bluuebunny/crossref_metadata_2025 huggingface dataset:
Dissociation of thermoregulation in cats with cytotoxic pontine lesions.
Authors: Amini-Sereshki L
Published: May 2006

Neurons of the pontine tegmentum of the cat were lesioned by microinjection of ibotenic acid into the brainstem. The threshold ambient temperatures for heat-gain (shivering) and for heat-loss (panting) responses, together with brain and skin temperatures, were measured in intact animals and after the neurotoxic lesioning. After the lesioning the shivering threshold was altered but the panting threshold did not change. The results indicate that certain neurons involved in the shivering response reside in the pontine tegmentum. Neurons involved in the panting response, however, may lie outside the lesioned areas.
Integration and expression of a rabbit liver cytochrome P-450 gene in transgenic Nicotiana tabacum.
Authors: Saito K , Noji M , Ohmori S , Imai Y , Murakoshi I
Published: May 2006

Cytochrome P-450 is involved in the oxidative metabolism of a broad range of substrates. We have made a chimeric construct, pSN002, containing the cDNA for rabbit liver cytochrome P-450 (IIC14) under the control of the TR2' promoter for mannopine synthase in the Agrobacterium Ti plasmid. Nicotiana tabacum was transformed with Agrobacterium tumefaciens harboring a cointegrated plasmid pSN002::pGV2260. The presence of mRNA and of the translated protein from the chimeric cytochrome P-450 gene in transgenic plants was confirmed by RNA blot hybridization and by Western blot and immunohistochemical analyses, respectively. The transformants in which the foreign cytochrome P-450 protein is expressed show marked phenotypic changes, notably a tendency rapidly to senesce. We detected 2-propenylpyrrolidine, a degradative metabolite of nicotine alkaloids, in transgenic tobacco showing this pronounced phenotypic change. Such metabolism is likely to be due to the effect of senescence and not directly to the presence of the cytochrome P-450.
Limited functional equivalence of phylogenetic variation in small nuclear RNA: yeast U2 RNA with altered branchpoint complementarity inhibits splicing and produces a dominant lethal phenotype.
Authors: Miraglia L , Seiwert S , Igel A H , Ares M
Published: May 2006

U2 is a highly conserved small nuclear RNA essential for pre-mRNA splicing in mammals and yeast and for trans-splicing in trypanosomes. To test the function of variant U2 RNA structures from different organisms, we conducted phylogenetic exchanges of U2 domains. Replacing nucleotides 1-120 of yeast U2 with the corresponding region of human U2 generates a U2 RNA that is correctly folded and functions in yeast. In contrast, replacement of the branchpoint interaction region of yeast U2 with the corresponding region from trypanosome is dominant lethal. Using a GAL-U2 promoter fusion, we show that the dominant phenotype can be made conditional and that the accumulation of mutant U2 is followed rapidly by inhibition of nuclear pre-mRNA splicing. The results suggest that U2 small nuclear ribonucleoprotein particles normally participate in stable complexes with a limiting splicing factor prior to formation of U2-intron branchpoint base pairs.
Antibodies to poly[(2----8)-alpha-N-acetylneuraminic acid] and poly[(2----9)-alpha-N-acetylneuraminic acid] are elicited by immunization of mice with Escherichia coli K92 conjugates: potential vaccines for groups B and C meningococci and E. coli K1.
Authors: Devi S J , Robbins J B , Schneerson R
Published: May 2006

Meningitis and other systemic infections caused by group B Neisseria meningitidis and Escherichia coli K1 remain important problems. The capsular polysaccharides (CPs) of these pathogens (poly[(2----8)-alpha-N-acetylneuraminic acid] or poly(alpha 2-8NeuNAc] are identical and are virulence factors and protective antigens for both. CP vaccines for these pathogens are not available because poly(alpha 2-8NeuNAc) alone, as a complex or a conjugate, is poorly immunogenic. Because oligomers of poly(alpha 2-8NeuNAc) in fetal brain and other tissues bind antibodies in vitro, it has been suggested that antibodies to this CP might be pathologic. We synthesized conjugates of this CP with tetanus toxoid under conditions that avoid lactone formation. Using this scheme, we also synthesized conjugates of group C meningococcal CP (poly[(2----9)-alpha-N-acetylneuraminic acid] or poly(alpha 2-9NeuNAc] and of E. coli K92 CP [poly(alpha 2-8, alpha 2-9NeuNAc)]. When injected s.c. in saline into mice, conjugates of poly(alpha 2-8NeuNAc) or poly(alpha 2-9NeuNAc) elicited homologous antibodies. E. coli K92 conjugates elicited both poly(alpha 2-8NeuNAc) and poly(alpha 2-9NeuNAc) antibodies. Both components of the conjugates expressed T-dependent immunologic properties under conditions and dosages acceptable for clinical evaluation. Poly(alpha 2-8NeuNAc) antibodies elicited by the homologous or the K92 conjugates had lower binding activities at 37 degrees C than at 22 degrees C. "Natural" poly(alpha 2-8NeuNAc) antibodies were present in almost all matched pairs of human maternal and cord sera; most cord levels were higher than in corresponding maternal sera. These findings suggest that increased levels of poly(alpha 2-8NeuNAc) IgG antibodies elicited by our conjugates will confer protective immunity to group B meningococci and E. coli K1 and will not be pathologic.
Mediation of the short-loop negative feedback of luteinizing hormone (LH) on LH-releasing hormone release by melatonin-induced inhibition of LH release from the pars tuberalis.
Authors: Nakazawa K , Marubayashi U , McCann S M
Published: May 2006

The pineal hormone melatonin is thought to mediate the effects of the pineal gland on seasonal reproduction by altering the release of gonadotropins. The mechanism by which melatonin controls gonadotropin secretion has been obscure. Recently, labeled 2-iodomelatonin was used to localize melatonin receptors in brain by radioautography. The highest concentration of melatonin receptors was found in the pars tuberalis of the pituitary gland of mammals. Pituitary hormones, in particular luteinizing hormone (LH), have been localized in cells of the pars tuberalis. Consequently, we hypothesized that melatonin might act on its receptors in the pars tuberalis to alter the release of LH. It would then be possible for this LH to diffuse into the overlying median eminence, there to alter the release of LH-releasing hormone (LHRH) from the axons of the LHRH neurons. To evaluate this hypothesis, we incubated median eminence-pars tuberalis tissue from male rats in vitro. After preincubation in Krebs-Ringer bicarbonate buffer for 30 min, test substances were added to fresh medium and the incubation was continued for 30 min. LHRH or LH released into the medium was measured by radioimmunoassay. Melatonin induced a dose-related release of LHRH with the maximum response at the greatest concentration tested (1 microM). This concentration of melatonin also significantly reduced the release of LH into the medium. The increased release of LHRH induced by melatonin (10 microM) was completely blocked by the addition of LH (50 ng/ml), which by itself had no significant effect on LHRH release. Rat LH antiserum (final dilution, 1:1800) significantly elevated LHRH output, whereas normal rabbit serum at a similar dilution had no effect. Finally, LHRH (0.1 microM) induced a significant release of LH from median eminence-pars tuberalis tissue that was completely blocked by melatonin (10 microM). The results support the hypothesis that LH released from the pars tuberalis diffuses to the LHRH terminals in the median eminence to suppress LHRH release. Melatonin acts on its receptors in the pars tuberalis to inhibit LH release, thereby stimulating the release of LHRH from its terminals in the median eminence. The negative short-loop feedback of LH inhibits basal LHRH release in vitro since antiserum against LH increased LHRH release. The results suggest a concept concerning the mechanism by which melatonin can affect the release of pituitary hormones from the pars tuberalis. It is likely that these pituitary hormones diffuse into the median eminence to modify the release of hypothalamic releasing and inhibiting peptides, thereby altering plasma pituitary hormone concentrations.
Protein-tyrosine-phosphatase CD45 is phosphorylated transiently on tyrosine upon activation of Jurkat T cells.
Authors: Stover D R , Charbonneau H , Tonks N K , Walsh K A
Published: May 2006

The leukocyte common antigen (CD45) is an abundant lymphocyte surface antigen that has been reported to be involved in signaling through the T-cell antigen receptor. CD45 is a transmembrane protein-tyrosine-phosphatase. An internal segment comprises two domains each of which is homologous to other protein-tyrosine-phosphatases; the extracellular segment has the hallmarks of a ligand-binding motif. Since tyrosine phosphorylation is an early signal resulting from stimulation of the T-cell antigen receptor and CD45 is required for proper activation through the receptor, we explored whether CD45 might be regulated by tyrosine phosphorylation. Treatment of a T-cell leukemia line (Jurkat) with either phytohemagglutinin or anti-CD3 antibodies induced phosphorylation of tyrosine residues in CD45; treatment with phorbol 12-myristate 13-acetate did not. Phosphorylation of CD45 was transient, disappearing within 40 min after phytohemagglutinin treatment. The requirement for stringent conditions of phosphatase inhibition suggests that CD45 is capable of autodephosphorylation in vivo. These observations support recent reports indicating CD45 is involved in an early step in the T-cell activation cascade. They also suggest that phosphorylation/dephosphorylation of tyrosine residues in CD45 should be explored further as a possible regulatory mechanism.
Molecular characterization of five human anti-human immunodeficiency virus type 1 antibody heavy chains reveals extensive somatic mutation typical of an antigen-driven immune response.
Authors: Andris J S , Johnson S , Zolla-Pazner S , Capra J D
Published: May 2006

We report the heavy chain variable region sequences from the cDNAs of five previously described monoclonal cell lines producing human antibodies specific for the human immunodeficiency virus type 1 and detail the molecular characteristics, germ-line origins, and extent of somatic mutation among these antibodies. Three of the five heavy chain variable regions derive from the VHIV gene family, but each has arisen from a different heavy chain variable region (VH) gene segment within the VHIV family. In addition, one is derived from a VHI gene segment, and one is derived from a VHV gene segment. Since four of the five antibodies arise from known germ-line VH elements, a precise determination of the extent of somatic variation is possible. The amount of variation from the closest germ-line sequence ranges from 4.5% to 14.8% among these antibodies, most of which is concentrated in the complementarity-determining regions. In general, the diversity (D) segments are long, characteristic of D-D fusions and/or extensive terminal deoxynucleotidyltransferase activity; however, definitive homologies cannot be found with the known germ-line D segments. Joining (JH) gene segment utilization appears random. The use of five different germ-line VH gene segments and extensive somatic mutation provides evidence that a polyclonal, antigen-driven immune response occurs during the natural infection with human immunodeficiency virus.
Integrative selection of human chromosome-specific yeast artificial chromosomes.
Authors: Pavan W J , Reeves R H
Published: May 2006

Human specific "integrative selection vectors" (ISVs) were designed to optimize integration of a yeast-selectable marker specifically into yeast artificial chromosomes (YACs) derived from human but not mouse DNA. ISVs were transformed into a YAC genomic library constructed from DNA of a human-mouse somatic cell hybrid containing chromosome 21 (HSA21) as the only human chromosome. One percent of the yeast in the original library contained HSA21-derived YACs; between 45% and 54% of the yeast recovered after transformation with ISV vectors contained human YACs. Integrative selection provides a rapid means of obtaining a highly enriched population of human chromosome-specific YACs by eliminating the labor-intensive steps of isolating and screening primary transformants. The procedure is biased toward the selection of YACs that contain a large number of targets for homologous recombinations; thus, libraries constructed by this procedure will be composed primarily of the largest YACs in the population.
Higher-order syzygies for the bracket algebra and for the ring of coordinates of the Grassmanian.
Authors: Anick D , Rota G C
Published: May 2006

A Poincaré resolution is given for the supersymmetric ring of brackets over a signed alphabet. As a consequence, a resolution is found for the ring of coordinates of the Grassmanian variety in projective space over any infinite field.
Ultraviolet irradiation of DNA complexed with alpha/beta-type small, acid-soluble proteins from spores of Bacillus or Clostridium species makes spore photoproduct but not thymine dimers.
Authors: Nicholson W L , Setlow B , Setlow P
Published: May 2006

UV irradiation of complexes of DNA and an alpha/beta-type small, acid-soluble protein (SASP) from Bacillus subtilis spores gave decreasing amounts of pyrimidine dimers and increasing amounts of spore photoproduct as the SASP/DNA ratio was increased. The yields of pyrimidine dimers and spore photoproduct were less than 0.2% and 8% of total thymine, respectively, when DNA saturated with SASP was irradiated at 254 nm with 30 kJ/m2; in the absence of SASP the yields were reversed-4.5% and 0.3%, respectively. Complexes of DNA with alpha/beta-type SASP from Bacillus cereus, Bacillus megaterium, or Clostridium bifermentans spores also gave spore photoproduct upon UV irradiation. However, incubation of these SASPs with DNA under conditions preventing complex formation or use of mutant SASPs that do not form complexes did not affect the photoproducts formed in vitro. These results suggest that the UV photochemistry of bacterial spore DNA in vivo is due to the binding of alpha/beta-type SASP, a binding that is known to cause a change in DNA conformation in vitro from the B form to the A form. The yields of spore photoproduct in vitro were significantly lower than in vivo, perhaps because of the presence of substances other than SASP in spores. It is suggested that as these factors diffuse out in the first minutes of spore germination, spore photoproduct yields become similar to those observed for irradiation of SASP/DNA complexes in vitro.
E1BF is an essential RNA polymerase I transcription factor with an intrinsic protein kinase activity that can modulate rRNA gene transcription.
Authors: Zhang J , Niu H F , Jacob S T
Published: May 2006

We previously described the purification and characterization of E1BF, a rat rRNA gene core promoter-binding factor that consists of two polypeptides of 89 and 79 kDa. When this factor was incubated in the absence of any exogenous protein kinase under conditions optimal for protein phosphorylation, the 79-kDa polypeptide of E1BF was selectively phosphorylated. The labeled phosphate could be removed from the E1BF polypeptide by treatment with calf intestinal alkaline phosphatase or potato acid phosphatase. Elution of the protein from the E1BF-promoter complex formed in an electrophoretic mobility-shift assay followed by incubation of the concentrated eluent with [gamma-32P] ATP resulted in the selective labeling of the 79-kDa band. The E1BF-associated protein kinase did not phosphorylate casein or histone H1. Fraction DE-B, a preparation containing RNA polymerase I and all polymerase I transcription factors (including E1BF), lost polymerase I transcriptional activity when treated with phosphatase. The phosphatase-induced inactivation of polymerase I activity associated with fraction DE-B could be reversed by the addition of purified E1BF. Treatment of purified E1BF with heat, SDS, or an ATP affinity analog eliminated its capacity to reactivate dephosphorylated fraction DE-B. These data demonstrate that (i) polymerase I promoter-binding factor E1BF contains an intrinsic substrate-specific protein kinase and (ii) E1BF is an essential polymerase I transcription factor that can modulate rRNA gene transcription by protein phosphorylation. Further, these studies have provided a direct means to identify a protein kinase or any other enzyme that can interact with a specific DNA sequence.
Human bone marrow non-B, non-T cells produce interleukin 4 in response to cross-linkage of Fc epsilon and Fc gamma receptors.
Authors: Piccinni M P , Macchia D , Parronchi P , Giudizi M G , Bani D , Alterini R , Grossi A , Ricci M , Maggi E , Romagnani S
Published: May 2006

Human bone marrow (BM) cells lacking T- and B-cell markers expressed RNA encoding interleukin (IL) 4 and secreted detectable amounts of IL-4 in supernatants in response to Fc epsilon or Fc gamma receptor (Fc epsilon R or Fc gamma R) cross-linking. In some experiments, IL-5 RNA expression in response to Fc epsilon R cross-linkage could also be detected. In contrast, RNA transcripts for, and secretion of, IL-2, IL-6, and interferon gamma were never observed. The presence of IL-3 in the cultures was essential for IL-4 production by non-B, non-T BM cells in response to Fc gamma R cross-linking and enhanced IL-4 RNA expression in response to Fc epsilon R cross-linking. Under the same experimental conditions, BM T and B lymphocytes, as well as peripheral blood T, B, and non-B, non-T cells, did not express IL-4 RNA. Prolonged incubation of non-B, non-T cells in IgE-free medium followed by extensive washing did not inhibit IL-4 production induced by anti-IgE antibodies, suggesting that the Fc epsilon R involved in the response has the characteristics of a high-affinity receptor. The Fc epsilon R+ cells were separated from the Fc epsilon R- cells by sorting non-B, non-T BM cell suspensions with fluorescein isothiocyanate-conjugated IgE and then assessed for both IL-4 RNA expression and alcian blue staining. Both IL-4-producing and alcian blue-positive cells segregated with the Fc epsilon R+ fraction. These data suggest that human BM cells, probably belonging to the mast cell and/or basophil lineage, are capable of producing IL-4 in response to Fc epsilon R or Fc gamma R cross-linkage.
Induction of plasmacytomas secreting antigen-specific monoclonal antibodies with a retrovirus expressing v-abl and c-myc.
Authors: Weissinger E M , Mischak H , Largaespada D A , Kaehler D A , Mitchell T , Smith-Gill S J , Risser R , Mushinski J F
Published: May 2006

ABL-MYC, a recombinant murine retrovirus that expresses v-abl and c-myc, rapidly induces transplantable mono- or oligoclonal plasmacytomas in BALB/c mice. To determine if the targets for transformation of this retrovirus are antigen-committed B lymphocytes and to explore this system as an alternative technique for producing antigen-specific monoclonal antibodies, plasmacytomas were induced in mice that had been immunized with two different types of immunogens, hen egg white lysozyme and sheep red blood cells. The majority of these plasmacytomas secreted immunogen-specific antibodies. Plasmacytomas induced in unimmunized mice did not react with hen egg white lysozyme or sheep red blood cells. The specific antibodies were comparable in concentration, specificity, and affinity to monoclonal antibodies obtained with conventional hybridoma technology, but, in addition to IgGs and IgMs, they included specific IgA antibodies, which are rare among splenic-derived hybridomas. Our results demonstrate that a principal target for ABL-MYC is an antigen-committed B lymphocyte. In addition this procedure provides an alternative method for the production of monoclonal antibodies, without a requirement for hetero-caryon formation by cell fusion techniques.
Amino acid microsequencing of internal tryptic peptides of heme-regulated eukaryotic initiation factor 2 alpha subunit kinase: homology to protein kinases.
Authors: Chen J J , Pal J K , Petryshyn R , Kuo I , Yang J M , Throop M S , Gehrke L , London I M
Published: May 2006

We have purified the heme-regulated eukaryotic initiation factor 2 alpha subunit (eIF-2 alpha) kinase (HRI) from rabbit reticulocytes for amino acid microsequencing. This kinase is a single 92-kDa polypeptide and migrates in perfect alignment with 32P-labeled HRI on SDS/PAGE. Its functions of binding ATP and of autophosphorylation and eIF-2 alpha phosphorylation are inhibited by hemin. The amino acid sequences of three tryptic peptides of HRI have been obtained. A search of the data base of the National Biomedical Research Foundation reveals that these amino acid sequences are unique and that two of these three sequences show homology to protein kinases. HRI peptide P-52 contains Asp-Phe-Gly, which is the most highly conserved short stretch of amino acids in catalytic domain VII of protein kinases. HRI peptide P-74 contains the conserved amino acid residues Asp-(Met)-Tyr-Ser-(Val)-Gly-Val found in catalytic domain IX of protein kinases [Hanks, S. K., Quinn, A. M. & Hunter, T. (1988) Science 241, 42-52]. These findings are consistent with the autokinase and eIF-2 alpha kinase activities of HRI. Synthetic HRI peptide P-74 is a very potent inhibitor of eIF-2 alpha phosphorylation by HRI. Since little is known about the function of conserved domain IX, P-74 peptide may be useful in elucidating the role of this domain of protein kinases.
Engagement of the natural killer cell IgG Fc receptor results in tyrosine phosphorylation of the zeta chain.
Authors: O'Shea J J , Weissman A M , Kennedy I C , Ortaldo J R
Published: May 2006

The zeta chain has emerged to be a key subunit of the T-cell antigen receptor with central roles not only in intracellular assembly of the multimeric receptor but also in mediating signal transduction events. This subunit is present in natural killer (NK) cells that lack the other subunits of the T-cell antigen receptor. In NK cells, the zeta chain appears to be associated with the NK Fc receptor [type 3 receptor for the Fc portion of IgG (Fc gamma RIII or CD16)] and may be necessary for efficient cell surface expression of this receptor complex. In T cells, the zeta chain is a prominent substrate that becomes phosphorylated on tyrosine residues after occupancy of the TCR; zeta chain phosphorylation was in fact the first evidence that the TCR was coupled to a protein-tyrosine kinase as well as to inositol phospholipid hydrolysis. To determine if Fc gamma RIII is coupled to a protein-tyrosine kinase in a manner analogous to the T-cell antigen receptor, we investigated ligand-dependent zeta-chain phosphorylation in NK cells. We observed that activation of NK cells with an anti-Fc gamma RIII monoclonal antibody induced tyrosine phosphorylation of the zeta chain whereas other activating stimuli, such as the combination of phorbol ester and ionomycin or a lymphokine, interleukin 2, did not result in phosphorylation of this protein. Perturbation of Fc gamma RIII by the more physiological stimulus, incubation of NK cells with antibody-coated target cells, also induced zeta-chain phosphorylation. Previous data have indicated that the NK-cell Fc gamma RIII is coupled to inositol phospholipid hydrolysis. This present finding that Fc gamma RIII is coupled to a protein-tyrosine kinase illustrates that there are significant similarities in the signaling pathways activated by Fc gamma RIII in NK cells and the T-cell antigen receptor in T cells; the zeta chain is a common element that may serve as a coupling protein for both of these receptors.
Scrapie-associated prion protein accumulates in astrocytes during scrapie infection.
Authors: Diedrich J F , Bendheim P E , Kim Y S , Carp R I , Haase A T
Published: May 2006

In the course of scrapie, a transmissible spongiform encephalopathy caused by an unconventional agent, a normal cellular protein is converted to an abnormal form that copurifies with infectivity and aggregates to form deposits of amyloid. We have used immunocytochemistry and methods that enhance detection of amyloidogenic proteins to investigate the types of cells in the central nervous system which are involved in the formation of the abnormal scrapie-associated protein. We show that this protein accumulates in astrocytes prior to the cardinal neuropathological changes in scrapie--astrogliosis, vacuolation, neuron loss, and amyloid deposition. These findings implicate the astrocyte in the formation of the scrapie isoform of the prion protein and amyloid in scrapie and suggest that this cell type might also be involved in the replication of the scrapie agent.
Expression of CD4 in transgenic mice alters the specificity of CD8 cells for allogeneic major histocompatibility complex.
Authors: Robey E , Ramsdell F , Elliott J , Raulet D , Kioussis D , Axel R , Fowlkes B J
Published: May 2006

We have generated a transgenic mouse line in which a CD4 transgene is expressed on a significant fraction of the mature CD8+ lymphocytes but is not expressed in the thymus. This provides an opportunity to examine the functional consequences of CD4 expression in a population of class I-selected CD8+ lymphocytes. CD8+ lymphocytes expressing the CD4 transgene proliferate in response to allogeneic class I and class II major histocompatibility complex, whereas CD8+ cells from control animals proliferate only to allogeneic class I gene products. These observations suggest that the ability of a T-cell population to react with class II allogeneic major histocompatibility complex is determined by the presence of CD4.
Immunoglobulins from animal models of motor neuron disease and from human amyotrophic lateral sclerosis patients passively transfer physiological abnormalities to the neuromuscular junction.
Authors: Appel S H , Engelhardt J I , García J , Stefani E
Published: May 2006

Amyotrophic lateral sclerosis (ALS) is a devastating human disease of upper and lower motoneurons of unknown etiology. In support of the potential role of autoimmunity in ALS, two immune-mediated animal models of motoneuron disease have been developed that resemble ALS with respect to the loss of motoneurons, the presence of IgG within motoneurons and at the neuromuscular junction, and with respect to altered physiology of the motor nerve terminal. To provide direct evidence for the primary role of humoral immunity, passive transfer with immunoglobulins from the two animal models and human ALS was carried out. Mice injected with serum or immunoglobulins from the animal disease models and human ALS but not controls demonstrated IgG in motoneurons and at the neuromuscular junction. The mice also demonstrated an increase in miniature end-plate potential (mepp) frequency, with normal amplitude and time course and normal resting membrane potential, indicating an increased resting quantal release of acetylcholine from the nerve terminal. The ability to transfer motoneuron dysfunction with serum immunoglobulins provides evidence for autoimmune mechanisms in the pathogenesis of both the animal models and human ALS.
Alteration from T- to B-cell tropism reduces thymic atrophy and cytocidal effects in thymocytes but not neurovirulence induced by ts1, a mutant of Moloney murine leukemia virus TB.
Authors: Wong P K , Szurek P F , Floyd E , Saha K , Brooks B R
Published: May 2006

The ts1 mutant of Moloney murine leukemia virus TB causes degenerative neurologic and immunologic disease in mice, characterized by development of spongiform encephalomyelopathy resulting in hindlimb paralysis, marked thymic atrophy associated with immunodeficiency, and generalized body wasting. To investigate the pathogenesis of the thymic atrophy caused by ts1, we constructed a chimeric virus, ts1-Cas(NS), in which a major portion of the U3 region of the long terminal repeat of ts1, a T-lymphotropic and neurovirulent murine leukemia virus, was replaced by the corresponding U3 region of Cas-Br-E, a B-lymphotropic and neurovirulent murine leukemia virus. In FVB/N mice, ts1-Cas(NS) induced paralytic and wasting disease with incidence, severity, and latency similar to that induced by ts1, but it failed to cause thymic atrophy as severe as that observed in ts1-infected mice. Furthermore, thymocytes cultured from ts1-Cas(NS)-infected mice died at a much slower rate than those of ts1-infected mice. The U3 substitution in ts1-Cas(NS) specifically diminished the ability of the virus to replicate in the thymus, whereas viral replication in the spinal cord was not significantly affected; thus, neurovirulence was not changed. The correlation of reduced thymic atrophy with decreased thymic viral titers and the decreased ability of ts1-Cas(NS) to cause thymocyte death in mice suggest strongly that the marked thymic atrophy in ts1-infected mice is not an indirect effect occurring secondary to neurodegenerative and wasting disease but is a direct cytopathic effect of high-level viral replication in the thymus.
Equivariant algebraic vector bundles over representations of reductive groups: theory.
Authors: Masuda M , Petrie T
Published: May 2006

Let G be a reductive algebraic group and let B be an affine variety with an algebraic action of G. Everything is defined over the field C of complex numbers. Consider the trivial G-vector bundle B x S = S over B where S is a G-module. From the endomorphism ring R of the G-vector bundle S a construction of G-vector bundles over B is given. The bundles constructed this way have the property that when added to S they are isomorphic to F + S for a fixed G-module F. For such a bundle E an invariant rho(E) is defined that lies in a quotient of R. This invariant allows us to distinguish nonisomorphic G-vector bundles. This is applied to the case where B is a G-module and, in that case, an invariant of the underlying equivariant variety is given too. These constructions and invariants are used to produce families of inequivalent G-vector bundles over G-modules and families of inequivalent G actions on affine spaces for some finite and some connected semisimple groups.
Direct physiological evidence for scene segmentation by temporal coding.
Authors: Engel A K , König P , Singer W
Published: May 2006

Theoretical studies have suggested that scene segmentation may be accomplished by a temporal coding mechanism using synchronization of neuronal responses. Here we report a direct experimental test of this hypothesis. Neuronal responses were recorded simultaneously from two to four sites with overlapping receptive fields in cat visual cortex. Correlation analysis revealed that all cells synchronized their responses irrespective of their orientation preference when they were activated by a single light bar. However, when stimulated with two superimposed light bars of different orientations, the same cells segregated into distinct assemblies according to their orientation preferences. Within each of these assemblies responses were synchronized, but correlation was absent between the two assemblies. These results are compatible with the hypothesis that responses to individual objects in a scene are distinguished by synchrony, whereas responses to different objects show no temporal correlation, thus allowing for the segmentation of superimposed stimuli. We conclude that stimulus-specific synchronization of spatially distributed neuronal responses may provide a physiological mechanism for scene segmentation.
Immunocytochemical localization of the cystic fibrosis gene product CFTR.
Authors: Crawford I , Maloney P C , Zeitlin P L , Guggino W B , Hyde S C , Turley H , Gatter K C , Harris A , Higgins C F
Published: May 2006

Antisera against two peptides, corresponding to different domains of the cystic fibrosis gene product CFTR, have been raised and extensively characterized. Both antisera recognize CFTR as a 165-kDa polypeptide in Western analysis of cells transfected with CFTR cDNA as well as in epithelial cell lines. The cell and tissue distribution of CFTR has been studied by immunocytochemistry. CFTR is abundant in epithelial cells, including those lining sweat ducts, small pancreatic ducts, and intestinal crypts. Unexpectedly, the level of CFTR in lung epithelia is relatively low, while it is abundant in the epithelia of kidney tubules. The protein appears to be restricted to the apical, rather than basolateral, regions of epithelial cells and at least a proportion is associated with the plasma membrane. The cell and tissue distributions of CFTR are consistent with a function for this protein as a chloride channel or as a regulator of channel activity.
Growth in high serum concentrations leads to rapid deadaptation of cells previously adapted to growth in an extremely low concentration of serum.
Authors: Yao A , Huang W , Rubin H
Published: May 2006

A subline of NIH 3T3 cells adapted to multiply in 0.25% calf serum (CS) by frequent passage (every 2-3 days) at low population density in 0.25% CS was deadapted by frequent successive passages of the cells in 10% CS for 3 weeks. The cells adapted to 0.25% CS multiplied with an average doubling time of 16.9 hr in 10% CS, and cells that had always been kept in 10% CS multiplied with an average doubling time of 14.6 hr, so there was weak selection for the latter in the higher serum concentration. When adapted cells were subjected to two passagers in 10% CS prior to assay of growth in 0.25% CS, a 4-day lag period was evident before commencement of exponential growth, and there was a decrease in saturation density. Further delay of growth in 0.25% CS developed as the number of passages of cells in 10% CS increased. The marked delay of growth in 0.25% CS of the bulk population after a few days in 10% CS argued against selection in 10% CS of rare nonadapted mutants from the adapted population and for an epigenetic origin of the change. Reconstruction experiments utilizing adapted cells mixed with non-adapted cells in 0.25% CS buttressed this explanation. Eight clones of the adapted population exhibited some loss of growth capacity in 0.25% CS after a single passage in 10% CS, though the extent of loss varied from clone to clone. The results support the idea that all cells in the adapted population respond to the lifting of growth constraints with loss of their growth potential under highly constrained conditions. They are consistent with the concept of progressive state selection in which selection operates on fluctuating metabolic states of individual cells rather than on genetic variants.
Do interhelical side chain-backbone hydrogen bonds participate in formation of leucine zipper coiled coils?
Authors: Tropsha A , Bowen J P , Brown F K , Kizer J S
Published: May 2006

The leucine zipper proteins are a group of transcriptional regulators that dimerize to form a DNA binding domain. It has been proposed that this dimerization results from the hydrophobic association of the alpha-helices of two leucine zipper monomers into a coiled coil. We propose a model for a coiled coil based on a periodic hydrophobic-hydrophilic amino acid motif found in the leucine zipper regions of 11 transcriptional regulatory proteins. This model predicts the symmetrical formation of secondary hydrogen bonds between the polar side chains of one helix and the peptide carbonyls of the opposite chain, supplementing the interactions between hydrophobic side chains. Physical modeling (CPK) and in vacuo molecular mechanics calculations of the stability of the GCN4 leucine zipper coiled coil configured in accordance with this model demonstrate a greater stability for this conformer than for a conformer configured according to a current hydrophobic model. Molecular dynamics simulations show similar stability of the two models in vacuo but a higher stability of the hydrophobic model in water.
Reduced amounts of cartilage collagen fibrils and growth plate anomalies in transgenic mice harboring a glycine-to-cysteine mutation in the mouse type II procollagen alpha 1-chain gene.
Authors: Garofalo S , Vuorio E , Metsaranta M , Rosati R , Toman D , Vaughan J , Lozano G , Mayne R , Ellard J , Horton W
Published: May 2006

We have generated transgenic mice harboring a glycine-to-cysteine mutation in residue 85 of the triple helical domain of mouse type II collagen. The offspring of different founders displayed a phenotype of severe chondrodysplasia characterized by short limbs and trunk, cranio-facial deformities, and cleft palate. The affected pups died of acute respiratory distress caused by an inability to inflate lungs at birth. Staining of the skeleton showed a severe retardation of growth for practically all bones. Light microscopic examination indicated a decrease in cartilage matrix density, a severe disorganization of growth plate architecture, and the presence of streaks of fibrillar material in the cartilage matrix. Electron microscopic analysis showed a pronounced decrease in the number of typical thin cartilage collagen fibrils, distension of the rough endoplasmic reticulum of chondrocytes, and the presence of abnormally large banded collagen fibril bundles. The level of expression of the mutant type II procollagen alpha 1 chain transgene in cartilage tissues was approximately equal to that of the endogenous gene in two of the strains. We propose that the principal consequence of the mutation is a considerable reduction in density of the typical thin cartilage collagen fibrils and that this phenomenon causes the severe disorganization of the growth plate. We also postulate that the abnormal thick collagen fibrils are probably related to a defect in crosslinking between the collagen molecules. The cartilage anomalies displayed by these transgenic mice are remarkably similar to those of certain human chondrodysplasias.
Further characterization of the receptor-binding region of the thyroid-stimulating hormone alpha subunit: a comprehensive synthetic peptide study of the alpha-subunit 26-46 sequence.
Authors: Leinung M C , Reed D K , McCormick D J , Ryan R J , Morris J C
Published: May 2006

Previously, using a synthetic peptide strategy, we determined that the region of the common glycoprotein hormone alpha subunit between residues 26 and 46 is a site of interaction of the hormone with the thyroid membrane-bound receptor for thyroid-stimulating hormone (TSH). We have undertaken to identify further the specific residues within this 21-amino acid span that are critical in hormone receptor binding. We synthesized three nested sets of peptide, two in which we systematically truncated the amino-terminal region of the sequence and another in which we truncated the carboxyl-terminal region, and we synthesized a fourth nested set in which we systematically substituted alanine for the native residues from the region of highest activity. Each peptide was tested in a TSH radioreceptor assay for its ability to inhibit binding of 125I-labeled bovine TSH to porcine thyroid membranes. Removal either by truncation or alanine substitution, of several specific residues resulted in a significant reduction in the ability of the sequence to interact with receptor; these residues included Cys31, Cys32, Phe33, Arg35, Arg42, Lys44, and Lys45, suggesting that they are crucial for binding activity. Loss of activity also occurred with substitution for Gly30 and Ser34, but the reduction was less pronounced. Amino-terminal truncation of the sequence through Arg35 (leaving the alpha-subunit peptide 36-46) resulted in greater than 98% loss of activity of the sequence. We conclude that two distinct receptor binding regions lie within the alpha-subunit 26-46 sequence. The first lies between residues Gly30 and Arg35 and includes Cys31, Cys32, and Phe33 as important constituents, and the second region lies between residues Arg42 and Lys45 and includes Lys44 as an important residue and Ser43 as a less important component.
Analysis of the energy of the first four excited states of the ground-state rotational bands of the even-even nuclei from 6C8 to 56Ba90 with the model of a single cluster of nucleons revolving about a sphere.
Authors: Pauling L
Published: May 2006

The results of the analysis of the first four energy levels of the ground-state rotational bands of even-even nuclei from 6C8 to 56Ba90 on the basis of the revolving-cluster model are reported. Values of the nucleon number of the revolving cluster are assigned on the basis in part of the shell model and in part of the expectation that the corresponding values of the radius of revolution would change only slightly from one energy level to an adjacent level or from one nucleus to an adjacent nucleus. The values of the radius of revolution are found to change gradually from about 5 to 6 fm for the lighter nuclei to 7 to 8 fm for the heavier nuclei in the sequence studied.
Evolution of compensatory substitutions through G.U intermediate state in Drosophila rRNA.
Authors: Rousset F , Pélandakis M , Solignac M
Published: May 2006

It has often been suggested that the frequently observed Watson-Crick base-pair compensatory substitutions in RNA helical structures occur mainly through a slightly deleterious G.U intermediate state. We have scored base substitutions in a set of 82 related Drosophila species for the D1 and D2 variable domains of the large rRNA subunit. In all locations where a G-C in equilibrium with A-U compensatory base change occurred, a G.U pair has been observed in one or several species. As this dominant process implies two transitions, their rate was far higher in paired regions (92%) than in unpaired regions (47%). The other types of compensation were rarer and no intermediate states were observed. Most of the G.U base pairs observed in a species are not slightly deleterious. The rate of evolution of compensatory substitution is close to that predicted by a simple model of compensatory substitution through slightly deleterious or slightly advantageous G.U pairs, although some exceptions are presented.
Does superoxide underlie the pathogenesis of hypertension?
Authors: Nakazono K , Watanabe N , Matsuno K , Sasaki J , Sato T , Inoue M
Published: May 2006

Although active oxygen species play important roles in the pathogenesis of various diseases, the molecular mechanism for oxygen toxicity in vascular diseases remains to be elucidated. Since endothelium-derived relaxing factor (EDRF) is inactivated by superoxide radicals in vitro, oxidative stress in and around vascular endothelial cells may affect the circulatory status of animals. To study the role of superoxide radicals and related enzymes, such as superoxide dismutase (SOD), in vascular diseases, we have developed a fusion protein (HB-SOD) consisting of human Cu/Zn-type SOD and a C-terminal basic peptide with high affinity for heparan sulfate on endothelial cells. When injected intravenously, HB-SOD bound to vascular endothelial cells, underwent transcellular transport, and localized within vascular walls by a heparin-inhibitable mechanism. The blood pressure of spontaneously hypertensive rats (SHR) but not normal animals was decreased significantly by HB-SOD. Heparin inhibited the depressor effect of HB-SOD. In contrast, native SOD had no effect on blood pressure of either SHR or normal rats. Neither H2O2-inactivated HB-SOD nor the C-terminal heparin-binding peptide showed such a depressor effect, suggesting that the catalytic function of HB-SOD is responsible for its depressor action. To know the source of superoxide radicals, we determined xanthine oxidase activity in the aorta and uric acid levels in the plasma. Although no appreciable difference in xanthine oxidase activity was found between the two animal groups, uric acid levels were significantly higher in SHR than in normal rats. Oxypurinol, a potent inhibitor of xanthine oxidase, also decreased the blood pressure of SHR but not of normal rats. These findings indicate that superoxide radicals in and around vascular endothelial cells play critical roles in the pathogenesis of hypertension of SHR.
Efficient trans cleavage and a common structural motif for the ribozymes of the human hepatitis delta agent.
Authors: Branch A D , Robertson H D
Published: May 2006

Cis-active ribozymes are potential therapeutic agents; however, to be used in this capacity, they must first be converted to trans-active ribozymes, a process facilitated by analysis of their structures. We present evidence that the genomic and antigenomic ribozymes of the human delta hepatitis agent share a structural ("axehead") motif that has conserved sequence elements and a stable hairpin. Guided by the features of the axehead, we divided each of the delta ribozymes into two subdomains, which we synthesized as separate RNA transcripts to give an enzyme and substrate for each ribozyme. Incubation of a substrate subdomain with its matching enzyme resulted in efficient and accurate trans cleavage. This work forms the basis for kinetic studies and for adapting the delta ribozymes for cleavage of selected target RNAs.
In vivo protein-DNA interactions at the beta-globin gene locus.
Authors: Ikuta T , Kan Y W
Published: May 2006

We have investigated in vivo protein-DNA interactions in the beta-globin gene locus by dimethyl sulfate (DMS) footprinting in K562 cells, which express epsilon- and gamma-globin but not beta-globin. In the locus control region, hypersensitive site 2 (HS-2) exhibited footprints in several putative protein binding motifs. HS-3 was not footprinted. The beta promoter was also not footprinted, while extensive footprints were observed in the promoter of the active gamma-globin gene. No footprints were seen in the A gamma and beta 3' enhancers. With several motifs, additional protein interactions and alterations in binding patterns occurred with hemin induction. In HeLa cells, some footprints were observed in some of the motifs in HS-2, compatible with the finding that HS-2 has some enhancer function in HeLa cells, albeit much weaker than its activity in K562 cells. No footprint was seen in B lymphocytes. In vivo footprinting is a useful method for studying relevant protein-DNA interactions in erythroid cells.
Relative differences in the binding free energies of human immunodeficiency virus 1 protease inhibitors: a thermodynamic cycle-perturbation approach.
Authors: Reddy M R , Viswanadhan V N , Weinstein J N
Published: May 2006

Peptidomimetic inhibitors of the human immunodeficiency virus 1 protease show considerable promise for treatment of AIDS. We have, therefore, been seeking computer-assisted drug design methods to aid in the systematic design of such inhibitors from a lead compound. Here we report thermodynamic cycle-perturbation calculations (using molecular dynamics simulations) to compute the relative difference in free energy of binding that results when one entire residue (valine) is deleted from one such inhibitor. In particular, we studied the "alchemic" mutation of the inhibitor Ac-Ser-Leu-Asn-(Phe-Hea-Pro)-Ile-Val-OMe (S1) to Ac-Ser-Leu-Asn-(Phe-Hea-Pro)-Ile-OMe (S2), where Hea is hydroxyethylamine, in two different (R and S) diastereomeric configurations of the hydroxyethylene group. The calculated (averaged for R and S) difference in binding free energy [3.3 +/- 1.1 kcal/mol (mean +/- SD); 1 cal = 4.184 J] is in good agreement with the experimental value of 3.8 +/- 1.3 kcal/mol, obtained from the measured Ki values for an equilibrium mixture of R and S configurations. Precise testing of our predictions will be possible when binding data become available for the two disastereomers separately. The observed binding preference for S1 is explained by the stronger ligand-protein interaction, which dominates an opposing contribution arising from the large desolvation penalty of S1 relative to S2. This calculation suggests that the thermodynamic cycle-perturbation approach can be useful even when a relatively large change in the ligand is simulated and supports the use of the thermodynamic cycle-perturbation algorithm for screening proposed derivatives of a lead inhibitor/drug prior to their synthesis.