bluuebunny/crossref_metadata_2025 huggingface dataset:
Mapping the determinants of human immunodeficiency virus 2 for infectivity, replication efficiency, and cytopathicity.
Authors: Talbott R , Kraus G , Looney D , Wong-Staal F
Published: May 2006

Human immunodeficiency virus 2 (HIV-2) ISY and the newly derived HIV-2KR are infectious molecular clones that yield viruses differing markedly in their abilities to infect and/or induce syncytia in various T- and monocytoid-cell lines. Chimeric viruses were constructed from these two viral genomes to localize the genetic determinants of some of these properties. Envelope sequences, particularly those spanning the CD4 binding site, appear to be critical for the ability of HIV-2KR to infect MOLT-4 clone 8 and SupT1 cells and to efficiently infect the H9 cell line. On the other hand, multiple determinants may contribute to cytopathicity (gp41 and nef) in H9 cells and replication efficiency in monocytic (THP-1) cells.
Characterization of a high-affinity galanin receptor in the rat anterior pituitary: absence of biological effect and reduced membrane binding of the antagonist M15 differentiate it from the brain/gut receptor.
Authors: Wynick D , Smith D M , Ghatei M , Akinsanya K , Bhogal R , Purkiss P , Byfield P , Yanaihara N , Bloom S R
Published: May 2006

Structure-activity studies demonstrate that galanin fragments 1-15 and 2-29 are fully active, whereas fragment 3-29 has been reported to be inactive, in a number of different in vivo models. M15, a chimeric peptide comprising galanin 1-13 and substance P5-11, has recently been found to be a potent galanin antagonist. Direct effects of galanin at the level of the pituitary have been defined, yet, paradoxically, a number of studies have been unable to demonstrate galanin binding to an anterior pituitary receptor. Porcine galanin stimulated prolactin release from dispersed rat anterior pituitary cells up to 180% +/- 12% (mean +/- SEM) of control secretion. The addition of a specific galanin antiserum caused a profound inhibition of basal prolactin release, maximal inhibition being 12% +/- 0.5% of control secretion. Addition of M15 produced no effect on basal or galanin-stimulated prolactin release. Galanin fragment 3-29 was fully active when compared to galanin 1-29. Fragments 5-29 and 8-29 stimulated prolactin release to a lesser extent and galanin 1-15, 10-29, and 20-29 had no significant prolactin-releasing activity. Using [mono(125I)iodo-Tyr26]galanin or porcine 125I-labeled Bolton-Hunter [mono(125I)iodo-Lys25]galanin, no anterior pituitary membrane binding was observed. In contrast, 125I-labeled Bolton-Hunter N-terminally labeled galanin allowed characterization of a single high-affinity anterior pituitary galanin receptor with a Kd of 4.4 +/- 0.34 nM and a Bmax of 79 +/- 8.3 fmol/mg of protein. The IC50 for porcine galanin was 0.51 +/- 0.04 nM but for M15 was in excess of 10 microM. Galanin 3-29 fully displaced the label with an IC50 of 0.96 +/- 0.7 nM. The IC50 for galanin 5-29 was 200 nM, whereas 8-29 and 1-15 were > 1 microM. Galanin 10-29 and 20-29 failed to displace the label. These data suggest the presence of a high-affinity pituitary galanin receptor, designated GAL-R2, in which region 3-10 and amino acid 25 are crucial for membrane binding and biological activity, in contrast to the known gut/brain galanin receptor (designated GAL-R1). A number of tissues known to bind or respond to galanin were screened. GAL-R2 would appear to be expressed only in the anterior pituitary and hypothalamus.
Ligand-induced activation of chimeric receptors between the erythropoietin receptor and receptor tyrosine kinases.
Authors: Ohashi H , Maruyama K , Liu Y C , Yoshimura A
Published: May 2006

Ligand-induced dimerization is a key step in the activation of receptor tyrosine kinases, including the epidermal growth factor receptor, stem cell factor receptor (c-kit), and colony-stimulating factor 1 receptor (c-fms). The erythropoietin receptor (EPOR), a member of the cytokine receptor family, contains no kinase motif and its activation mechanism remains unclear. Here we show that chimeric receptors carrying the extracellular domain of the epidermal growth factor receptor or c-kit linked to the cytoplasmic domain of the EPOR, transmitted epidermal growth factor or stem cell factor-dependent proliferation signals in an interleukin 3-dependent cell line. The chimeric receptors as well as the wild-type EPOR also mediated the ligand-induced tyrosine phosphorylation of a set of similar proteins. Moreover, erythropoietin triggered mitogenic signals of chimeric receptors carrying the extracellular domain of the EPOR linked to the tyrosine kinase of c-fms. These data demonstrate the interchangeability of domains between two distinct receptor families and suggest that ligand-induced dimerization is a key step in activating the EPOR.
Homeostatic regulation of hepatocyte nuclear transcription factor 1 expression in cultured hepatoma cells.
Authors: Pietrangelo A , Shafritz D A
Published: May 2006

Serum colloid osmotic pressure is believed to control the hepatic output of plasma proteins, including albumin. The present study was aimed at identifying the molecular basis for feedback control of albumin gene expression in highly differentiated hepatoma cells. The steady-state level of albumin mRNA and the activity of a 282-bp albumin promoter-chloramphenicol acetyltransferase reporter gene in cells incubated in the presence of increasing amounts of serum albumin or dextran were significantly and selectively decreased. When nuclear extracts from cells exposed to 5% (wt/vol) serum albumin were tested in a gel-retardation assay with six oligonucleotide probes containing DNA elements of the albumin promoter, only the element B-retarded band, which contains the nucleotide recognition sequence for hepatocyte nuclear transcription factor 1 alpha (HNF-1 alpha), was consistently decreased as compared to nuclear extract from cells not exposed to serum albumin. Moreover, the activity of a reporter gene with a basal TATA-promoter driven by multiple HNF-1 alpha recognition elements was selectively inhibited in cells incubated in the presence of 5% serum albumin. A reduction of HNF-1 alpha mRNA appears to be responsible for this response to a change in the level of macromolecules in the incubation medium. These results indicate that activity of a dominant liver transcription factor, HNF-1 alpha, controlling the transcription of several liver-specific genes, is modulated by a fluctuation in the level of oncotically active macromolecules.
Epithelial autotoxicity of nitric oxide: role in the respiratory cytopathology of pertussis.
Authors: Heiss L N , Lancaster J R , Corbett J A , Goldman W E
Published: May 2006

Bordetella pertussis releases a specific peptidoglycan fragment known as tracheal cytotoxin (TCT) that reproduces the respiratory epithelial cytopathology of whooping cough (pertussis). In vitro, TCT inhibits DNA synthesis in hamster trachea epithelial cells and causes specific destruction of ciliated cells in explants of human and hamster respiratory epithelium. We have recently demonstrated that TCT triggers production of intracellular interleukin 1 by respiratory epithelial cells, and this cytokine may act as an intermediate signal in the generation of TCT toxicity. Here we report the identification of a subsequent critical step in this pathway: induction of nitric oxide synthesis in the respiratory epithelium. The toxic effects of nitric oxide are consistent with spectroscopic evidence of the formation of iron-dinitrosyl-dithiolate complexes in TCT-treated cells. Aconitase, with its iron-sulfur center, is one expected target of nitric oxide, and TCT inhibited 80% of the activity of this enzyme in respiratory epithelial cells. The deleterious effects of TCT and interleukin 1 were dramatically attenuated by the nitric oxide synthase inhibitors NG-monomethyl-L-arginine and aminoguanidine. These results indicate that nitric oxide mediates the toxicity of TCT for the respiratory epithelium, thus implicating a central role for nitric oxide in the pathogenesis of pertussis.
Quantitative magnetic resonance imaging of human brain perfusion at 1.5 T using steady-state inversion of arterial water.
Authors: Roberts D A , Detre J A , Bolinger L , Insko E K , Leigh J S
Published: May 2006

We report our experience using a noninvasive magnetic resonance technique for quantitative imaging of human brain perfusion at 1.5 T. This technique uses magnetically inverted arterial water as a freely diffusible blood flow tracer. A perfusion image is calculated from magnetic resonance images acquired with and without arterial blood inversion and from an image of the apparent spin-lattice relaxation time. Single-slice perfusion maps were obtained from nine volunteers with approximately 1 x 2 x 5-mm resolution in an acquisition time of 15 min. Analysis yielded average perfusion rates of 93 +/- 16 ml.100 g-1.min-1 for gray matter, 38 +/- 10 ml.100 g-1.min-1 for white matter, and 52 +/- 8 ml.100 g-1.min-1 for whole brain. Significant changes in perfusion were observed during hyperventilation and breath holding. This technique may be used for quantitative measurement of perfusion in human brain without the risks and expense of methods which use exogenous tracers.
Gamma delta T cells contribute to immunity against the liver stages of malaria in alpha beta T-cell-deficient mice.
Authors: Tsuji M , Mombaerts P , Lefrancois L , Nussenzweig R S , Zavala F , Tonegawa S
Published: May 2006

The functional role of gamma delta T cells (expressing the gamma delta heterodimeric T-cell receptor for antigen) in infectious diseases remains largely unknown. We have therefore attempted to define the possible role of these T cells in the immune response against the various developmental stages of malaria parasites. For this purpose, we monitored the immune response and the development of liver and blood stages of Plasmodium yoelii, a rodent malaria parasite, in immunized and nonimmunized alpha beta T-cell-deficient and gamma delta T-cell-deficient mice. Immunization of alpha beta T-cell-deficient mice with irradiated sporozoites induced an immune response that significantly inhibited the development of the parasite's liver stages. This inhibitory immune response was abolished by an antibody-mediated transient in vivo depletion of gamma delta T cells. Two gamma delta T-cell clones were derived from malaria-immunized alpha beta T-cell-deficient mice. The adoptive transfer of one of these gamma delta T-cell clones to normal mice inhibited the development of liver stages, following sporozoite inoculation. These results provide evidence for gamma delta T-cell-mediated protective immunity against parasites, in the absence of alpha beta T cells. As for the blood phase of the infection, both normal mice and gamma delta T-cell-deficient mice cleared the blood stages of the nonlethal strain of P. yoelii, while alpha beta T-cell-deficient mice failed to control the parasitemia.
Transplantation of glial cells enhances action potential conduction of amyelinated spinal cord axons in the myelin-deficient rat.
Authors: Utzschneider D A , Archer D R , Kocsis J D , Waxman S G , Duncan I D
Published: May 2006

A central issue in transplantation research is to determine how and when transplantation of neural tissue can influence the development and function of the mammalian central nervous system. Of particular interest is whether electrophysiological function in the traumatized or diseased mammalian central nervous system can be improved by the replacement of cellular elements that are missing or damaged. Although it is known that transplantation of neural tissue can lead to functional improvement in models of neurological disease characterized by neuronal loss, less is known about results of transplantation in disorders of myelin. We report here that transplantation of glial cells into the dorsal columns of neonatal myelin-deficient rat spinal cords leads to myelination and a 3-fold increase in conduction velocity. We also show that impulses can propagate into and out of the transplant region and that axons myelinated by transplanted cells do not have impaired frequency-response properties. These results demonstrate that myelination following central nervous system glial cell transplantation enhances action potential conduction in myelin-deficient axons, with conduction velocity approaching normal values.
A reinvestigation of a synthetic peptide (TrPepz) designed to mimic trypsin.
Authors: Wells J A , Fairbrother W J , Otlewski J , Laskowski M , Burnier J
Published: May 2006

Recently, a 29-residue cyclic peptide was synthesized (TrPepz) that was reported to possess nearly the same catalytic activity and specificity as the pancreatic serine protease, trypsin, for hydrolysis of a small ester substrate, N-tosyl-L-arginine methyl ester (TAME), and small and large peptides [Atassi, M. Z. & Manshouri, T. (1993) Proc. Natl. Acad Sci. USA 90, 8282-8286]. To study these results we have resynthesized TrPepz and a related cyclic peptide reported to possess some trypsin-like activity. The authenticity of each peptide was confirmed by mass spectrometry, peptide sequencing, compositional analysis, and 1H NMR spectroscopy. However, neither peptide exhibited any detectable esterase activity or amidase activity under a variety of conditions tested. Molecular modeling studies indicated it was possible for TrPepz to be nearly superimposed upon the active site of trypsin. However, NMR experiments showed the structure of the cyclic peptide to be disordered. Thus, we were unable to confirm the results of Atassi and Manshouri. Our results are consistent with the view that serine protease activity depends not only on the presence of catalytic groups but also on their precise and stable alignment.
Participation of target Fas protein in apoptosis pathway induced by CD4+ Th1 and CD8+ cytotoxic T cells.
Authors: Ju S T , Cui H , Panka D J , Ettinger R , Marshak-Rothstein A
Published: May 2006

The results presented here provide evidence that the presence of Fas protein in target cells is essential to permit cytotoxicity (resulting in apoptosis) mediated by cloned CD4+ Th1 cells. Using mitogen-activated B cells as targets, antigen-dependent lysis by CD4+ Th1 effectors was observed with MRL/MpJ+ but not with MRL/MpJ-lpr targets. The congenic MRL/MpJ-lpr strain is defective in Fas expression. Target cells from various lymphoid tissues of C3H.MRL-lpr mice were also resistant to the lectin-dependent cytotoxicity of Th1 effectors, whereas C3H/HeJ targets were sensitive. Moreover, a rapid DNA fragmentation prior to 51Cr release was induced only in C3H/HeJ targets. Thus, cytotoxicity induced by Th1 effectors correlates with target Fas expression. In contrast to Th1 effectors, CD8+ cytotoxic T lymphocytes (CTLs) killed C3H.MRL-lpr targets. When cytotoxicity was assayed in the presence of EGTA and MgCl2, which chelates extracellular Ca2+ [(Ca2+)ext], only C3H.MRL-lpr targets became resistant to CD8+ CTLs. This (Ca2+)ext-independent cytotoxicity of both Th1 and CD8+ effectors could be inhibited with unlabeled C3H/HeJ thymocytes or with a transfectoma carrying a murine Fas-human mu gene construct. In comparison, C3H.MRL-lpr thymocytes and the nontransfected parental cell line were poor inhibitors. Our study demonstrates that CD4+ Th1 cells and CD8+ CTLs differ in their (Ca2+)ext-dependent cytotoxicity but share a (Ca2+)ext-independent cytotoxicity that requires participation of Fas molecules for cytotoxic signal transduction leading to target apoptosis.
Tyrosine phosphorylation of Blk and Fyn Src homology 2 domain-binding proteins occurs in response to antigen-receptor ligation in B cells and constitutively in pre-B cells.
Authors: Aoki Y , Isselbacher K J , Cherayil B J , Pillai S
Published: May 2006

Proteins that bind to discrete domains of the Blk, Fyn, Lyn, and Btk protein tyrosine kinases were examined in pre-B cells that had not been subjected to any external stimulation, as well as in nonstimulated and antigen-receptor-ligated B cells. Proteins that bind to the Src homology 2 domains of Blk and Fyn were identified in B cells that had been activated with anti-IgM but were not identified in unstimulated B cells. A number of Blk and Fyn Src homology 2 domain-binding phosphoproteins were also observed in pre-B cells that had not been stimulated in vitro. The phosphoproteins seen in activated B cells potentially represent substrates that play a role in the pathway of antigen-receptor-mediated signaling. Distinct signaling pathways involving distinguishable kinase substrates may be relevant in pre-B-cell-receptor-mediated cell survival during ontogeny. These results indirectly support models that predict constitutive ligand-independent signaling by the pre-antigen receptor during lymphoid ontogeny.
Endothelial nitric oxide synthase localized to hippocampal pyramidal cells: implications for synaptic plasticity.
Authors: Dinerman J L , Dawson T M , Schell M J , Snowman A , Snyder S H
Published: May 2006

Using antibodies that react selectively with peptide sequences unique to endothelial nitric oxide synthase (eNOS), we demonstrate localizations to neuronal populations in the brain. In some brain regions, such as the cerebellum and olfactory bulb, eNOS and neuronal NOS (nNOS) occur in the same cell populations, though in differing proportions. In the hippocampus, localizations of the two enzymes are strikingly different, with eNOS more concentrated in hippocampal pyramidal cells than in any other brain area, whereas nNOS is restricted to occasional interneurons. In many brain regions NADPH diaphorase staining reflects NOS catalytic activity. Hippocampal pyramidal cells do not stain for diaphorase with conventional paraformaldehyde fixation but stain robustly with glutaraldehyde fixatives, presumably reflecting eNOS catalytic activity. eNOS in hippocampal pyramidal cells may generate the NO that has been postulated as a retrograde messenger of long-term potentiation.
Growth arrest by induction of p53 in DNA damaged keratinocytes is bypassed by human papillomavirus 16 E7.
Authors: Demers G W , Foster S A , Halbert C L , Galloway D A
Published: May 2006

Cellular tumor suppressors p53 and Rb play an important role in controlling cell proliferation. Inactivation of these tumor suppressor proteins can occur by gene mutation or by association with oncoproteins from the small DNA tumor viruses. One function of p53 is in regulating cell cycle check-point control after DNA damage. To dissect the pathways by which p53 and Rb may act, the E6 and E7 oncogenes of human papillomavirus (HPV) types 6 and 16 were introduced into primary human epithelial cells by retroviral transfer vector, and cells were assayed for growth arrest after DNA damage induced by actinomycin D. The E6 or E7 oncogenes from the low-risk HPV6 had no affect on growth arrest, p53 protein levels increased, Rb protein levels decreased, and Rb was predominantly in the hypophosphorylated state similar to vector-infected cells. Either the E6 or the E7 oncogene from the high-risk HPV16 abrogated growth arrest. Cells expressing HPV16 E6 (16E6) were severely reduced in p53 protein levels that did not increase detectably after DNA damage, Rb protein levels did not decrease, and hyperphosphorylated Rb was present. After DNA damage in cells expressing 16E7 p53 levels increased, and Rb protein levels decreased; however, Rb was predominantly in the hyperphosphorylated state. Even though p53 protein levels increased in response to DNA damage in cells expressing 16E7, G1 growth arrest was bypassed. This suggests that the circuitry controlling the growth arrest signal after DNA damage may be interconnected between the p53 and Rb pathways.
Calorie restriction prevents the occlusive coronary vascular disease of autoimmune (NZW x BXSB)F1 mice.
Authors: Mizutani H , Engelman R W , Kinjoh K , Kurata Y , Ikehara S , Matsuzawa Y , Good R A
Published: May 2006

Male (NZW x BXSB)F1 (W/BF1) mice develop systemic autoimmunity involving autoantibodies, thrombocytopenia, lupus nephritis, and coronary vascular disease (CVD) with myocardial infarction. To determine whether this murine lupus-associated CVD can be prevented by the reduction of dietary calories, male W/BF1 mice were separated into five experimental groups and fed either ad libitum (designated group A, n = 50), fed 32% fewer calories of an otherwise comparable diet (designated group B6, n = 20), or initially fed ad libitum and then switched to reduced calorie intake (RCI) feeding at ages 14, 17, or 22 weeks (designated B14, n = 10; B17, n = 20; or B22, n = 20). Occlusive CVD was prevented by RCI. Life-span was significantly extended among the early onset RCI cohorts, B6 and B14 (P = 0.0001 and P = 0.005), compared to group A mice. Mean anti-cardiolipin autoantibody titers and mean levels of circulating immune complexes were also lowered in RCI mice when all RCI mice were compared to ad libitum fed group A mice. Histological grades of both coronary vascular and glomerular lesions were significantly less than those of group A mice (P < 0.001). Immunoprecipitates indicative of immunoglobulin deposition within coronary or glomerular vascular walls were also substantially less than those of group A mice. These findings indicate a possible causal role for anti-cardiolipin autoantibody in development of autoimmune CVD in W/BF1 mice and suggest that regulating dietary calories can influence the mechanism involved in pathogenesis of autoimmune-associated CVD development.
Excision close to matrix attachment regions of the entire chicken alpha-globin gene domain by nuclease S1 and characterization of the framing structures.
Authors: Targa F R , Razin S V , de Moura Gallo C V , Scherrer K
Published: May 2006

Nuclease S1-hypersensitive sites in a 40-kb region of the chicken genome including the domain of the alpha-globin genes were mapped. Brief treatment of isolated chicken erythroid cell nuclei with nuclease S1 allowed separation of an approximately 20-kb genomic DNA fragment containing the whole alpha-globin gene cluster. No S1-hypersensitive sites were observed in the internal part of the domain. The upstream S1 site was found in a DNA fragment of 1.7 kb where the origin of replication and several protein binding sites were identified previously. Precise mapping of the positions of S1 cleavage in this fragment and "in vivo" footprinting of DNA-protein interactions in isolated nuclei showed a correspondence with some of these protein binding sites. The possible significance of all these observations is discussed in connection with the replication origin and the nuclear matrix attachment regions in the framing structures.
Ion transport and methane production in Methanobacterium thermoautotrophicum.
Authors: Sauer F D , Blackwell B A , Kramer J K
Published: May 2006

In Methanobacterium thermoautotrophicum, the protonmotive force for the H+-translocating ATPase consists mainly of a transmembrane electrical gradient (Deltapsi). These cells do not establish a significant transmembrane pH gradient (inside alkaline) and, in fact, if the suspending medium is of pH >/= 7.0, the pH gradient may be reversed-i.e., inside acid with respect to the extracellular pH. These studies show by both 23Na NMR and 22Na+ distribution that Na+ extrusion with the generation of Deltapsi precedes methanogenesis in Mb. thermoautotrophicum. It is calculated that the newly established Na+ gradients increase Deltapsi by approximately 50 mV (inside negative). There is no detectable H+ extrusion during methane synthesis; instead there is a high rate of H+ consumption for methane synthesis and an increase in internal pH. This was supported by 31P NMR experiments, which showed an internal pH shift from 6.8 to 7.6. With the cells maintained at an external pH of 7.2, the initial transmembrane pH gradient of -0.4 (inside acid) at 60 degrees C is equivalent to Deltapsi of + 27 mV (inside positive); after 20 min of incubation, the transmembrane pH gradient is + 0.4 (inside alkaline), which at 60 degrees C is equivalent to Deltapsi of -27 mV (inside negative). Actively respiring cells generated a protonmotive force of -198 mV. It is proposed that energy for CO2 reduction to the level of formaldehyde (the first step in methane synthesis) in Mb. thermoautotrophicum is derived from the Deltapsi generated by electrogenic Na+ extrusion. The protonmotive force required for ATP synthesis consists primarily of Deltapsi and appears to be the result of both an electrogenic Na+ extrusion and a pH gradient (inside alkaline) which develops during methanogenesis.
Glial-specific cAMP response of the glial fibrillary acidic protein gene cell lines.
Authors: Kaneko R , Hagiwara N , Leader K , Sueoka N
Published: May 2006

Expression of the rat glial fibrillary acidic protein (GFAP) gene is responsive to the intracellular level of cAMP. We have examined the sequence 5'-upstream of the transcription start site of the rat GFAP-encoding gene to determine the elements responsible for regulating the cAMP response. The RT4 cell lines consist of a neural stem-cell type RT4-AC and its three derivative cell types, one glial-cell type, RT4-D, and two neuronal-cell types, RT4-B and RT4-E. GFAP is expressed in the stem-cell type and the glial-cell type but is not expressed in the neuronal-cell types. Luciferase expression vectors containing various areas of the 10.8-kb region upstream of the transcription start site of the GFAP gene were transiently transfected into these RT4 cells. The effect of cAMP was examined by quantitating the transient expression of luciferase. We found that (i) the 5'-upstream region alone (up to 10.8 kb) allows expression of the GFAP gene in the stem-cell type, the glial-cell type, and a neuronal-cell type; (ii) there are negative and positive cAMP-responsive elements that are juxtaposed within the region between -240 bp and -110 bp upstream and are functional in the stem-cell and glial-cell types but are not functional in the neuronal-cell type RT4-E; (iii) there may be elements that respond to dibutyryl-cAMP in all three RT4 cell types within the region from 2 kb to 10.8 kb upstream of the transcription start site; and (iv) a regulatory luciferase plasmid pRLgfap-1, containing both the upstream and downstream regulatory regions of the GFAP gene, not only expresses luciferase but also responds to forskolin in the stem-cell type and the glial-cell type. This regulatory plasmid, however, does not express in the neuronal-cell type with or without the forskolin treatment.
Gamma interferon activates a previously undescribed Ca2+ influx in T lymphocytes from patients with multiple sclerosis.
Authors: Martino G , Clementi E , Brambilla E , Moiola L , Comi G , Meldolesi J , Grimaldi L M
Published: May 2006

Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the central nervous system. The etiology of the disease is still unknown. Activated T lymphocytes are considered essential in mediating the inflammatory process leading to demyelination in MS. They operate through a complex network of cytokines among which gamma interferon (gamma-IFN) plays a key role. Here we report that exposure to gamma-IFN of T lymphocytes from patients with MS activates, by a protein kinase C-mediated pathway, a previously undescribed gamma-IFN-activated Ca2+ influx, functionally coupled to the gamma-IFN receptor. The influx mainly expressed by CD4+ T lymphocytes, was found in 12 of 15 (80%) patients with clinically active MS and in 14 of 30 (46%) patients with stable MS. The influx was found in only 3 of 24 (12%) control patients and in none of the 15 healthy subjects studied. Our results document the appearance in MS lymphocytes of a gamma-IFN-activated, protein kinase C-dependent, Ca2+ influx that might be due to the expression of a new cation-specific plasmalemma channel. This finding suggests that at least part of gamma-IFN's contribution to the pathogenesis of MS is exerted through a Ca(2+)-dependent regulation of T lymphocyte activity.
LR1 regulates c-myc transcription in B-cell lymphomas.
Authors: Brys A , Maizels N
Published: May 2006

LR1 is a 106-kDa sequence-specific DNA-binding protein first identified as a potential regulator of immunoglobulin class switch recombination in B lymphocytes. Here we report that LR1 binds to a site 310 nt upstream of the human c-myc P1 promoter. Mutation of this site decreases reporter gene expression 5.5-fold in the Burkitt lymphoma line Raji and 3.8-fold in the lymphoma line BJAB. These experiments show that LR1 can function as a transcription factor and identify it as a cell type-specific activator of c-myc expression. There are multiple matches to the LR1 recognition consensus at the immunoglobulin heavy-chain locus and at c-myc, which further suggests that LR1 may play a dual role, facilitating c-myc translocation as well as regulating c-myc transcription.
Light induces rapid changes of the phosphorylation pattern in the cytosol of evacuolated parsley protoplasts.
Authors: Harter K , Frohnmeyer H , Kircher S , Kunkel T , Mühlbauer S , Schäfer E
Published: May 2006

The fractionation of cells of a parsley suspension culture [Petroselinum crispum (Mill.) A. Hill] by protoplasting and subsequent removal of the vacuoles led to physiologically intact evacuolated protoplasts retaining light inducibility of chalcone synthase expression. Lysis of the evacuolated protoplasts permitted the isolation of a pure, highly concentrated cytosolic fraction containing major cytosolic membranes but only minor contamination by proplastids, mitochondria, and nuclei. Short-time irradiations of the cytosol with red or UV-containing white light resulted in very fast changes of the phosphorylation pattern of 18-, 40-, 48-, 55- to 70-, and 120-kDa proteins. Major differences were observed between the phosphorylation patterns obtained by red or UV-containing white light treatment, indicating a different primary action of the excited photoreceptors in vitro. Separation of the microsomal fraction from the cytosolic matrix established the localization of these proteins. Chase and photoreversibility experiments revealed that phytochrome in vitro regulates the phosphorylation of the 40-kDa protein by modifying a soluble cytosolic kinase/phosphatase system.
Molecular cloning of the gene encoding the mouse parathyroid hormone/parathyroid hormone-related peptide receptor.
Authors: McCuaig K A , Clarke J C , White J H
Published: May 2006

The parathyroid hormone/parathyroid hormone-related peptide receptor (PTHR) is a G-protein-coupled receptor containing seven predicted transmembrane domains. We have isolated and characterized recombinant bacteriophage lambda EMBL3 genomic clones containing the mouse PTHR gene, including 10 kilobases of the promoter region. The gene spans > 32 kilobases and is divided into 15 exons, 8 of which contain the transmembrane domains. The PTHR exons containing the predicted membrane-spanning domains are heterogeneous in length and three of the exon-intron boundaries fall within putative transmembrane sequences, suggesting that the exons did not arise from duplication events. This arrangement is closely related to that of the growth hormone releasing factor receptor gene, particularly in the transmembrane region, providing strong evidence that the two genes evolved from a common precursor. Transcription is initiated principally at a series of sites over a 15-base-pair region. The proximal promoter region is highly (G+C)-rich and lacks an apparent TATA box or initiator element homologies but does contain CCGCCC motifs. The presumptive amino acid sequence of the encoded receptor is 99%, 91%, and 76% identical to those of the rat, human, and opossum receptors, respectively. There is no consensus polyadenylation signal in the 3' untranslated region. The poly(A) tail of the PTHR transcript begins 32 bases downstream of a 35-base-long A-rich sequence, suggesting that this region directs polyadenylylation.
Mitochondrial creatine kinase: a major constituent of pathological inclusions seen in mitochondrial myopathies.
Authors: Stadhouders A M , Jap P H , Winkler H P , Eppenberger H M , Wallimann T
Published: May 2006

Overaccumulation of abnormally organized mitochondria in so-called "ragged-red" skeletal muscle fibers is a morphological hallmark of mitochondrial myopathies, in particular of mitochondrial encephalomyopathies. Characteristic for the abnormal mitochondria is the occurrence of highly ordered crystalline inclusions. Immuno-electron microscopy revealed that these inclusions react heavily with specific antibodies against mitochondrial creatine kinase (Mi-CK). Image processing of selected crystalline inclusions, sectioned along the crystallographic b, c planes, resulted in an averaged picture displaying an arrangement of regular, square-shaped particles with a central cavity. The overall appearance, dimensions, and symmetry of these building blocks are very reminiscent of single isolated Mi-CK octamers. Taking these findings together, it is concluded that Mi-CK octamers indeed represent the major, if not the only, component of these mitochondrial inclusions.
DNA fingerprinting analysis of parent-offspring conflict in a bee.
Authors: Mueller U G , Eickwort G C , Aquadro C F
Published: May 2006

Demonstrating the importance of haplodiploidy in the evolution of eusociality among the Hymenoptera (bees, wasps, and ants) requires estimation of four parameters: relatedness between cooperating individuals, effective mating frequency, sex ratio, and rates of worker reproduction. Multilocus DNA fingerprinting techniques permitted the precise determination of these parameters for the primitively eusocial bee Augochlorella striata (Halictidae). DNA fingerprints revealed an unprecedented resolution of genetic relationships within colonies, detecting factors such as intraspecific nest parasitism and diploid males that confounded estimates of relatedness and sex ratio, respectively. Parameter estimates (i) corroborate recent evidence for queen-worker conflict over the sex ratio and (ii) implicate the role of haplodiploidy in the evolution of worker behavior.
Natural sleep modifies the rat electroretinogram.
Authors: Galambos R , Juhász G , Kékesi A K , Nyitrai G , Szilágyi N
Published: May 2006

We show here electroretinograms (ERGs) recorded from freely moving rats during sleep and wakefulness. Bilateral ERGs were evoked by flashes delivered through a light-emitting diode implanted under the skin above one eye and recorded through electrodes inside each orbit near the optic nerve. Additional electrodes over each visual cortex monitored the brain waves and collected flash-evoked cortical potentials to compare with the ERGs. Connections to the stimulating and recording instruments through a plug on the head made data collection possible at any time without physically disturbing the animal. The three major findings are (i) the ERG amplitude during slow-wave sleep can be 2 or more times that of the waking response; (ii) the ERG patterns in slow-wave and REM sleep are different; and (iii) the sleep-related ERG changes closely mimic those taking place at the same time in the responses evoked from the visual cortex. We conclude that the mechanisms that alter the visual cortical-evoked responses during sleep operate also and similarly at the retinal level.
Arrest of Listeria movement in host cells by a bacterial ActA analogue: implications for actin-based motility.
Authors: Southwick F S , Purich D L
Published: May 2006

Upon entering the host cell's cytoplasm, the pathogen Listeria monocytogenes can subvert the normal contractile system of the host cell; subsequent assembly of polar actin-filament structures is likely to provide the force for rapid intracellular bacterial movement and its cell-to-cell spread. We have now investigated the functional consequences of microinjecting Listeria-infected PtK2 cells with a synthetic peptide, CFEFPPPPTDE. This peptide represents one of four related oligoproline stretches in ActA, a bacterial surface protein necessary for Listeria-induced actin assembly. Over an estimated intracellular concentration range of 80 nM to 0.8 microM, this analogue rapidly blocks the formation of the actin-filament tails and arrests intracellular bacterial motility. Over the same time scale and concentration range, introduction of the ActA analogue also causes host cell membrane retraction. Bodipyphallacidin staining reveals that microinjection of the ActA analogue results in massive retraction of the actin cytoskeleton. Microinjection of 1-20 microM poly(L-proline) (intracellular concentration) fails to block Listeria intracellular movement or polar actin-filament assembly. As observed with ActA, however, poly(L-proline) does cause membrane retraction. Our findings demonstrate the efficacy of low molecular weight peptides in efforts to distinguish mechanistic features in Listeria motility and PtK2 host cell membrane reorganization. These observations also suggest that a cytoskeletal component sensitive to specific oligoproline peptides may participate in protein-protein interactions essential for both of these actin-associated processes.
Calcium transients in dendrites of neocortical neurons evoked by single subthreshold excitatory postsynaptic potentials via low-voltage-activated calcium channels.
Authors: Markram H , Sakmann B
Published: May 2006

Simultaneous recordings of membrane voltage and concentration of intracellular Ca2+ ([Ca2+]i) were made in apical dendrites of layer 5 pyramidal cells of rat neocortex after filling dendrites with the fluorescent Ca2+ indicator Calcium Green-1. Subthreshold excitatory postsynaptic potentials (EP-SPs), mediated by the activation of glutamate receptor channels, caused a brief increase in dendritic [Ca2+]i. This rise in dendritic [Ca2+]i was mediated by the opening of low-voltage-activated Ca2+ channels in the dendritic membrane. The results provide direct evidence that dendrites do not function as passive cables even at low-frequency synaptic activity; rather, a single subthreshold EPSP changes the dendritic membrane conductance by opening Ca2+ channels and generating a [Ca2+]i transient that may propagate towards the soma. The activation of these Ca2+ channels at a low-voltage threshold is likely to influence the way in which dendritic EPSPs contribute to the electrical activity of the neuron.
Molecular cloning of an essential subunit of RNA polymerase II elongation factor SIII.
Authors: Garrett K P , Tan S , Bradsher J N , Lane W S , Conaway J W , Conaway R C
Published: May 2006

A transcription factor designated SIII was recently purified from mammalian cells and shown to regulate the activity of the RNA polymerase II elongation complex. SIII is a heterotrimer composed of approximately 110-, 18-, and 15-kDa polypeptides and is capable of increasing the overall rate of RNA chain elongation by RNA polymerase II by suppressing transient pausing of polymerase at multiple sites on the DNA template. Here we describe the molecular cloning and characterization of a cDNA encoding the functional 15-kDa subunit (p15) of SIII. The p15 cDNA encodes a 112-amino-acid polypeptide with a calculated molecular mass of 12,473 Da and an electrophoretic mobility indistinguishable from that of the natural p15 subunit. When combined with the 110- and 18-kDa SIII subunits, bacterially expressed p15 efficiently replaces the natural p15 subunit in reconstitution of transcriptionally active SIII. A homology search revealed that the amino-terminal half of the SIII p15 subunit shares significant sequence similarity with a portion of the RNA-binding domain of Escherichia coli transcription termination protein rho and with the E. coli NusB protein, suggesting that SIII may be evolutionarily related to proteins involved in the control of transcription elongation in eubacteria.
Exo-endocytosis and closing of the fission pore during endocytosis in single pituitary nerve terminals internally perfused with high calcium concentrations.
Authors: Rosenboom H , Lindau M
Published: May 2006

An increase in free Ca2+ triggers exocytosis in pituitary nerve terminals leading to an increase in membrane area and membrane capacitance. When Ca2+ is increased by step depolarization, an instantaneous capacitance increase during the first 80 ms is followed by a slow increase extending over several seconds. We measured capacitance changes associated with exocytosis and endocytosis in single pituitary nerve terminals internally perfused with high Ca2+. At 50 microM Ca2+ the capacitance increased by up to 2%/s, similar to the slow phase observed during depolarization. Our results indicate that at the site of fusion very high Ca2+ is required. Following exocytosis, large downward capacitance steps were measured, reflecting endocytosis of large vacuoles. These events were not abrupt but reflected a gradual decrease of fission pore conductance from 8 nS to < 40 pS during 500 ms, revealing the dynamics of individual fission pore closures. Above 300 pS, narrowing of the endocytotic fission pore was approximately 10 times slower than the previously reported expansion of the exocytotic fusion pore. The transition between 300 pS and 0 pS took approximately 200 ms, whereas it has been reported that the exocytotic fusion pore measured in mast cells opens from 0 to 280 pS in < 100 microseconds. The time course of closing of the fission pore may be explained by an exponential decrease in pore diameter occurring at a constant rate.
Role of Src homology 3 domains in assembly and activation of the phagocyte NADPH oxidase.
Authors: Sumimoto H , Kage Y , Nunoi H , Sasaki H , Nose T , Fukumaki Y , Ohno M , Minakami S , Takeshige K
Published: May 2006

The phagocyte NADPH oxidase, dormant in resting cells, is activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants. The activated oxidase is a complex of membrane-integrated cytochrome b558, composed of 91-kDa (gp91phox) and 22-kDa (p22phox) subunits, and two cytosolic factors (p47phox and p67phox), each containing two Src homology 3 (SH3) domains. Here we show that the region of the tandem SH3 domains of p47phox (p47-SH3) expressed as a glutathione S-transferase fusion protein inhibits the superoxide production in a cell-free system, indicating involvement of the domains in the activation. Furthermore, we find that arachidonic acid and sodium dodecyl sulfate, activators of the oxidase in vitro, cause exposure of p47-SH3, which has probably been masked by the C-terminal region of this protein in a resting state. The unmasking of p47-SH3 appears to play a crucial role in the assembly of the oxidase components, because p47-SH3 binds to both p22phox and p67phox but fails to interact with a mutant p22phox carrying a Pro-156-->Gln substitution in a proline-rich region, which has been found in a patient with chronic granulomatous disease. Based on the observations, we propose a signal-transducing mechanism whereby normally inaccessible SH3 domains become exposed upon activation to interact with their target proteins.
Insulin promotes the biosynthesis and secretion of apolipoprotein B-48 by altering apolipoprotein B mRNA editing.
Authors: Thorngate F E , Raghow R , Wilcox H G , Werner C S , Heimberg M , Elam M B
Published: May 2006

Long-term insulin treatment selectively stimulates secretion of the truncated form of apolipoprotein B (apoB), apoB-48, from primary rat hepatocytes in culture. Chronic treatment with insulin at 400 ng/ml causes a 3-fold increase in total apoB secretion, with apoB-48 making up about 75% of that increase. apo-B-48 is the protein product generated by translation of full-length apoB mRNA which has been modified by a posttranscriptional editing mechanism. Editing changes codon 2153 in the middle of the apoB-100 coding region from CAA, coding for glutamine, to UAA, a translation stop signal. We therefore examined the effect of insulin treatment on the ratio of edited to nonedited apoB mRNA in RNA isolated from primary rat hepatocyte cultures. There was a dramatic shift in the ratio of edited versus nonedited forms of apoB mRNA, from about 1:1 in untreated cells to 7:1 in insulin-treated cells. Insulin exerted a dose-dependent effect on apoB secretion and apoB mRNA editing over the range of insulin concentrations studied (0.4-400 ng/ml). In contrast, oleic acid, which also increased apoB (B-48 and B-100) secretion, had no significant effect on the ratio of apoB-48 to apoB-100 particles secreted and no effect on the proportion of edited apoB mRNA. Neither insulin nor oleic acid affects total apoB mRNA levels as assayed by Northern blot analysis. These data strongly suggest that insulin stimulates biosynthesis and secretion of apoB-48 in rat hepatocytes by regulating the proportion of edited apoB mRNA.
A net +1 frameshift permits synthesis of thymidine kinase from a drug-resistant herpes simplex virus mutant.
Authors: Hwang C B , Horsburgh B , Pelosi E , Roberts S , Digard P , Coen D M
Published: May 2006

Clinical resistance to antiviral drugs requires that a virus evade drug therapy yet retain pathogenicity. Thymidine kinase (TK)-negative mutants of herpes simplex virus are resistant to the drug, acyclovir, but are attenuated for pathogenicity in animal models. However, numerous cases of clinical resistance to acyclovir have been associated with viruses that were reported to express no TK activity. We studied an acyclovir-resistant clinical mutant that contains a single-base insertion in its tk gene, predicting the synthesis of a truncated TK polypeptide with no TK activity. Nevertheless, the mutant retained some TK activity and the ability to reactivate from latent infections of mouse trigeminal ganglia. The mutant expressed both the predicted truncated polypeptide and a low level of a polypeptide that comigrated with full-length TK on polyacrylamide gels and reacted with anti-TK antiserum, providing evidence for a frameshifting mechanism. In vitro transcription and translation of mutant tk genes, including constructs in which reporter epitopes could be expressed only if frameshifting occurred, also gave rise to truncated and full-length polypeptides. Reverse transcriptase-polymerase chain reaction analysis coupled with open reading frame cloning failed to detect alterations in tk transcripts that could account for the synthesis of full-length polypeptide. Thus, synthesis of full-length TK was due to an unusual net +1 frameshift during translation, a phenomenon hitherto confined in eukaryotic cells to certain RNA viruses and retrotransposons. Utilization of cellular frameshifting mechanisms may permit an otherwise TK-negative virus to exhibit clinical acyclovir resistance.
Senescence of immortal human fibroblasts by the introduction of normal human chromosome 6.
Authors: Sandhu A K , Hubbard K , Kaur G P , Jha K K , Ozer H L , Athwal R S
Published: May 2006

In these studies we show that introduction of a normal human chromosome 6 or 6q can suppress the immortal phenotype of simian virus 40-transformed human fibroblasts (SV/HF). Normal human fibroblasts have a limited life span in culture. Immortal clones of SV/HF displayed nonrandom rearrangements in chromosome 6. Single human chromosomes present in mouse/human monochromosomal hybrids were introduced into SV/HF via microcell fusion and maintained by selection for a dominant selectable marker gpt, previously integrated into the human chromosome. Clones of SV/HF cells bearing chromosome 6 displayed limited potential for cell division and morphological characteristics of senescent cells. The loss of chromosome 6 from the suppressed clones correlated with the reappearance of immortal clones. Introduced chromosome 6 in the senescing cells was distinguished from those of parental cells by the analysis for DNA sequences specific for the donor chromosome. Our results further show that suppression of immortal phenotype in SV/HF is specific to chromosome 6. Introduction of individual human chromosomes 2, 8, or 19 did not impart cellular senescence in SV/HF. In addition, introduction of chromosome 6 into human glioblastoma cells did not lead to senescence. Based upon these results we propose that at least one of the genes (SEN6) for cellular senescence in human fibroblasts is present on the long arm of chromosome 6.