally hydrolyzed before force generation. In the presence of Ca2+, photolysis of caged ATP[gamma S] within rigor fibers caused tension to relax fully but significant stiffness remained. Stiffness also developed without concomitant tension when Ca2+ concentration was raised from less than 1 nM to 30 microM in the presence of ATP[gamma S]. The amplitude of the tension response to ramp stretches in the presence of Ca2+ and ATP[gamma S] increased with ramp stretch velocity, suggesting that the cross-bridges have detachment rate constants extending into the 10(3) s-1 range. The results provide evidence that the Ca2+-regulatory system can directly control attachment of cross-bridges into states before the power stroke.
Restriction fragment length polymorphism linkage map for Arabidopsis thaliana.
Authors: Chang C , Bowman J L , DeJohn A W , Lander E S , Meyerowitz E M
Published: May 2006

We have constructed a restriction fragment length polymorphism linkage map for the nuclear genome of the flowering plant Arabidopsis thaliana. The map, containing 90 randomly distributed molecular markers, is physically very dense; greater than 50% of the genome is within 1.9 centimorgans, or approximately 270 kilobase pairs, of the mapped DNA fragments. The map was based on the meiotic segregation of markers in two different crosses. The restriction fragment length polymorphism linkage groups were integrated with the five classically mapped linkage groups by virtue of mapped mutations included in these crosses. Markers consist of both cloned Arabidopsis genes and random low-copy-number genomic DNA clones that are able to detect polymorphisms with the restriction enzymes EcoRI, Bgl II, and/or Xba I. These cloned markers can serve as starting points for chromosome walking, allowing for the isolation of Arabidopsis genes of known map location. The restriction fragment length polymorphism map also can associate clones of unknown gene function with mutant phenotypes, and vice versa.
IgE production by normal human lymphocytes is induced by interleukin 4 and suppressed by interferons gamma and alpha and prostaglandin E2.
Authors: Pène J , Rousset F , Brière F , Chrétien I , Bonnefoy J Y , Spits H , Yokota T , Arai N , Arai K , Banchereau J
Published: May 2006

The effect of human recombinant interleukin 4 (IL-4) on antibody production by normal peripheral blood mononuclear cells enriched for B cells was investigated. IL-4 preferentially induced IgE synthesis in vitro. In addition, a low induction of IgG production was observed, whereas IL-4 had no effect on IgA and IgM synthesis. The IL-4-induced IgE production by B cells required T cells and monocytes but was specifically inhibited by an anti-IL-4 antiserum indicating that, although IL-4 acts indirectly, it is responsible for the induction of IgE synthesis. IL-4-induced IgE production was blocked in a dose-dependent way by interferon gamma (IFN-gamma), interferon alpha (IFN-alpha), and prostaglandin E2. IFN-gamma also inhibited IL-4-induced IgG production. These inhibitory effects of IFN-gamma and IFN-alpha on IgE production cannot be attributed to toxic effects since IFN-alpha induced IgM production in the presence of IL-4, whereas IFN-gamma was ineffective in inhibiting IgG production induced by IL-2. IFN-gamma, IFN-alpha, and prostaglandin E2 also inhibited IL-4-induced expression of the low-affinity receptor for the Fc portion of IgE (CD23) on B cells, indicating that there is an association between CD23 expression and IL-4-induced IgE production. This theory was supported by the finding that IL-4-induced IgE production was inhibited by F(ab')2 fragments of an anti-CD23 monoclonal antibody.
Identification and arrangement of the DNA sequence recognized in specific transformation of Neisseria gonorrhoeae.
Authors: Goodman S D , Scocca J J
Published: May 2006

DNA segments from Neisseria gonorrhoeae, cloned and propagated in Escherichia coli, were tested for the ability to competitively inhibit gonococcal transformation. The nucleotide sequences of active segments were determined and compared; these sequences contained the sequence 5' GCCGTCTGAA 3' in common. Subcloning studies confirmed the identity of this sequence as the gonococcal DNA recognition site. The three instances of the recognition sequence isolated from N. gonorrhoeae chromosomal DNA contain the sequence in the immediate neighborhood of its inverted repeat. Because a single copy of the sequence functions as a recognition site, the inverted duplication is not required for specific binding. The dyad symmetric arrangements of the chromosomal recognition sequences may form stable stem-loop structures that can function as terminators or attenuators of transcription. These inverted repeats are located at the boundaries of long open reading frames. The recognition sequence also constitutes part of two other probable terminators of gonococcal genes. We conclude that the signal for recognition of transforming DNA by gonococci is a frequent component of transcriptional terminator sequences. This regulatory function might account for the origin and maintenance of recognition sequences in the chromosomes of Gram-negative transformable bacteria.
A critical period for estrogen action on neurons of the song control system in the zebra finch.
Authors: Konishi M , Akutagawa E
Published: May 2006

The song nuclei of the male zebra finch (Poephila guttata) contain larger neurons than those of the female. This gender difference arises after hatching as a result of cell atrophy in the female and cell growth in the male. Implantation of estrogen in female chicks induces masculine differentiation of neurons in their song nuclei. The effects of estrogen on neuron size decline steeply after posthatching day 35 when neuronal atrophy begins. Estrogen loses its masculinizing effects completely after day 45 when the adult level of neuronal atrophy is reached. Thus, the end and the intensity of hormone action appear to be correlated with the timing of neuronal atrophy.
Duck lens epsilon-crystallin and lactate dehydrogenase B4 are identical: a single-copy gene product with two distinct functions.
Authors: Hendriks W , Mulders J W , Bibby M A , Slingsby C , Bloemendal H , de Jong W W
Published: May 2006

To investigate whether or not duck lens epsilon-crystallin and duck heart lactate dehydrogenase (LDH) B4 are the product of the same gene, we have isolated and sequenced cDNA clones of duck epsilon-crystallin. By using these clones we demonstrate that there is a single-copy Ldh-B gene in duck and in chicken. In the duck lens this gene is overexpressed, and its product is subject to posttranslational modification. Reconstruction of the evolutionary history of the LDH protein family reveals that the mammalian Ldh-C gene most probably originated from an ancestral Ldh-A gene and that the amino acid replacement rate in LDH-C is approximately 4 times the rate in LDH-A. Molecular modeling of LDH-B sequences shows that the increased thermostability of the avian tetramer might be explained by mutations that increase the number of ion pairs. Furthermore, the replacement of bulky side chains by glycines on the corners of the duck protein suggests an adaptation to facilitate close packing in the lens.
Transient administration of estradiol-17 beta establishes an autoregulatory loop permanently inducing estrogen receptor mRNA.
Authors: Barton M C , Shapiro D J
Published: May 2006

A single transient dose of estradiol-17 beta is sufficient to elicit the permanent induction of hepatic estrogen receptor mRNA, which is induced 18-fold (from 0.13 to 2.4 molecules per cell) and then remains fully induced for at least 125 days. In primary liver cultures, extremely low concentrations of estradiol-17 beta, which are below the Kd of the Xenopus laevis estrogen receptor, maintain persistent induction of estrogen receptor mRNA but not of estrogen-inducible vitellogenin mRNA. These data and the ability of the antiestrogen, hydroxytamoxifen, to reverse persistent induction of estrogen receptor mRNA, support a model in which transient doses of estradiol-17 beta induce the estrogen receptor and thereby establish an autoregulatory loop. The low levels of estradiol-17 beta normally circulating in male X. laevis and the elevated level of receptor provide sufficient hormone-receptor complex to permanently maintain the induced level of expression of the estrogen receptor gene. The permanent induction of the estrogen receptor may be the regulatory switch that results in the persistent expression of a recently identified class of proteins that exhibit long-term responses to estrogen.
Phosphorylation of serum response factor, a factor that binds to the serum response element of the c-FOS enhancer.
Authors: Prywes R , Dutta A , Cromlish J A , Roeder R G
Published: May 2006

Serum and growth factor regulation of c-FOS protooncogene transcription is mediated by the serum response element. A factor, serum response factor, binding to this element has already been identified. We demonstrate that serum response factor is phosphorylated in vivo on serine residues and that phosphatase treatment of this factor in vitro abolishes its DNA-binding activity. These results show phosphorylation of serum response factor to be required for its DNA-binding activity. The importance of serum response factor phosphorylation for the regulation of c-FOS expression is discussed.
Ly-1 B-cell clones similar to human chronic lymphocytic leukemias routinely develop in older normal mice and young autoimmune (New Zealand Black-related) animals.
Authors: Stall A M , Fariñas M C , Tarlinton D M , Lalor P A , Herzenberg L A , Strober S , Herzenberg L A
Published: May 2006

Studies presented here demonstrate that individually expanded clones of murine Ly-1 B cells, perhaps analogous to the expanded neoplastic Leu-1 B-cell clones in human chronic lymphocytic leukemias, are universally detectable in young New Zealand Black (NZB)-related autoimmune mice and in senescent normal mice (greater than 18 months old). These clones are visible as phenotypically homogeneous cell populations in multiparameter fluorescence-activated cell sorter analyses of peritoneal and splenic B cells; they show unique immunoglobulin heavy- and light-chain gene rearrangements in Southern gel analyses of peritoneal and splenic DNA; and, like the self-replenishing Ly-1 B-cell population from which they are drawn, they tend to grow readily in irradiated or unirradiated syngeneic or allotype congenic hosts. Furthermore, they develop and generalize in primary and secondary hosts in a characteristic pattern (peritoneum much greater than spleen greater than lymph node greater than bone marrow) that suggests that their initial growth is controlled by the mechanisms that normally control Ly-1 B-cell distribution in lymphoid organs. The universal emergence of these clones within the Ly-1 B-cell lineage may be explained by the substantially greater opportunity for hyperplastic and neoplastic transformation events in this long-lived self-replenishing Ly-1 B-cell population, which must divide relatively frequently to maintain its normal size throughout adulthood. Repeated exposure to internal or environmental antigens (with which Ly-1 B cells are known to react) may also play a role in driving the development of these clones.
Characterization of the cDNA encoding a protein binding to the major histocompatibility complex class II Y box.
Authors: Didier D K , Schiffenbauer J , Woulfe S L , Zacheis M , Schwartz B D
Published: May 2006

The expression of HLA class II genes is regulated by a series of cis-acting elements and trans-acting factors. Several cis-acting elements have been identified and have been termed the Z box, X box, Y box, octamer, and "TATA" box. The Y box contains an inverted CCAAT box. By probing a phage lambda gt11 library with double-stranded oligonucleotides, we have directly isolated a cDNA encoding a Y box-binding protein designated YB-1. YB-1 binding has an absolute requirement for the CCAAT box and relative specificity for the Y box. It has a Mr of 35,414, contains 18% basic residues, and contains putative nuclear localization signals. An inverse correlation of YB-1 and HLA-DR beta chain mRNA levels suggests that YB-1 is a negative regulatory factor.
Characterization of dihydropyridine-sensitive calcium channels in rat brain synaptosomes.
Authors: Woodward J J , Cook M E , Leslie S W
Published: May 2006

We examined the effects of dihydropyridine Ca2+-channel agonists on synaptosomal voltage-dependent Ca2+ entry and endogenous dopamine release. The (-) isomer of Bay K 8644 and the (+) isomer of Sandoz compound 202-791 were 100-1000 times more potent than their respective opposite enantiomers in enhancing Ca2+ uptake and dopamine release from striatal synaptosomes. The active isomer of each of these compounds increased Ca2+ entry and dopamine release to the same extent at a concentration of 1 nM. Fast-phase Ca2+ entry into synaptosomes isolated from cerebellum, cortex, and hippocampus was sensitive to nanomolar concentrations of Bay K 8644. No effect of Bay K 8644 was observed in synaptosomes isolated from brainstem. Bay K 8644 increased synaptosomal Ca2+ uptake and endogenous dopamine release from striatal synaptosomes only during the initial seconds of KCl-induced depolarization. The greatest increase was observed during the first second of depolarization. No effect was observed after greater than or equal to 5 sec of depolarization. Bay K 8644 did not alter Ca2+ uptake or dopamine release under resting conditions (5 mM KCl) or in response to KCl at greater than 15 mM. The activity of Bay K 8644 was also attenuated by lowering the concentrations of divalent cations in the incubation medium. Agonist activity was observed at Mg2+ concentrations greater than 500 microM (Ca2+ held at 100 microM) and Ca2+ concentrations greater than 100 microM (Mg2+ held at 1000 microM). These results suggest that the Ca2+ channels present in synaptosomes are sensitive to nanomolar concentrations of dihydropyridine agonists under a narrow range of experimental conditions.
Conserved quaternary structure of ligand-gated ion channels: the postsynaptic glycine receptor is a pentamer.
Authors: Langosch D , Thomas L , Betz H
Published: May 2006

The postsynaptic glycine receptor of rat spinal cord is a glycosylated membrane protein that, after affinity purification, contains membrane-spanning subunits of Mr 48,000 and 58,000 and an associated peripheral polypeptide of Mr 93,000. Here, the quaternary structure of the transmembrane core of the receptor was investigated by chemically crosslinking its subunits. Upon treatment with crosslinking reagents of different side-chain specificities and lengths, a consistent set of adducts up to Mr 260,000 was detected after separation by NaDodSO4/PAGE. The observed pattern of adducts was similar irrespective of whether purified receptor protein or synaptosomal membranes were crosslinked. Compositional analysis revealed that the crosslinked adducts contained the Mr 48,000 and 58,000 subunits in varying ratios but not the peripheral Mr 93,000 polypeptide. Thus adducts of intermediate molecular weight represent dimers, trimers, and tetramers of the transmembrane subunits, whereas the major adduct of Mr 260,000 corresponds to a pentameric assembly of subunits forming the ion channel of the glycine receptor. This subunit arrangement is similar to that reported for the nicotinic acetylcholine receptor of fish electric organ and skeletal muscle. Hence, we suggest that the different ligand-gated ion channels of excitable membranes share a similar quaternary structure.
Neonatal administration of prolactin antiserum alters the developmental pattern of T- and B-lymphocytes in the thymus and spleen of BALB/c female mice.
Authors: Russell D H , Mills K T , Talamantes F J , Bern H A
Published: May 2006

We have evaluated the effect of neonatal administration of mouse prolactin (PRL) antiserum on the developmental expression of T- and B-lymphocytes in the thymus and spleen of female BALB/c mice. Newborn female mice were injected subcutaneously with a 50-microliters aliquot of PRL antiserum or normal rabbit serum on days 1, 2, and 3. On neonatal day 5, the PRL antiserum-treated group had a significantly (P less than 0.05) increased population of cells in the thymus and the spleen that were positive for Thy-1.2 and for L3T4. Increases in Thy-1.2- and L3T4-positive cells in the thymus were detectable also on days 8 and 14 in mice that received the PRL antiserum and in mice injected with bromocriptine, a dopamine agonist that inhibits PRL release from the anterior pituitary. On neonatal days 21, 28, and 32, there were no significant differences in the percentage of cells positive for Thy-1.2, Ly-2 (formerly Lyt-2), or L3T4 antigens in the thymus. However, there were significant increases in the percentage of Thy-1.2- and L3T4-positive spleen cells in the bromocriptine-treated group at all times monitored and in the PRL antiserum-treated group except on day 14. In addition, the percentage of splenocytes that were positive for IgG was significantly increased in the PRL antiserum-treatment group on days 8-28, although not on neonatal day 32. Of tissues known to contain PRL receptors, neonatal administration of PRL antiserum or bromocriptine resulted in a significant alteration in the wet weight of spleen and liver, with no significant effect in thymus, heart, and kidney. Pituitary implants also resulted in a significant increase in both concanavalin A- and lipopolysaccharide-stimulated thymidine incorporation into murine splenic lymphocytes prepared from 45-day-old female mice. These data extend the role of PRL as an immunomodulator of adult lymphocyte function to a role in the developmental expression of T- and B-lymphocyte populations in the thymus and spleen of mice.
Galanin is an estrogen-inducible, secretory product of the rat anterior pituitary.
Authors: Kaplan L M , Gabriel S M , Koenig J I , Sunday M E , Spindel E R , Martin J B , Chin W W
Published: May 2006

Galanin is a peptide widely distributed throughout vertebrate central and peripheral nervous systems. Although its precise physiologic role is unknown, it can stimulate the pituitary secretion of prolactin and growth hormone. We examined the control of rat galanin (rGal) gene expression in the anterior pituitary using RNA blot and in situ hybridization analyses and using specific RIA. Pituitaries of normal male and ovariectomized female rats contained little detectable rGal mRNA. Treatment of these animals with 17 beta-estradiol increased pituitary rGal mRNA up to 4000-fold. These increases depended on time and dose of estrogen administration and correlated with up to 50-fold increases in pituitary galanin-like immunoreactivity. Galanin-like immunoreactivity was detectable in the plasma of estrogen-treated animals. Pituitary levels of rGal mRNA in female rats varied greater than 30-fold during the estrous cycle, with a peak on estrus and a nadir on diestrus. Estrogen-induced rGal gene expression was also observed in transplantable MtTW15 prolactin- and growth hormone-containing tumors but not in neuronal tissues expressing this gene. These data demonstrate that rGal is a secreted product of rat anterior pituitary cells, where its gene expression is strongly affected by physiologic levels of circulating estrogen.
Tumor promoter induces reorganization of actin filaments and calspectin (fodrin or nonerythroid spectrin) in 3T3 cells.
Authors: Sobue K , Fujio Y , Kanda K
Published: May 2006

We have used immunofluorescence, differential-interference-contrast, and interference-reflection microscopy to examine the translocation of actin filaments and calspectin (fodrin or nonerythroid spectrin) in 3T3 cells induced by phorbol 12-myristate 13-acetate (PMA). The two cytoskeletal proteins were observed to localize in dot structures that corresponded to the cell-substratum contact sites (focal contact) of the cytoplasmic surface of the plasma membrane. The induction of these cytoskeletal changes was specific for tumor promoters. High-resolution microscopy revealed that calspectin was intensely concentrated in ring-like structures surrounding actin dots. It was also located within the areas of actin dots, but to a lesser extent. Trifluoperazine and other phenothiazine derivatives inhibited the formation of those dot structures that appeared after the addition of PMA. Some serine protease inhibitors were also demonstrated to influence cytoskeletal changes by PMA. Our results provide evidence that calspectin is closely associated with actin filaments in dot structures induced by PMA. Possible mechanisms for these cytoskeletal changes produced by PMA are discussed.
Enhancement of antibody responses by IgD-binding factors induced by anti-IgD treatment of spleen cells.
Authors: Adachi M , Ishizaka K
Published: May 2006

Treatment of normal BALB/c splenocytes with anti-mouse IgD antibodies at 0 degrees C followed by incubation of the cells at 37 degrees C resulted in the formation of soluble factors that selectively inhibit rosette formation of lymphocytes bearing Fc delta receptors with IgD-coated erythrocytes (i.e., IgD-binding factors). Treatment of the same cells with anti-IgM antibodies failed to induce the formation of the factors. Analysis of cellular mechanisms indicated that polymerized surface IgD on B cells, as well as surface IgD-anti-IgD complexes shed from the B cells, induced T cells bearing Fc delta receptors to form IgD-binding factors. The factors formed by the anti-IgD treatment of splenic lymphocytes are composed of molecular mass species of 70 and 34 kDa, as estimated by gel filtration. Both the 70- and 34-kDa IgD-binding factors enhanced IgM and IgG1 plaque-forming cell responses of sheep erythrocyte-primed mouse spleen cells to the antigen.
Identification of a cDNA encoding a parathyroid hormone-like peptide from a human tumor associated with humoral hypercalcemia of malignancy.
Authors: Mangin M , Webb A C , Dreyer B E , Posillico J T , Ikeda K , Weir E C , Stewart A F , Bander N H , Milstone L , Barton D E
Published: May 2006

Humoral hypercalcemia of malignancy is a common paraneoplastic syndrome that appears to be mediated in many instances by a parathyroid hormone-like peptide. Poly(A)+ RNA from a human renal carcinoma associated with this syndrome was enriched by preparative electrophoresis and used to construct an enriched cDNA library in phage lambda gt10. The library was screened with a codon-preference oligonucleotide synthesized on the basis of a partial N-terminal amino acid sequence from a human tumor-derived peptide, and a 2.0-kilobase cDNA was identified. The cDNA encodes a 177 amino acid protein consisting of a 36 amino acid leader sequence and a 141 amino acid mature peptide. The first 13 amino acids of the deduced sequence of the mature peptide display strong homology to human PTH, with complete divergence thereafter. RNA blot-hybridization analysis revealed multiple transcripts in mRNA from tumors associated with the humoral syndrome and also in mRNA from normal human keratinocytes. Southern blot analysis of genomic DNA from humans and rodents revealed a simple pattern compatible with a single-copy gene. The gene has been mapped to chromosome 12.
Autoradiographic localization of supraspinal kappa-opioid receptors with [125I-Tyr1, D-Pro10]dynorphin A-(1-11).
Authors: Jomary C , Gairin J E , Cros J , Meunier J C
Published: May 2006

[125I-Tyr1, D-Pro10]dynorphin A-(1-11) (125I-DP-DYN), an opioid peptide analogue that has previously been shown to be kappa selective, displays specific, saturable, and high-affinity (Kd = 0.3 nM) binding in slide-mounted sections from nerve tissue. We have used 125I-DPDYN to autoradiographically visualize supraspinal kappa-opioid receptor sites in rats, guinea pigs, and rabbits. The autoradiographic dispositions of 125I-DPDYN in sections from cerebellum are clearly different in guinea pig and rabbit, suggesting that kappa receptors have different functions in this organ of the two species. Autoradiograms from 125I-DPDYN-labeled brain sections also reveal major species differences, in particular in thalamus, which is densely labeled in rabbit and considerably less so in rat and guinea pig. The data show that 125I-DPDYN is a useful probe to visualize kappa-opioid receptor sites in nerve tissue sections directly and rapidly.
Thermodynamics of cosmological matter creation
Authors: Prigogine I. , Geheniau J. , Gunzig E. , Nardone P.
Published: May 2006

A type of cosmological history that includes large-scale entropy production is proposed. These cosmologies are based on reinterpretation of the matter-energy stress tensor in Einstein's equations. This modifies the usual adiabatic energy conservation laws, thereby including irreversible matter creation. This creation corresponds to an irreversible energy flow from the gravitational field to the created matter constituents. This point of view results from consideration of the thermodynamics of open systems in the framework of cosmology. It is shown that the second law of thermodynamics requires that space-time transforms into matter, while the inverse transformation is forbidden. It appears that the usual initial singularity associated with the big bang is structurally unstable with respect to irreversible matter creation. The corresponding cosmological history therefore starts from an instability of the vacuum rather than from a singularity. This is exemplified in the framework of a simple phenomenological model that leads to a three-stage cosmology: the first drives the cosmological system from the initial instability to a de Sitter regime, and the last connects with the usual matter-radiation Robertson-Walker universe. Matter as well as entropy creation occurs during the first two stages, while the third involves the traditional cosmological evolution. A remarkable fact is that the de Sitter stage appears to be an attractor independent of the initial fluctuation. This is also the case for all the physical predictions involving the present Robertson-Walker universe. Most results obtained previously, in the framework of quantum field theory, can now be obtained on a macroscopic basis. It is shown that this description leads quite naturally to the introduction of primeval black holes as the intermediate stage between the Minkowski vacuum and the present matter-radiation universe. The instability at the origin of the universe is the result of fluctuations of the vacuum in which black holes act as membranes that stabilize these fluctuations. In short, black holes will be produced by and “inverse” Hawking radiation process and, once formed, will decompose into “real” matter through the usual Hawking radiation. In this way, the irreversible transformation of space-time into matter can be described as a phase separation between matter and gravitation in which black holes play the role of “critical nuclei.”
DNA topoisomerase-targeting antitumor drugs can be studied in yeast.
Authors: Nitiss J , Wang J C
Published: May 2006

The antitumor drugs camptothecin and an anilinoacridine, 4'-(9-acridinylamino)-methanesulfon-m-anisidide (mAMSA), which act on DNA topoisomerase I and II, respectively, are shown to inhibit the growth of Saccharomyces cerevisiae mutants selected for their permeability to other inhibitors. In addition to growth inhibition, these drugs induce high levels of homologous recombination and induce the expression of a DNA damage-inducible gene DIN3. Cytotoxicity of the drugs is more pronounced in strains that also carry a rad52 mutation. An analog of mAMSA), which is ineffective as an inhibitor of DNA topoisomerase II in mammalian cells, is also ineffective in eliciting physiological responses in these yeast strains. The physiological effects of camptothecin, but not those of mAMSA, disappear if the TOP1 gene encoding DNA topoisomerase I is disrupted. This shows that DNA topoisomerase I is the sole target of camptothecin cytotoxicity and illustrates that a nonessential enzyme can nevertheless be the target for a cytotoxic drug.
A missense mutation in the human liver/bone/kidney alkaline phosphatase gene causing a lethal form of hypophosphatasia.
Authors: Weiss M J , Cole D E , Ray K , Whyte M P , Lafferty M A , Mulivor R A , Harris H
Published: May 2006

Hypophosphatasia is an inherited disorder characterized by defective bone mineralization and a deficiency of serum and tissue liver/bone/kidney alkaline phosphatase (L/B/K ALP) activity. Clinical severity is variable, ranging from death in utero (due to severe rickets) to pathologic fractures first presenting in adult life. Affected siblings, however, are phenotypically similar. Severe forms of the disease are inherited in an autosomal recessive fashion; heterozygotes often show reduced serum ALP activity. The specific gene defects in hypophosphatasia are unknown but are thought to occur either at the L/B/K ALP locus or within another gene that regulates L/B/K ALP expression. We used the polymerase chain reaction to examine L/B/K ALP cDNA from a patient with a perinatal (lethal) form of the disease. We observed a guanine-to-adenine transition in nucleotide 711 of the cDNA that converts alanine-162 of the mature enzyme to threonine. The affected individual, whose parents are second cousins, is homozygous for the mutant allele. Introduction of this mutation into an otherwise normal cDNA by site-directed mutagenesis abolishes the expression of active enzyme, demonstrating that a defect in the L/B/K ALP gene results in hypophosphatasia and that the enzyme is, therefore, essential for normal skeletal mineralization.
Identification of specific residues of human interleukin 2 that affect binding to the 70-kDa subunit (p70) of the interleukin 2 receptor.
Authors: Collins L , Tsien W H , Seals C , Hakimi J , Weber D , Bailon P , Hoskings J , Greene W C , Toome V , Ju G
Published: May 2006

Analogs of interleukin 2 containing defined amino acid substitutions and deletions were assayed for bioactivity and for competitive binding to the high-affinity human interleukin 2 receptor complex and its two component subunits, a 55-kDa subunit (p55 or TAC) and a 70-kDa subunit (p70). Substitution of Asp20 or deletion of Phe124 resulted in inactive analog proteins that were unable to interact with the high-affinity p55/p70 complex or the intermediate-affinity p70 subunit of the interleukin 2 receptor. These analogs, however, retained the capacity to compete for binding to the low-affinity p55 subunit. The presence of the carboxylic acid in the side chain of Asp20 was necessary for effective binding to the p70 protein. In contrast, substitution of Trp121 and Leu17 created analogs that were inactive in the bioassay and all three binding assays. The effects of these mutations on protein conformation were assessed by circular dichroism. These results demonstrate that specific residues in the NH2 and COOH termini of interleukin 2 are crucial for its structure and activity.
A single antigen-specific B cell can conjugate to either a type 1 or a type 2 helper T cell.
Authors: Sanders V M , Fernandez-Botran R , Coffman R L , Mosmann T R , Vitetta E S
Published: May 2006

Keyhole-limpet-hemocyanin-specific I-Ad-restricted T helper cells type 1 (TH1 cells) and type 2 (TH2 cells) were studied for their ability to physically conjugate to trinitrophenyl-specific antigen-binding B cells (TNP-ABCs). The same TNP-ABCs (which had already processed TNP-keyhole limpet hemocyanin) formed conjugates with one or the other T helper (TH) cell type, and conjugation was antigen-specific and major histocompatibility complex-restricted. The conjugation of a fixed number of one type of TH cell to the TNP-ABCs was inhibited by co-incubation with increasing numbers of cells of the other TH cell type. Thus, the vast majority of TNP-ABCs can conjugate to either type of TH cell. A similar pattern of inhibition of the conjugation of one TH cell type to the TNP-ABCs was seen when cells were co-incubated with increasing numbers of cells from an alloreactive T-cell clone. In all cases, conjugates contained only one T cell bound to one B cell, suggesting that interaction of an antigen-presenting B cell with one TH cell prevents the simultaneous binding of another TH cell.
Endothelial cell hyperplasia in human glioblastoma: coexpression of mRNA for platelet-derived growth factor (PDGF) B chain and PDGF receptor suggests autocrine growth stimulation.
Authors: Hermansson M , Nistér M , Betsholtz C , Heldin C H , Westermark B , Funa K
Published: May 2006

The genes for platelet-derived growth factor (PDGF) A chain, B chain/c-sis, and the PDGF receptor are expressed in human malignant glioma cell lines. In the present investigation we have studied the expression of these genes in biopsy specimens from human glioblastomas. Hyperplasia of the vascular endothelium is a prominent characteristic of human glioblastoma multiforme and simian sarcoma virus-induced gliomas in primates. RNA transfer blot analysis of biopsies from glioblastoma multiforme showed transcripts for PDGF A and B chains and the PDGF receptor. Tissue sections from this tumor examined by in situ hybridization techniques revealed that the proliferating vascular endothelial cells contained large quantities of mRNA for PDGF B chain/c-sis and its receptor and, to a lesser extent, for PDGF A chain. In contrast, the tumor cells expressed more mRNA for PDGF A chain than for PDGF B chain and PDGF receptor. The latter two were also expressed at higher levels in glioma cells than in glial cells of nontumorous human brain tissue. Thus, an autocrine stimulation by the PDGF B chain/c-sis product via its receptor, evoked by interaction with surrounding glioma cells, could be the mechanism behind the pathological proliferation of endothelial cells characteristically found in this type of malignancy.
Intracellular pH in human skeletal muscle by 1H NMR.
Authors: Pan J W , Hamm J R , Rothman D L , Shulman R G
Published: May 2006

We report here the in vivo observation of the imidazole protons (C-2 and C-4) of carnosine (beta-Ala-His) by 1H NMR at 4.7 T in human skeletal muscle. The relationship between the pH and chemical shift of the C-2 and C-4 resonances was determined analytically. These titration curves were used to measure the resting pH of human muscle in vivo, 7.01 x 0.04 (C-2 proton) and 6.97 +/- 0.10 (C-4 proton). An in vivo titration curve of the C-2 proton resonance was determined by interleaving 1H and 31P NMR spectra after exhaustive exercise, during which muscle pH recovers from an acidic value of 6.1. We observed excellent agreement between the pH values as determined by the C-2 resonance of carnosine and that of inorganic phosphate. Carnosine provides an excellent pH indicator since (i) its concentration is relatively stable and (ii) it allows measurement of proton metabolites and pH to be achieved through the same coil, thus enabling a better signal/noise ratio and better localization.
A gene activated in mouse 3T3 cells by serum growth factors encodes a protein with "zinc finger" sequences.
Authors: Christy B A , Lau L F , Nathans D
Published: May 2006

We have recently identified by cDNA cloning a set of genes that are rapidly activated in mouse 3T3 cells by serum or purified growth factors. Here we report that the cDNA (clone 268) derived from one of these immediate early genes (zif/268) encodes a protein with three tandem "zinc finger" sequences typical of a class of eukaryotic transcription factors. The mRNA of zif/268 is present in many organs and tissues of the mouse and is especially abundant in the brain and thymus tissue. The 5' genomic flanking sequence of zif/268 has sequences related to binding sites for known regulatory proteins, including four sequences that resemble the core of the serum response elements (SREs) upstream of the c-fos and actin genes. The SRE-like sequences could be responsible for the coordinate activation of zif/268 and fos after serum stimulation of 3T3 cells.
Thermotolerance and synthesis of heat shock proteins: these responses are present in Hydra attenuata but absent in Hydra oligactis.
Authors: Bosch T C , Krylow S M , Bode H R , Steele R E
Published: May 2006

Organisms respond to environmental stress by synthesizing a small number of highly conserved heat shock proteins. In organisms as diverse as bacteria, plants, invertebrates, and vertebrates, synthesis of these proteins is directly correlated with the acquisition of thermotolerance. While studying the freshwater coelenterate hydra, we observed that Hydra oligactis was extremely sensitive to thermal stress. In contrast, the related species Hydra attenuata survives short-term exposure to high temperatures. Furthermore, after incubation at an elevated but nonlethal temperature, H. oligactis did not become thermotolerant. H. attenuata, however, acquired thermotolerance after such a preincubation. In H. attenuata the major heat shock protein was found to be 60 kDa in size. H. oligactis did not synthesize detectable levels of this protein or any new species of proteins in response to stress. Several other species of hydra were found to behave like H. oligactis in response to stress. Thus, these findings provide direct support for the hypothesis that heat shock proteins are required for stress tolerance and that the major heat shock protein in hydra does not have any effects on normal growth or physiology. The findings also indicate that the presence of a heat shock response might be related to the natural environment in which an organism lives.
Bafilomycins: a class of inhibitors of membrane ATPases from microorganisms, animal cells, and plant cells.
Authors: Bowman E J , Siebers A , Altendorf K
Published: May 2006

Various membrane ATPases have been tested for their sensitivity to bafilomycin A1, a macrolide antibiotic. F1F0 ATPases from bacteria and mitochondria are not affected by this antibiotic. In contrast, E1E2 ATPases--e.g., the K+-dependent (Kdp) ATPase from Escherichia coli, the Na+,K+-ATPase from ox brain, and the Ca2+-ATPase from sarcoplasmic reticulum--are moderately sensitive to this inhibitor. Finally, membrane ATPases from Neurospora vacuoles, chromaffin granules, and plant vacuoles are extremely sensitive. From this we conclude that bafilomycin A1 is a valuable tool for distinguishing among the three different types of ATPases and represents the first relatively specific potent inhibitor of vacuolar ATPases.
Numerous transcription initiation sites exist for the maize mitochondrial genes for subunit 9 of the ATP synthase and subunit 3 of cytochrome oxidase.
Authors: Mulligan R M , Lau G T , Walbot V
Published: May 2006

Transcripts for plant mitochondrial genes are frequently present as multiple size classes. In maize, these differences often result from variation in the 5' noncoding region. To determine where transcription initiates, primary (unprocessed) transcripts were specifically labeled in vitro by the capping reaction catalyzed by guanylyltransferase. Direct mapping of transcription initiation sites was accomplished by hybridization of in vitro-capped RNA with the 5' flanking sequences of mitochondrial genes and subsequent digestion with single-strand-specific RNases. The RNase protection experiments identified three transcription initiation sites for subunit 3 of cytochrome oxidase and at least six transcription initiation sites for subunit 9 of ATP synthase. Thus, transcript size heterogeneity is primarily the result of multiple transcription initiation sites for these genes rather than RNA processing. Primer extension analyses of maize mitochondrial RNA were used to precisely establish the sequences at the initiation sites. Comparison of sequences at transcription initiation sites suggests that some homology exists at these sites, although no highly conserved consensus sequence is obvious.
Asynchronous DNA replication within the human beta-globin gene locus.
Authors: Epner E , Forrester W C , Groudine M
Published: May 2006

The timing of DNA replication of the human beta-globin gene locus has been studied by blot hybridization of newly synthesized BrdUrd-substituted DNA from cells in different stages of the S phase. Using probes that span greater than 120 kilobases across the human beta-globin gene locus, we show that the majority of this domain replicates in early S phase in the human erythroleukemia cell line K562 and in middle-to-late S phase in the lymphoid cell line Manca. However, in K562 cells three small regions display a strikingly different replication pattern than adjacent sequences. These islands, located in the inter-gamma-globin gene region and approximately 20 kilobases 5' to the epsilon-globin gene and 20 kilobases 3' to the beta-globin gene, replicate later and throughout S phase. A similar area is also present in the alpha-globin gene region in K562 cells. We suggest that these regions may represent sites of termination of replication forks.
The 43-kDa neuronal growth-associated protein (GAP-43) is present in plasma membranes of rat astrocytes.
Authors: Vitković L , Steisslinger H W , Aloyo V J , Mersel M
Published: May 2006

One of the neuronal growth-associated proteins, GAP-43 (molecular mass, approximately 43 kDa; pI 4.3), is abundant in growth-cone membranes and corresponds to a major protein kinase C substrate, the 46-kDa phosphoprotein (pp46), of a growth-cone-enriched subcellular fraction. This protein has the following additional designations (depending on context): B-50 (phospholipid metabolism), F1 (synaptic plasticity), and p57 (calmodulin binding). We show that a protein with the same molecular mass and isoelectric point as GAP-43, which interacts with anti-GAP-43 antibodies on immunoblots, is present in the plasma membranes of cultured neonatal rat cortical astrocytes. Double-immunofluorescence labeling of cells with a serum against glial fibrillary acidic protein and anti-GAP-43 antibody was observed. Furthermore, astrocytic protein was phosphorylated in vitro by protein kinase C and comigrated in two-dimensional PAGE with GAP-43. The data indicate that GAP-43, heretofore believed to be neuron-specific, is present in at least one class of glial cells.