bluuebunny/crossref_metadata_2025 huggingface dataset:
Frameshifting is required for production of the transposase encoded by insertion sequence 1.
Authors: Sekine Y , Ohtsubo E
Published: May 2006

Insertion sequence IS1 has two coding frames, insA and insB, which are essential for its transposition. Here, we show that a frameshifting event in the -1 direction from the 3' end region of the insA frame to an open reading frame (B' frame), extending from the 5' end of the insB frame, is involved in production of the InsA-B'-InsB fusion protein that has IS1 transposase activity. The frameshifting event is likely to have occurred at the sequence AAAAAC where the insA frame overlaps the B' frame. Interestingly, this sequence is also present in one of the two sequences identified in retroviruses as frameshift signals for production of transframe polyproteins from the overlapping genes gag-pro or gag-pro-pol.
Sphingosine, an inhibitor of protein kinase C, suppresses the insulin-like effects of growth hormone in rat adipocytes.
Authors: Smal J , De Meyts P
Published: May 2006

Insulin, human growth hormone (hGH), and phorbol 12-myristate 13-acetate all stimulate lipogenesis in rat adipocytes preincubated without hGH for 4 hr. As previous data suggested that protein kinase C plays an important role in the action of insulin and in the insulin-like effects of hGH in rat adipocytes, we tested the effects of sphingosine, a potent inhibitor of protein kinase C, on the lipogenic activity of both hormones. At 50 microM, sphingosine had no effect on basal lipogenesis but completely abolished the action of phorbol 12-myristate 13-acetate and decreased by 65% and 89%, respectively, the effects of hGH and insulin. At higher concentrations (100 microM), sphingosine abolished both basal and hormone-stimulated lipogenesis; this effect was partially reversible after washing the cells. Similar effects of sphingosine on basal and stimulated glucose uptake were seen in parallel, suggesting that sphingosine inhibits lipogenesis at the glucose-uptake step in rat adipocytes. N-Acetylsphingosine and sphingomyelin, two analogs of sphingosine that are inactive on protein kinase C, did not inhibit lipogenesis induced by hGH, insulin, or phorbol 12-myristate 13-acetate. Sphingosine did not inhibit insulin binding to rat adipocytes at concentrations up to 200 microM but decreased hGH binding to its receptors by 44% at 50 microM. These data suggest a direct link between the inhibition of protein kinase C and that of lipogenesis and provide new evidence for the involvement of protein kinase C in the mechanism of action of growth hormone and insulin in rat adipocytes.
A central nervous system defect in biosynthesis of corticotropin-releasing hormone is associated with susceptibility to streptococcal cell wall-induced arthritis in Lewis rats.
Authors: Sternberg E M , Young W S , Bernardini R , Calogero A E , Chrousos G P , Gold P W , Wilder R L
Published: May 2006

We have recently found that susceptibility to streptococcal cell wall (SCW)-induced arthritis in Lewis (LEW/N) rats is due, in part, to defective inflammatory and stress mediator-induced activation of the hypothalamic-pituitary-adrenal (HPA) axis. Conversely, the relative arthritis resistance of histocompatible Fischer (F344/N) rats is related to their intact responses to the same stimuli. Specifically, LEW/N rats, in contrast to F344/N rats, have markedly impaired plasma corticotropin and corticosterone responses to SCW, recombinant human interleukin 1 alpha, the serotonin agonist quipazine, or synthetic rat/human corticotropin-releasing hormone (CRH). To explore the mechanism of this defect, we examined the functional integrity of the hypothalamic CRH neuron in LEW/N rats compared to F344/N rats. LEW/N rats, in contrast to F344/N rats, showed profoundly deficient paraventricular nucleus CRH mRNA levels and hypothalamic CRH content in response to SCW. Compared to F344/N rats, there was no increase in LEW/N hypothalamic CRH content or CRH release from explanted LEW/N hypothalami in organ culture in response to recombinant interleukin 1 alpha. These data provide strong evidence that the defective LEW/N corticotropin and corticosterone responses to inflammatory and other stress mediators, and the LEW/N susceptibility to experimental arthritis, are due in part to a hypothalamic defect in the synthesis and secretion of CRH. The additional finding of deficient expression in LEW/N rats of the hypothalamic enkephalin gene, which is coordinately regulated with the CRH gene in response to stress, suggests that the primary defect is not in the CRH gene but is instead related to its inappropriate regulation.
Export of an N-terminal fragment of Escherichia coli flagellin by a flagellum-specific pathway.
Authors: Kuwajima G , Kawagishi I , Homma M , Asaka J , Kondo E , Macnab R M
Published: May 2006

Flagellin and several other external components of the bacterial flagellum are thought to be exported, not by the general N-terminal signal peptide-dependent pathway, but by a flagellum-specific pathway involving a central channel in the flagellum itself. We have constructed a variety of mutant alleles of the Escherichia coli flagellin gene. Mutant flagellins with large internal deletions or truncations of their C-terminal region could still be exported, even though they could not assemble into filament. The most extreme example was a fragment containing only the N-terminal 183 residues of the 497-residue wild-type flagellin. This result suggests that the N-terminal region of flagellin contains a signal that enables the protein to be recognized and exported by the flagellum-specific pathway.
Functional role of human immunodeficiency virus type 1 vpu.
Authors: Terwilliger E F , Cohen E A , Lu Y C , Sodroski J G , Haseltine W A
Published: May 2006

To investigate the role of vpu in the replication and cytopathicity of human immunodeficiency virus type 1 (HIV-1), infectious proviruses were constructed that were isogenic except for the ability to produce the protein product of vpu. The vpu-encoded protein is shown to decrease the rate of syncytium formation and cell killing in infected CD4+ human T cells, to increase greatly the export of virus particles from infected cells, and to reduce the rate of accumulation of cell-associated viral proteins. The vpu protein complements in trans the defect in a vpu- HIV-1 provirus but does not affect the simian immunodeficiency virus, which lacks vpu. These observations suggest that vpu may contribute to the AIDS epidemic by increasing the transmission efficiency of the virus.
Voltage-gated calcium channels: direct observation of the anomalous mole fraction effect at the single-channel level.
Authors: Friel D D , Tsien R W
Published: May 2006

Voltage-gated Ca channels are very efficient pores: even while exhibiting strong ionic selectivity, they are highly permeant to divalent cations. Studies of the mechanism of selectivity and ion permeation have demonstrated that whole-cell Ca channel current in mixtures of Ca and Ba ions can be smaller than with equimolar concentrations of either ion alone. This anomalous mole fraction effect (AMFE) has provided an important impetus for proposed mechanisms of ion selectivity and permeation that invoke multiple ion binding sites. However, recordings of unitary L-type Ca currents did not demonstrate the AMFE [Marban, E. & Yue, D.T. (1988) Biophys. J. 55, 594a (abstr.)], raising doubts about whether it is an expression of ion permeation through open Ca channels. We have made patch-clamp recordings from single L-type Ca channels in PC-12 pheochromocytoma cells. Our results demonstrate a significant AMFE at the single-channel level but also indicate that the AMFE can only be found under restrictive conditions of permeant ion concentration and membrane potential. While the AMFE is clear at 0 mV when permeant ions are present at 10 mM, it is not evident when the divalent cation concentration is increased to 110 mM or the membrane potential is hyperpolarized to -40 mV. We compared our experimental observations with predictions of a single-file, two-binding-site model of the Ca channel. The model accounts for our experimental results. It predicts an AMFE under conditions that favor ion-ion interactions, as long as the outer binding site is not saturated due to high permeant ion concentration or negative membrane potential.
Drosophila ninaA gene encodes an eye-specific cyclophilin (cyclosporine A binding protein).
Authors: Schneuwly S , Shortridge R D , Larrivee D C , Ono T , Ozaki M , Pak W L
Published: May 2006

Mutations in the ninaA gene of Drosophila severely reduce the amount of rhodopsin specifically in R1-6 photoreceptors. Isolation of the ninaA gene by chromosomal walking revealed that it is expressed only in the eye and encodes a 237-amino acid polypeptide that shows strong sequence similarity to cyclophilin, a putative molecular target for cyclosporine A, a potent immunosuppressant used in human organ transplantations. Unlike most cyclophilins characterized to date, the ninaA-encoded protein has a putative signal sequence and a transmembrane domain. Each of the three ehtyl methanesulfonate-induced ninaA mutant alleles analyzed shows a single nucleotide change in the mRNA coding region leading to either a nonsense or a missense mutation. We find no evidence that the ninaA-encoded protein is directly involved in phototransduction. The only detectable mutant phenotype that correlates with the severity of molecular defects in the three mutants is the amount of depletion of R1-6 rhodopsin. The above results and the recent findings that cyclophilin is a peptidylprolyl cis-trans-isomerase suggest that the ninaA-encoded protein may be required for proper folding and stability of R1-6 rhodopsin.
Adrenalectomy decreases nerve growth factor in young adult rat hippocampus.
Authors: Aloe L
Published: May 2006

The effect of adrenalectomy on the level of nerve growth factor (NGF) in the hippocampus and on the distribution of choline acetyltransferase immunoreactivity in forebrain cholinergic neurons of developing rats was studied. Biological and immunohistochemical determinations indicated that in 40-day-old rats, adrenalectomy reduced the NGF level in the hippocampus and the choline acetyltransferase immunoreactivity in the septal lateral bands. Furthermore, autoradiographic studies showed that adrenalectomy causes changes in the distribution and expression of NGF receptors in the hippocampus. These results suggest that adrenal hormones are involved in the regulation of the NGF level in the hippocampus and of NGF receptors in the septum.
Cloning of the immunological repertoire in Escherichia coli for generation of monoclonal catalytic antibodies: construction of a heavy chain variable region-specific cDNA library.
Authors: Sastry L , Alting-Mees M , Huse W D , Short J M , Sorge J A , Hay B N , Janda K D , Benkovic S J , Lerner R A
Published: May 2006

Efficient generation of catalytic antibodies is uniquely dependent on the exact nature of the binding interactions in the antigen-antibody complex. Current methods for generation of monoclonal antibodies do not efficiently survey the immunological repertoire and, therefore, they limit the number of catalysts that can be obtained. We are exploring methods to clone and express the immunological repertoire in Escherichia coli. As the essential first step, we present here a method for the establishment of a highly diverse heavy chain variable region library. Consequently, it should now be possible to express and recombine the heavy and light chain variable region fragments to generate a large array of functional combining portions of the antibody molecule. This technology may provide an alternative to the hybridoma methodology for accessing the monoclonal antibody specificity of the immune system.
Delineation of three functional domains of the transcriptional activator encoded by the c-myb protooncogene.
Authors: Sakura H , Kanei-Ishii C , Nagase T , Nakagoshi H , Gonda T J , Ishii S
Published: May 2006

The c-myb protooncogene encodes a sequence-specific DNA-binding protein (c-Myb) that induces transcriptional activation or repression. We have identified three functional domains of the mouse c-Myb protein that are responsible for DNA binding, transcriptional activation, and negative regulation, respectively. In addition to the DNA-binding domain, which is located near the N terminus, an adjacent region (the transcriptional activation domain) containing about 80 amino acids was found to be essential for transcriptional activation. Deletion of a region spanning about 175 amino acids of the C-proximal portion increased transcriptional activation markedly, revealing that this domain normally represses activation. Differences between the transcriptional activation and repression functions of c-Myb and v-Myb are discussed in the light of these functional domains. Our results suggest that transcriptional activation may be involved in transformation by myb gene products.
Cellular responses to Pyrularia thionin are mediated by Ca2+ influx and phospholipase A2 activation and are inhibited by thionin tyrosine iodination.
Authors: Evans J , Wang Y D , Shaw K P , Vernon L P
Published: May 2006

Pyrularia thionin, isolated from nuts of Pyrularia pubera, is a strongly basic peptide of 47 amino acids. The amino acid sequence and configuration of its four disulfide bonds place this plant peptide, known to be hemolytic, cytotoxic, and neurotoxic, among the thionins. We report and compare several cellular responses mediated by Pyrularia thionin: hemolysis of human erythrocytes, activation of an endogenous phospholipase A2 in Swiss 3T3 cells, cytotoxicity toward HeLa and mouse B16 melanoma cells in culture, viability of rat hepatocytes and lymphocytes measured by trypan blue exclusion, and lethality in mice. Cellular responses related to ion movement include a toxin-mediated influx of Ca2+ into mouse P388 cells measured by Fura-2 fluorescence, depolarization of mouse P388 plasma membrane measured by fluorescence of bis(1,3-diethylthiobarbituric acid)trimethine oxonol (bisoxonol), and depolarization of frog (Rana pipiens) sartorius muscle determined by direct measurement of membrane potential. Graded iodination of Pyrularia thionin leads to a related loss of activity for hemolysis, phospholipase A2 activation, cytotoxicity, and lethality in mice. The mediated Ca2+ influx into and depolarization of P388 cells require Ca2+ in the external medium and are inhibited by 100 microM Ni2+. Depolarization of sartorius muscle by Pyrularia thionin also requires a functional Ca2+ channel, as shown by verapamil inhibition. This muscle depolarization also involves phospholipase A2 activation because dexamethasone and quinacrin, but not indomethacin, protect against depolarization. The IC50 values for viability of rat hepatocytes and splenic lymphocytes measured by trypan blue exclusion were 0.17 and 40 microM, respectively. The general response of cells to Pyrularia thionin involves a membrane alteration leading to depolarization and a channel-mediated influx of Ca2+. There is a related activation of phospholipase A2 that results in loss of membrane integrity, hemolysis in the case of erythrocytes, and eventually cell death. Iodination of Pyrularia thionin leads to a corresponding inhibition of all three cellular responses, which indicates an essential role for tyrosine in either maintenance of peptide structure or interaction of the peptide with cellular membranes.
Highly potent analogues of luteinizing hormone-releasing hormone containing D-phenylalanine nitrogen mustard in position 6.
Authors: Bajusz S , Janaky T , Csernus V J , Bokser L , Fekete M , Srkalovic G , Redding T W , Schally A V
Published: May 2006

The nitrogen mustard derivatives of 4-phenylbutyric acid and L-phenylalanine, called chlorambucil (Chl) and melphalan (Mel), respectively, have been incorporated into several peptide hormones, including luteinizing hormone-releasing hormone (LH-RH). The alkylating analogues of LH-RH were prepared by linking Chl, as an N-acyl moiety, to the complete amino acid sequence of agonistic and antagonistic analogues. These compounds, in particular the antagonistic analogues, showed much lower potency than their congeners carrying other acyl groups. To obtain highly potent alkylating analogues of LH-RH, the D enantiomer of Mel was incorporated into position 6 of the native hormone and some of its antagonistic analogues. Of the peptides prepared, [D-Mel6]LH-RH (SB-05) and [Ac-D-Nal(2)1,D-Phe(pCl)2,D-Pal(3)3,Arg5,D-Mel6,D-Ala10++ +]LH-RH [SB-86, where Nal(2) is 3-(2-naphthyl)alanine and Pal(3) is 3-(3-pyridyl)alanine] possessed the expected high agonistic and antagonistic activities, respectively, and also showed high affinities for the membrane receptors of rat pituitary cells, human breast cancer cells, human prostate cancer cells, and rat Dunning R-3327 prostate tumor cells. These two analogues exerted cytotoxic effects on human and rat mammary cancer cells in vitro. Thus these two D-Mel6 analogues seem to be particularly suitable for the study of how alkylating analogues of LH-RH could interfere with intracellular events in certain cancer cells.
Direct transfer of NADH between alpha-glycerol phosphate dehydrogenase and lactate dehydrogenase: fact or misinterpretation?
Authors: Srivastava D K , Smolen P , Betts G F , Fukushima T , Spivey H O , Bernhard S A
Published: May 2006

Following the criticism by Chock and Gutfreund [Chock, P.B. & Gutfreund, H. (1988) Proc. Natl. Acad. Sci. USA 85, 8870-8874], that our proposal of direct transfer of NADH between glycerol-3-phosphate dehydrogenase (alpha-glycerol phosphate dehydrogenase, alpha-GDH; EC 1.1.1.8) and L-lactate dehydrogenase (LDH; EC 1.1.1.27) was based on a misinterpretation of the kinetic data, we have reinvestigated the transfer mechanism between this enzyme pair. By using the "enzyme buffering" steady-state kinetic technique [Srivastava, D.K. & Bernhard, S.A. (1984) Biochemistry 23, 4538-4545], we examined the mechanism (random diffusion vs. direct transfer) of transfer of NADH between rabbit muscle alpha-GDH and pig heart LDH. The steady-state data reveal that the LDH-NADH complex and the alpha-GDH-NADH complex can serve as substrate for the alpha-GDH-catalyzed reaction and the LDH-catalyzed reaction, respectively. This is consistent with the direct-transfer mechanism and inconsistent with a mechanism in which free NADH is the only competent substrate for either enzyme-catalyzed reaction. The discrepancy between this conclusion and that of Chock and Gutfreund comes from (i) their incorrect measurement of the Km for NADH in the alpha-GDH-catalyzed reaction, (ii) inadequate design and range of the steady-state kinetic experiments, and (iii) their qualitative assessment of the prediction of the direct-transfer mechanism. Our transient kinetic measurements for the transfer of NADH from alpha-GDH to LDH and from LDH to alpha-GDH show that both are slower than predicted on the basis of free equilibration of NADH through the aqueous environment. The decrease in the rate of equilibration of NADH between alpha-GDH and LDH provides no support for the random-diffusion mechanism; rather, it suggests a direct interaction between enzymes that modulates the transfer rate of NADH. Thus, contrary to Chock and Gutfreund's conclusion, all our experimental data compel us to propose, once again, that NADH is transferred directly between the sites of alpha-GDH and LDH.
Genome organization of the anaerobic pathogen Clostridium perfringens.
Authors: Canard B , Cole S T
Published: May 2006

A physical map of the genome of Clostridium perfringens, an important human pathogen, has been established by pulsed-field gel electrophoresis. Recognition sites for six rare-cutting endonucleases were situated on a single circular chromosome of approximately 3.6 million base pairs thus defining 50 arbitrary genetic intervals of between 10 and 250 kilobase pairs. This considerably facilitated the chromosomal localization of some 24 genes and loci for which probes were available and allowed the construction of the genome map of a clostridial species.
The octamer motif is a B-lymphocyte-specific regulatory element of the HLA-DR alpha gene promoter.
Authors: Sherman P A , Basta P V , Heguy A , Wloch M K , Roeder R G , Ting J P
Published: May 2006

The human class II gene, HLA-DR alpha, contains an octanucleotide sequence ATTTGCAT located approximately 40 base pairs upstream of the transcription initiation site. We have investigated the transcriptional function of the DR alpha octamer in human B-lymphoblastoid cells and non-B cells. Deletion and substitution mutagenesis of the octamer sequence greatly reduced the activity of the DR alpha promoter in both in vivo and in vitro cell-free transcription systems of B-cell origin. Conversely, these mutations did not affect promoter activity in several non-B-cell lines that express the DR alpha gene. Removal of octamer-binding proteins by in vivo titration with an octamer-containing competitor plasmid reduced DR alpha promoter activity in B-lymphoblastoid cells. These results suggest that a protein-octamer interaction, most likely involving the B-cell-specific octamer binding protein (OTF-2), is required for DR alpha promoter function in B-lymphoblastoid cells but not in non-B cells.
Immunogenicity and efficacy testing in chimpanzees of an oral hepatitis B vaccine based on live recombinant adenovirus.
Authors: Lubeck M D , Davis A R , Chengalvala M , Natuk R J , Morin J E , Molnar-Kimber K , Mason B B , Bhat B M , Mizutani S , Hung P P
Published: May 2006

As a major cause of acute and chronic liver disease as well as hepatocellular carcinoma, hepatitis B virus (HBV) continues to pose significant health problems world-wide. Recombinant hepatitis B vaccines based on adenovirus vectors have been developed to address global needs for effective control of hepatitis B infection. Although considerable progress has been made in the construction of recombinant adenoviruses that express large amounts of HBV gene products, preclinical immunogenicity and efficacy testing of candidate vaccines has remained difficult due to the lack of a suitable animal model. We demonstrate here that chimpanzees are susceptible to enteric infection by human adenoviruses type 7 (Ad7) and type 4 (Ad4) following oral administration of live virus. Moreover, after sequential oral immunization with Ad7- and Ad4-vectored vaccines containing the hepatitis B surface antigen (HBsAg) gene, significant antibody responses to HBsAg (anti-HBs) were induced in two chimpanzees. After challenge with heterologous HBV, one chimpanzee was protected from acute hepatitis and the other chimpanzee experienced modified HBV-induced disease. These data demonstrate the feasibility of using orally administered recombinant adenoviruses as a general approach to vaccination.
Significance of abnormal serum binding of insulin-like growth factor II in the development of hypoglycemia in patients with non-islet-cell tumors.
Authors: Daughaday W H , Kapadia M
Published: May 2006

We reported that serum and tumor from a hypoglycemic patient with a fibrosarcoma contained insulin-like growth factor II (IGF-II), mostly in a large molecular form designated "big IGF-II." We now describe two additional patients with non-islet-cell tumor with hypoglycemia (NICTH) whose sera contained big IGF-II. Removal of the tumor eliminated most of the big IGF-II from the sera of two patients. Because specific IGF-binding proteins modify the bioactivity of IGFs, the sizes of the endogenous IGF-binding protein complexes were determined after neutral gel filtration through Saphadex G-200. Normally about 75% of IGFs are carried as a ternary complex of 150 kDa consisting of IGF, a growth hormone (GH)-dependent IGF-binding protein, and an acid-labile complexing component. The three patients with NICTH completely lacked the 150-kDa complex. IGF-II was present as a 60-kDa complex with variable contributions of smaller complexes. In the immediate postoperative period, a 110-kDa complex appeared rather than the expected 150-kDa complex. Abnormal IGF-II binding may be important in NICTH because the 150-kDa complexes cross the capillary membrane poorly. The smaller complexes present in our patients' sera would be expected to enter interstitial fluid readily, and a 4- to 5-fold increase in the fraction of IGFs reaching the target cells would result.
5-HT1c receptor is a prominent serotonin receptor subtype in the central nervous system.
Authors: Molineaux S M , Jessell T M , Axel R , Julius D
Published: May 2006

Neurons in rat central nervous system (CNS) that express 5-HT1c receptor mRNA have been localized by in situ hybridization histochemistry. The 5-HT1c receptor is expressed in a wide variety of cortical and subcortical neurons including hippocampal pyramidal neurons, neurons within most of the central monoaminergic cell groups, neurons in thalamic sensory relay nuclei, and neurons involved in the central processing and regulation of nociceptive transmission. Therefore, the 5-HT1c receptor is a prominent but poorly characterized central subclass of serotonin (5-HT) receptor. The distribution of the 5-HT1c receptor within the CNS is considerably more widespread than that of the structurally and functionally related 5-HT2 receptor.
Voltage-activated K+ conductances in freshly isolated embryonic chicken osteoclasts.
Authors: Ravesloot J H , Ypey D L , Vrijheid-Lammers T , Nijweide P J
Published: May 2006

Patch-clamp measurements on freshly isolated embryonic chicken osteoclasts revealed three distinct types of voltage-dependent K+ conductance. The first type of conductance, present in 72% of the cells, activated at membrane potentials less negative than -30 to -20 mV and reached full activation at +40 mV. It activated with a delay, reached a peak value, and then inactivated with a time constant of approximately 1.5 s. Inactivation was complete or almost so. Recovery from inactivation, at -70 mV, had a time constant of roughly 1 s. The conductance could be blocked, at least partly, by 4 mM 4-aminopyridine. The second type of conductance (present in all cells) activated at membrane potentials more negative than -40 to -80 mV and reached full activation at -130 mV. Activation potential and maximal conductance were dependent on the extracellular K+ concentration. Inactivation of the conductance first became apparent at membrane potentials more negative than -100 mV and was a two-exponential process. The conductance could be blocked by external 5 mM Cs+ ions. The third type of conductance (present in all cells) activated at membrane potentials more positive than +30 mV. Generally, the conductance did not inactivate.
Expression of major histocompatibility complex class I antigens in rat muscle cultures: the possible developmental role in myogenesis.
Authors: Honda H , Rostami A
Published: May 2006

The expression of major histocompatibility complex class I antigens was demonstrated on aneurally cultured rat muscle cells. Myoblasts showed constitutive expression of class I antigens on their cell surfaces. The presence of the antigens was transitory, disappearing as myoblasts fused and differentiated into multinucleate myotubes. Furthermore, antibody against rat class I antigens showed an inhibitory effect on the generation of myotubes during muscle development. Although mature myotubes did not show any detectable levels of class I antigens on their cell surface, soluble factors from concanavalin A-activated spleen cells or interferon gamma could induce the expression of class I antigens on muscle fibers. These results suggest that the expression of class I antigens on muscle cells is not only immunologically modulated but also developmentally regulated and that the antigens may play a role in cell recognition and interactions during the fusion process of myogenesis.
Interferon gamma-resistant mutants are defective in the induction of indoleamine 2,3-dioxygenase.
Authors: Feng G S , Taylor M W
Published: May 2006

Several mutants of the human cell line ME-180 resistant to the cytotoxic effect of interferon gamma (IFN-gamma) were isolated after mutagenesis with nitrosoguanidine. Two of the mutant lines (ME-IR3b and ME-IR6g) examined had significantly lower induction levels of the L-tryptophan (L-Trp) degradative enzyme indoleamine 2,3-dioxygenase activity in response to IFN-gamma. Moreover, culture medium supplemented with low concentrations of L-Trp reversed the cytotoxic effect of IFN-gamma, whereas higher concentrations of L-Trp in the medium were extremely toxic to both parental and mutant cells. These mutants were still protected against herpes simplex virus infection by IFN-gamma and expressed the HLA-DR alpha gene normally in the presence of this lymphokine. Thus, the mutation in these cells is specific to indoleamine 2,3-dioxygenase and not a global effect of an IFN-gamma-induced gene. This genetic evidence indicates that the major pathway of IFN-gamma cytotoxicity in this cell line is mediated primarily by induction of indoleamine 2,3-dioxygenase and deprivation of L-Trp.
Transcription of thrombomodulin mRNA in mouse hemangioma cells is increased by cycloheximide and thrombin.
Authors: Dittman W A , Kumada T , Majerus P W
Published: May 2006

We have measured mRNA levels for thrombomodulin, an endothelial membrane cofactor for the activation of protein C by thrombin, in a mouse hemangioma cell line. Cycloheximide, an inhibitor of protein synthesis, increased levels of thrombomodulin mRNA, as measured in an S1 nuclease protection assay, to 2.5-4.0 times control levels. Thrombomodulin transcription in response to cycloheximide treatment, as determined by nuclear run-on analysis, was 3.9 +/- 1.3 (mean +/- SD) times that found in untreated cells. Thrombin also increased thrombomodulin mRNA levels to 151 +/- 21% (mean +/- SD) of control levels after 2 hr. Transcription increased in response to thrombin by 2.1- to 7.3-fold. The combination of thrombin and cycloheximide had no additive effect on thrombomodulin mRNA levels. Thrombin treatment of hemangioma cells also caused an increase in thrombomodulin protein synthesis to 142 +/- 17% (mean +/- SD) of control levels as determined by immunoprecipitation of [32S]methionine-labeled thrombomodulin. We conclude that thrombomodulin expression is determined in part by the rate of transcription and that thrombomodulin mRNA levels in hemangioma cells are increased by treatment with cycloheximide or thrombin. The increased transcription in response to cycloheximide suggests the existence of a labile protein repressor of thrombomodulin transcription.
Changes in brain gene expression shared by scrapie and Alzheimer disease.
Authors: Duguid J R , Bohmont C W , Liu N G , Tourtellotte W W
Published: May 2006

We have isolated two recombinant cDNAs whose corresponding RNAs have an increased abundance in scrapie-infected hamster brain. DNA sequence analysis has shown that these two recombinants represent the genes for sulfated glycoprotein 2 and transferrin. The abundance of sulfated glycoprotein 2 RNA is increased in hippocampus from patients with Alzheimer disease and Pick disease, whereas transferrin RNA is not strongly modulated in these conditions. Expression of two previously identified scrapie-modulated genes, encoding glial fibrillary acidic protein and metallothionein, is also increased in both of these neurodegenerative diseases.
Isolation and characterization of a vascular endothelial cell mitogen produced by pituitary-derived folliculo stellate cells.
Authors: Gospodarowicz D , Abraham J A , Schilling J
Published: May 2006

A growth factor with specificity for vascular endothelial cells has been identified in conditioned medium of pituitary-derived folliculo stellate cells. This factor, named folliculo stellate-derived growth factor (FSdGF), was purified to homogeneity by a combination of heparin-Sepharose affinity chromatography, Bio-Gel P-60 exclusion chromatography, Mono S ion-exchange chromatography, and hydrophobic chromatography on a C4 reverse-phase HPLC column. FSdGF was characterized as a homodimer composed of two subunits with a molecular mass of 23 kDa. FSdGF was a potent mitogen for vascular endothelial cells with activity detectable at 25 pg/ml and saturation at 500 pg/ml. It did not stimulate the proliferation of other cell types such as bovine vascular smooth muscle cells, corneal endothelial cells, adrenal cortex cells, granulosa cells, BALB/MK cells, or BHK-21 cells. Microsequencing revealed an N-terminal sequence having no significant homology to any known protein. The release of FSdGF by pituitary cells and its target cell specificity raise the possibility that FSdGF may play a role in angiogenesis.
Diversity of the G-protein family: sequences from five additional alpha subunits in the mouse.
Authors: Strathmann M , Wilkie T M , Simon M I
Published: May 2006

Biochemical analysis has revealed a number of guanine nucleotide-binding regulatory proteins (G proteins) that mediate signal transduction in mammalian systems. Characterization of their cDNAs uncovered a family of proteins with regions of highly conserved amino acid sequence. To examine the extent of diversity of the G protein family, we used the polymerase chain reaction to detect additional gene products in mouse brain and spermatid RNA that share these conserved regions. Sequences corresponding to six of the eight known G protein alpha subunits were obtained. In addition, we found sequences corresponding to five newly discovered alpha subunits. Our results suggest that the complexity of the G protein family is much greater than previously suspected.
Primary structure of prion protein may modify scrapie isolate properties.
Authors: Carlson G A , Westaway D , DeArmond S J , Peterson-Torchia M , Prusiner S B
Published: May 2006

Scrapie is an infectious neurodegenerative disease caused by unusual pathogens called prions, in which no scrapie-specific nucleic acid has been detected to date. The only known component of the prion is the scrapie isoform of prion protein (PrPSc), which is encoded by a host gene (Prn-p). Isolates of scrapie agent were prepared by passage of infectivity through inbred strains of mice that differ in scrapie incubation time and produce PrPSc molecules differing by two amino acids. Both the length and variability of the incubation period were increased by inocula containing allogeneic PrPSc. For example, Prn-pb I/Ln mice inoculated with scrapie isolate passaged through Prn-pa NZW mice had incubation times of 283 +/- 21 days compared with a 193 +/- 6 day incubation time seen with isolate passaged once through isologous I/Ln mice. No further shortening of incubation time was observed following further isologous passage. NZW incubation times were prolonged by inoculation with prions from I/Ln mice. Results from (NZW x I/Ln)F2 mice and from using inocula from donors isologous for Prn-p but otherwise allogeneic with respect to the recipient suggest that the primary structure of PrPSc is responsible for these incubation time results. Incubation times in (NZW x I/Ln)F1 mice were constant regardless of the passage histories of the scrapie isolates and were equivalent to those of I/Ln mice inoculated with I/Ln prions, contending that prolongation of scrapie incubation time by the prion incubation time gene Prn-i is fully dominant. I/Ln incubation times longer than those in F1 hybrids may reflect a reduced efficiency of allogeneic PrPSc in initiating disease. Although some investigators propose that differences in behavior among scrapie isolates reflect host selection and argue for a nucleic acid genome, we suggest that the variation observed among our scrapie isolates is epigenetic, reflecting host-directed differences in the amino acid sequence of PrPSc.
Genetic analysis of the imperfect association of H-2 haplotype with lupus-like autoimmune disease.
Authors: Babcock S K , Appel V B , Schiff M , Palmer E , Kotzin B L
Published: May 2006

Unlike parental New Zealand Black (NZB) or New Zealand White (NZW) mice, (NZB x NZW)F1 mice exhibit a lupus-like disease characterized by high serum levels of IgG anti-nuclear antibodies and a fatal immune complex glomerulonephritis. Previous results from studying [(NZB x NZW)F1 x NZB] backcross mice indicated that the NZW major histocompatibility complex (MHC) or gene(s) closely linked to this locus provides the major dominant NZW genetic contribution to the F1 disease. A surprising feature of the results was the 12% frequency of discordance between the autoimmune phenotype and the presence of the NZW H-2z haplotype. In the current study, we attempted to precisely define the position of the NZW gene(s) required for lupus-like renal disease by mapping genes in individual backcross mice that are both centromeric and telomeric to the MHC and then correlating genotypes for each locus with disease. The data indicate that an adjacent NZW locus does not provide a more accurate correlation with the autoimmune phenotype compared with MHC genes themselves. Thus, the imperfect association of MHC haplotype with disease in this murine model is not explained by genetic recombination with linked genes. These data may provide insight into the mechanisms by which MHC antigens increase the probability of developing autoimmune disease and may help explain the difficulty of defining MHC relationships in human systemic lupus erythematosus.
Monoclonal antibodies to the apical chloride channel in Necturus gallbladder inhibit the chloride conductance.
Authors: Finn A L , Tsai L M , Falk R J
Published: May 2006

Monoclonal antibodies raised by injecting Necturus gallbladder cells into mice were tested for their ability to inhibit the apical chloride conductance induced by elevation of cellular cAMP. Five of these monoclonal antibodies bound to the apical cells, as shown by indirect immunofluorescence microscopy, and inhibited the chloride conductance; one antibody that bound only to subepithelial smooth muscle, by indirect immunofluorescence microscopy, showed no inhibition of chloride transport. The channel or a closely related molecule is present in the membrane whether or not the pathway is open, since, in addition to inhibiting the conductance of the open channel, the antibody also bound to the membrane in the resting state and prevented subsequent opening of the channel. The antibody was shown to recognize, by ELISA, epitopes from the Necturus gallbladder and small intestine. Finally, by Western blot analysis of Necturus gallbladder homogenates, the antibody was shown to recognize two protein bands of Mr 219,000 and Mr 69,000. This antibody should permit isolation and characterization of this important ion channel.
Molecular analysis of a t(14;14) translocation in leukemic T-cells of an ataxia telangiectasia patient.
Authors: Russo G , Isobe M , Gatti R , Finan J , Batuman O , Huebner K , Nowell P C , Croce C M
Published: May 2006

We have detected and cloned two rearrangements in the T-cell receptor alpha locus from a clone of somatic cell hybrids carrying a t(14;14)(q11;q32) chromosomal translocation derived from an ataxia telangiectasia patient with T-cell chronic lymphocytic leukemia. The T-cell clone carrying the t(14;14) chromosomal translocation was known to be present for greater than 10 years before the onset of overt leukemia. One molecular rearrangement of the T-cell receptor alpha locus corresponded to a functional variable-joining region (V-J) joining, whereas the other derived from the breakpoint of the t(14;14)(q11;q32) translocation. Chromosomal in situ hybridization of the probe derived from the t(14;14) breakpoint localized the breakpoint region to 14q32.1, apparently the same region that is involved in another ataxia telangiectasia characteristic chromosome translocation, t(7;14)(q35;q32). The 14q32.1 breakpoint is at least 10,000 kilobase pairs (kbp) centromeric to the immunoglobulin heavy chain locus. Sequence analysis of the breakpoint indicates the involvement of a J alpha sequence during the translocation. Comigration of high-molecular weight DNA fragments involved with t(7;14) and t(14;14) translocations suggests the presence of a cluster of breakpoints in the 14q32.1 region, the site of a putative oncogene, TCL1.
Mononuclear phagocytes of blood and bone marrow: comparative roles as viral reservoirs in human immunodeficiency virus type 1 infections.
Authors: McElrath M J , Pruett J E , Cohn Z A
Published: May 2006

We examined human immunodeficiency virus (HIV) production in cultured mononuclear cells from 36 seropositive homosexual males. Production was detected by using an HIV p24 antigen ELISA assay in blood mononuclear cells in 54% of asymptomatic, 71% of acquired immunodeficiency syndrome (AIDS)-related complex, and 100% of AIDS patients. When the peripheral blood mononuclear cells were separated into monocytes and CD4+ T cells, we found that the CD4+ T-cell fraction was preferentially infected in the three clinical stages. The ability to isolate HIV from blood monocyte-derived macrophages was similar in the three stages (24-33%) and required coculture with phytohemagglutinin-stimulated lymphoblasts. Bone marrow and blood mononuclear cells cultured simultaneously yielded virus from both sources in 13 individuals. Again the prime source of virus was the nonadherent bone marrow mononuclear cells, which contained CD4+ T cells, and not the adherent monocyte-enriched fraction. We conclude that blood mononuclear cells yield productive virus more readily as disease progresses and that infection is detected in association with CD4+ T-cell-enriched fractions. In our large sample of patients, monocyte infection was detected in only a small fraction, suggesting that this cell type is neither a primary nor an exclusive reservoir of HIV infection.
Improved predictions of secondary structures for RNA.
Authors: Jaeger J A , Turner D H , Zuker M
Published: May 2006

The accuracy of computer predictions of RNA secondary structure from sequence data and free energy parameters has been increased to roughly 70%. Performance is judged by comparison with structures known from phylogenetic analysis. The algorithm also generates suboptimal structures. On average, the best structure within 10% of the lowest free energy contains roughly 90% of phylogenetically known helixes. The algorithm does not include tertiary interactions or pseudoknots and employs a crude model for single-stranded regions. The only favorable interactions are base pairing and stacking of terminal unpaired nucleotides at the ends of helixes. The excellent performance is consistent with these interactions being the primary interactions determining RNA secondary structure.
Differential regulation of two distinct glucose transporter species expressed in 3T3-L1 adipocytes: effect of chronic insulin and tolbutamide treatment.
Authors: Tordjman K M , Leingang K A , James D E , Mueckler M M
Published: May 2006

The HepG2-type glucose transporter (HepG2-GT) is expressed in 3T3-L1 fibroblasts and adipocytes. In contrast, the acutely insulin-regulatable glucose transporter (IRGT) is expressed only in the adipocytes. In the present study, the expression of the IRGT was shown to increase in parallel with the acquisition of acutely insulin-stimulated glucose uptake during differentiation of these cells, whereas the level of the HepG2-GT decreased during the course of differentiation in parallel with a decline in basal glucose uptake. We examined the effects of chronic insulin and tolbutamide treatment on glucose transporter activity in conjunction with the expression of these two glucose transporter species in 3T3-L1 adipocytes. Treatment of adipocytes with insulin, tolbutamide, or both agents in combination increased 2-deoxyglucose uptake, HepG2-GT protein, and HepG2-GT mRNA levels in parallel. The effect of combined insulin/tolbutamide administration on these three parameters was greater than the effect of either treatment alone. In contrast, these treatments either had no significant effect or decreased levels of IRGT protein and mRNA. We conclude that chronic treatment of 3T3-L1 adipocytes with insulin or tolbutamide increases glucose uptake primarily by means of a selective increase in the expression of the HepG2-GT. We suggest that part of the in vivo hypoglycemic effect of insulin and sulfonylureas may involve an increased expression of the HepG2-GT.
Human carboxyl-terminal variant of alpha-type c-erbA inhibits trans-activation by thyroid hormone receptors without binding thyroid hormone.
Authors: Lazar M A , Hodin R A , Chin W W
Published: May 2006

Multiple thyroid hormone (T3; L-3,3',5-triiodothyronine) receptors related to the viral oncogene v-erbA have been identified in mammalian tissues and have been shown to mediate certain actions of T3. The role of a carboxyl-terminal variant of alpha-type c-erbA (c-erbA alpha-2) is controversial, since the human form has been reported to be a T3 receptor, while the rat form has been shown to not bind T3. In fact, the rat homolog of c-erbA alpha-2 has been reported to be an inhibitor of T3 action. We have compared the properties of human c-erbA alpha-2 with those of its rat homolog and with other forms of c-erbA. Neither form of c-erbA alpha-2 binds T3 with high affinity whether synthesized in reticulocyte lysates or by transient expression in mammalian cells. Also, neither form increases the expression of a T3-responsive gene. However, human c-erbA alpha-2 inhibits T3 action mediated by a T3-receptor form of c-erbA to an extent similar to that of rat c-erbA alpha-2. Our data strongly suggest that human c-erbA alpha-2 has a biological function similar to that of its rat homolog. Thus, the modulation of T3 action by an endogenous inhibitor related to T3 receptors is likely a general regulatory mechanism.
On the ion selectivity in Ca-binding proteins: the cyclo(-L-Pro-Gly-)3 peptide as a model.
Authors: Sussman F , Weinstein H
Published: May 2006

Calcium plays a crucial role in many cellular processes. Its functions are directly dependent on the high specificity for Ca2+ exhibited by the proteins and ion carriers that bind divalent ions. To elucidate the basis for this specificity we have calculated the relative energies of solvation of calcium and magnesium ions in complexes with cyclo(-L-Pro-Gly-)3, a small synthetic peptide that binds Ca2+ with an affinity comparable to those of the naturally occurring proteins. The results show that the ion selectivity of the peptide resides in the difference in the solvation energies of the competing ions in water. Although the peptide is able to complex Mg2+ better than Ca2+ in the stoichiometries in which cyclo(-L-Pro-Gly-)3 binds divalent ions, it is not always able to provide as much stabilization for Mg2+ as water does. These results also explain why cyclo(-L-Pro-Gly-)3 binds Ca2+ and Mg2+ with different stoichiometries and indicate the source for expected differences in the structures of complexes of the two ions.
Vertebrate and yeast calmodulin, despite significant sequence divergence, are functionally interchangeable.
Authors: Davis T N , Thorner J
Published: May 2006

Yeast strains relying solely on vertebrate (Xenopus laevis) calmodulin, expressed under control of a yeast (GAL1) promoter, grew at the same rate as yeast cells containing their endogenous calmodulin. Therefore, the ability to perform essential functions has been conserved between yeast and vertebrate calmodulins, suggesting that calmodulin performs the same (or overlapping) roles in yeast as it does in higher eukaryotes. Successful substitution of vertebrate for yeast calmodulin also suggests that the two proteins can adopt similar conformations in vivo, despite the large number of amino acid differences between them (60 out of 148 residues). Strains overproducing either vertebrate or yeast calmodulin about 100-fold and a strain producing a normal level of yeast calmodulin were essentially indistinguishable in many characteristics, including microtubule distribution, rate of secretion, response to mating pheromone, sporulation, and adaptation to nutrient limitation. Calmodulin overproduction did not confer elevated resistance to a phenothiazine drug, trifluoperazine, thought to be a calmodulin-specific inhibitor. These results have important implications for understanding the role of calmodulin in intracellular calcium signaling.