bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of the type 1 human immunodeficiency virus Nef protein in T cells prevents antigen receptor-mediated induction of interleukin 2 mRNA.
Authors: Luria S , Chambers I , Berg P
Published: May 2006

Stable transformants of the Jurkat T-cell line have been obtained that express either of two distinct forms of the type 1 human immunodeficiency virus nef gene: the nef-1-encoded protein (Nef-1) contains alanine, glycine, and valine at positions 15, 29, and 33, respectively; the protein specified by nef-2 (Nef-2) has threonine, arginine, and alanine at the corresponding positions. When Jurkat cells or their Nef-2-expressing transformants are treated with phorbol 12-myristate 13-acetate (PMA) plus either phytohemagglutinin (PHA) or antibodies against CD3 epsilon, T-cell receptor beta chain, or CD2, there is a prompt increase in interleukin 2 (IL-2) mRNA and intracellular calcium and in the IL-2 receptor alpha chain on the cell surface. Although cells expressing Nef-1 also induce calcium mobilization and the production of IL-2 receptor alpha chain, the formation of IL-2 mRNA is blocked in response to these stimuli. Moreover, Nef-1-expressing cells transfected with a plasmid in which the IL-2 promoter is fused to the chloramphenicol acetyltransferase (CAT) gene fail to induce CAT following treatment with PMA and PHA. By contrast, the parental and Nef-2-containing cells induce CAT normally. Nef-1-expressing cells can produce IL-2 mRNA in response to a combination of PMA and ionomycin, although much less efficiently than the parental Jurkat cells or Nef-2-expressing cells. These findings, and others described herein, suggest that the virally encoded Nef protein interferes with a signal emanating from the T-cell receptor complex that induces IL-2 gene transcription.
In vivo analysis of the promoter structure of the influenza virus RNA genome using a transfection system with an engineered RNA.
Authors: Yamanaka K , Ogasawara N , Yoshikawa H , Ishihama A , Nagata K
Published: May 2006

A system for the expression of a foreign gene derived from negative polarity RNA was developed using influenza virus, a negative-stranded RNA virus. From cDNA for the influenza virus RNA genome segment 8, the region coding for the nonstructural protein was deleted and replaced by the chloramphenicol acetyltransferase (CAT) gene. The resulting DNA sequence was placed under the control of the promoter of T7 RNA polymerase such that the antisense RNA to CAT mRNA was produced when transcribed by T7 RNA polymerase. Transfection of HeLa cells with this antisense CAT RNA in the presence of the helper ribonucleoprotein cores led to no significant production of the CAT. In contrast, when the RNA was covered with purified nucleoprotein prior to transfection, the CAT gene was efficiently expressed. This indicated that the viral RNA polymerase transcribed the RNA transfected as the RNA-nucleoprotein complexes. In addition, this system was used for analysis of the cis-acting region in transcription and the promoter structure of the viral RNA genome.
p53 mutations in human lymphoid malignancies: association with Burkitt lymphoma and chronic lymphocytic leukemia.
Authors: Gaidano G , Ballerini P , Gong J Z , Inghirami G , Neri A , Newcomb E W , Magrath I T , Knowles D M , Dalla-Favera R
Published: May 2006

We have investigated the frequency of p53 mutations in B- and T-cell human lymphoid malignancies, including acute lymphoblastic leukemia, the major subtypes of non-Hodgkin lymphoma, and chronic lymphocytic leukemia. p53 exons 5-9 were studied by using genomic DNA from 197 primary tumors and 27 cell lines by single-strand conformation polymorphism analysis and by direct sequencing of PCR-amplified fragments. Mutations were found associated with (i) Burkitt lymphoma (9/27 biopsies; 17/27 cell lines) and its leukemic counterpart L3-type B-cell acute lymphoblastic leukemia (5/9), both of which also carry activated c-myc oncogenes, and (ii) B-cell chronic lymphocytic leukemia (6/40) and, in particular, its stage of progression known as Richter's transformation (3/7). Mutations were not found at any significant frequency in other types of non-Hodgkin lymphoma or acute lymphoblastic leukemia. In many cases, only the mutated allele was detectable, implying loss of the normal allele. These results suggest that (i) significant differences in the frequency of p53 mutations are present among subtypes of neoplasms derived from the same tissue; (ii) p53 may play a role in tumor progression in B-cell chronic lymphocytic leukemia; (iii) the presence of both p53 loss/inactivation and c-myc oncogene activation may be important in the pathogenesis of Burkitt lymphoma and its leukemic form L3-type B-cell acute lymphoblastic leukemia.
Transcriptional antagonist cAMP-responsive element modulator (CREM) down-regulates c-fos cAMP-induced expression.
Authors: Foulkes N S , Laoide B M , Schlotter F , Sassone-Corsi P
Published: May 2006

Protooncogene c-fos is induced by activation of adenylate cyclase through the major cAMP-responsive element (CRE) centered at position -60 of the promoter. cAMP induction is followed by a rapid decrease in transcriptional rate, reminiscent of down-regulation after serum stimulation. Fos protein is known to negatively autoregulate serum-induced transcription of c-fos promoter, but whether Fos is responsible for down-regulation of cAMP-induced transcription is unclear. Here we show that Fos is unable to down-regulate CRE-mediated activation. We present evidence that the transcriptional antagonist CRE modulator (CREM) can bind to c-fos CRE and heterodimerize with activator CRE-binding protein, thereby blocking cAMP induction. Furthermore, expression of antisense CREM enhances c-fos basal and cAMP-induced transcription. CREM does not antagonize serum-induced transcription; therefore, we conclude that down-regulation of c-fos is exerted by different effectors, depending upon which signal transduction pathway is activated. We speculate that, by its c-fos down-regulatory function, CREM may act as an antioncogene.
Electron crystallography of linear polymers: direct structure analysis of poly(epsilon-carprolactone).
Authors: Dorset D L
Published: May 2006

Three-dimensional electron diffraction intensity data from poly(epsilon-caprolactone) are used for an ab initio phase determination based on the probabilistic estimate of three- and four-phase structure invariants in space group P212121. From the initial phase set of 30 reflections (two of which are incorrect) of the 47 in the total data set, computed electrostatic potential maps produce a recognizable structure that can be refined by Fourier techniques and atomic displacement. The final structure (R = 0.21), which is very similar to one found in an earlier fiber x-ray study, shows that the polymer chain must adopt a nonplanar conformation around the ester linkage.
Observation of the light-triggered binding of pyrone to chymotrypsin by Laue x-ray crystallography.
Authors: Stoddard B L , Koenigs P , Porter N , Petratos K , Petsko G A , Ringe D
Published: May 2006

Crystals of gamma-chymotrypsin inhibited with the photodissociable group trans-p-diethylamino-o-hydroxy-alpha-methylcinnamate were irradiated with a 1-msec flash from a high-energy xenon flashlamp in the presence of the mechanism-based inhibitor 3-benzyl-6-chloro-2-pyrone. The ensuing reaction was monitored by collection of sequential, single-exposure Laue x-ray diffraction patterns. The experiment was also performed in solution to verify the regeneration of catalytic activity and the subsequent inhibition of the enzyme by pyrone after photolysis. The resulting crystallographic structures show the presence of covalently bound cinnamate prior to photolysis, the generation of "free" enzyme after irradiation of the crystal, and the slow formation of a pyrone-inhibited complex several hours after photolysis. The structure of the free enzyme shows a significant proportion of the active sites in the crystal to contain a naturally occurring, noncovalently bound tetrapeptide inhibitor [Dixon, M.M. & Matthews, B.W. (1989) Biochemistry 28, 7033-7038], even after cinnamate acylation and photolysis. Data collected simultaneously with irradiation show the crystal to be slightly disordered during photolysis, leading to streaked x-ray photos. The resulting maps are suggestive of a bicyclic coumarin species produced by photolysis and deacylation; however, the electron density is difficult to model unambiguously by one unique chemical state. Nevertheless, Laue crystallography is shown to be capable of visualizing time-dependent chemical changes in the active site of an enzyme.
Gene mapping of the putative structural region of the hepatitis C virus genome by in vitro processing analysis.
Authors: Hijikata M , Kato N , Ootsuyama Y , Nakagawa M , Shimotohno K
Published: May 2006

Processing of the putative structural proteins of hepatitis C virus was examined by using an in vitro expression system. An RNA transcript for cell-free translation was prepared from a cDNA construct that encompasses the region encoding the 980 amino-terminal residues of the viral polyprotein precursor. Processing of the in vitro translation product proceeded cotranslationally in the presence of microsomal membranes and generated four major membrane-associated products. Two of these four major products, named gp35 and gp70, were shown to be transported into microsomes and heavily glycosylated, suggesting that the processing events are partly mediated by the signal peptidase of the endoplasmic reticulum. The other two products, p19 and p21, were probably associated with the outer surface of the microsomal membrane. Analysis of processed proteins translated from a series of truncated forms of the cDNA construct as well as determination of amino-terminal amino acid sequences of gp35 and gp70 indicated that these four products are arranged from the amino-terminal end of the polyprotein precursor in the order: NH2-p22-gp35-gp70-p19. Both gp35 and gp70 could be candidates of initially processed forms of envelope proteins of the hepatitis C virus.
Cooperativity in axonemal motion: analysis of a four-state, two-site kinetic model.
Authors: Omoto C K , Palmer J S , Moody M E
Published: May 2006

A kinetic model for axonemal motion based upon a four-state mechanochemical cycle of dynein with two active sites is described. Our model analysis determines the pseudo-steady-state concentrations of enzyme species for specified rate constants, most of which are experimentally determined, with given substrate and product concentrations. The proportion of enzyme species in which both active sites are detached from the microtubule (denoted as "both detached"), numerically calculated from the model, appears to be proportional to experimental observations of flagellar beat frequency. This correlation between beat frequency and the both-detached enzyme species is maintained over a wide range of substrate concentrations and exhibited an apparent positive cooperativity at low substrate concentrations, which we call "obligate cooperativity." The unusual obligate cooperativity exhibited by flagellar beat frequency parallels that seen in the calculated proportion of the both-detached enzyme species and is interpreted as a requirement for a molecule of substrate to bind to each active site in a multimeric dynein in order to produce oscillatory motion. Furthermore, the proportion of the both-detached enzyme species correlates with experimentally observed changes in beat frequency with a nucleotide analog and with product inhibition.
Sequence conservation in avian CR1: an interspersed repetitive DNA family evolving under functional constraints.
Authors: Chen Z Q , Ritzel R G , Lin C C , Hodgetts R B
Published: May 2006

CR1 is a short interspersed repetitive DNA element originally identified in the domestic chicken (Gallus gallus). However, unlike virtually all other such sequences described to date, CR1 is not confined to one or a few closely related species. It is probably a ubiquitous component of the avian genome, having been detected in representatives of nine orders encompassing a wide spectrum of the class Aves. This identification was made possible by using the polymerase chain reaction (PCR), which revealed interspecific similarities not detected by conventional Southern analysis. DNA sequence comparisons between a CR1 element isolated from a sarus crane (Grus antigone) and those isolated from an emu (Dromaius novaehollandiae) showed that two short highly conserved regions are present. These are included within two regions previously characterized in the CR1 units of domestic fowl. One of these behaves as a transcriptional silencer and the other is a binding site for a nuclear protein. Our observations suggest that CR1 has evolved under functional constraints and that interspersed repetitive sequences as a class may constitute a more significant component of the eukaryotic genome than is generally acknowledged.
Rostrocaudal gradient of transgene expression in adult skeletal muscle.
Authors: Donoghue M J , Merlie J P , Rosenthal N , Sanes J R
Published: May 2006

Transgenic mice were produced in which expression of the reporter gene chloramphenicol acetyltransferase (CAT) is controlled by regulatory elements of a rodent myosin light chain gene. CAT activity was readily detectable in muscles of these mice but negligible in a variety of nonmuscle tissues. Unexpectedly, levels of CAT expression varied greater than 100-fold from muscle to muscle, forming a gradient in which a muscle's position in the rostrocaudal axis was correlated with its level of CAT enzyme activity and abundance of CAT mRNA. Thus, rostral muscles (innervated by cranial nerves) had the lowest levels of CAT, thoracic muscles had intermediate levels, and caudal muscles (innervated through lumbar and sacral roots) had the highest levels. We established that myosin light chain sequences are responsible for the gradient of CAT expression but observed no strong gradient of endogenous myosin light chain expression. We argue that elements that are silent or masked by other sequences in their native context are revealed in the transgene and that the rostrocaudal gradient of gene expression they produce reveals the existence of a positionally graded endogenous regulator of gene expression. These transgenic mice provide evidence that cells in adult mammals retain "positional information" of a sort hitherto studied largely in embryos. The transgene they express may provide a means for determining how such positional values are generated and maintained.
Absence of the 40-kDa form of (2'-5')oligoadenylate synthetase and its corresponding mRNA from skin fibroblasts derived from Alzheimer disease patients.
Authors: Khanna K K , An S J , Wu J M , Landolfo S , Hovanessian A G
Published: May 2006

Cultured skin fibroblasts derived from Alzheimer disease patients fail to express the 1.6-kilobase (kb) mRNA and the corresponding 40-kDa form of (2'-5')oligoadenylate (2-5A) synthetase when exposed to interferon. In addition, the 3.6-kb mRNA, which is present in normal fibroblasts, is barely detectable in the Alzheimer disease counterpart. The deficiency of the 2-5A synthetase 1.6-kb mRNA and its corresponding protein is not related to an impairment of interferon receptors but most probably represents an alteration in the expression of the 2-5A synthetase gene. The data have potential implications for the diagnosis of Alzheimer disease and demonstrate that the absence of a specific form of 2-5A synthetase is linked to a disease state.
Heparin stimulates epidermal growth factor receptor-mediated phosphorylation of tyrosine and threonine residues.
Authors: Revis-Gupta S , Abdel-Ghany M , Koland J , Racker E
Published: May 2006

We have described previously that in extracts of A431 cells epidermal growth factor (EGF) stimulates the phosphorylation of tyrosine as well as of threonine residues in the EGF receptor and in lipocortin 1. We now report that heparin at low concentrations also stimulates the autophosphorylation of the EGF receptor and of the recombinant 56-kDa domain of the EGF receptor that lacks the EGF binding site. To study the stimulations of phosphorylation of threonine residues, a fusion protein was prepared with glutathione S-transferase (GST) and an EGF receptor fragment, TK8 (residues 647-688), that contains the threonine phosphorylation site but no tyrosine. We show that the phosphorylation of threonine residues in GST-TK8 by extracts of A431 cells is stimulated by heparin but not by EGF. These and other results suggest that heparin acts as a chaperone, a substrate modulator, that enhances the susceptibility of the substrate to phosphorylation by protein kinases.
Class I-induced resistance to natural killing: identification of nonpermissive residues in HLA-A2.
Authors: Storkus W J , Salter R D , Alexander J , Ward F E , Ruiz R E , Cresswell P , Dawson J R
Published: May 2006

Structural characteristics of major histocompatibility complex class I antigens associated with natural killer (NK)-resistance phenomena were examined. Previous research has shown that transfection of class I genomic DNA clones into class I-deficient, NK-sensitive target cell lines results in transfectants exhibiting class I+, NK-resistant phenotypes. In contrast to the HLA-A3, -B7, -B27, and -Bw58 class I molecules, the HLA-A2 class I molecules were shown not to protect target cells from NK activity. Here we show that this nonprotective phenotype maps to the alpha 1 domain of the HLA-A2 molecule by examining the NK-protective capacity of the natural interdomain recombinant HLA-Aw69 molecule. HLA-Aw69, which consists of an alpha 1 domain exhibiting homology with HLA-Aw68, and alpha 2/alpha 3/transmembrane-cytoplasmic domains, exhibiting homologies with HLA-A2, mimics HLA-Aw68 and provides HLA-A,B null target cell (C1R) transfectants with increased resistance to NK. Further, the inability of transfected HLA-A2 to confer protection against NK activity can be completely attributed to the expression of a "nonpermissive" residue at position 74 in the alpha 1 domain. Site-directed mutation of the His-74 residue in HLA-A2 to the Asp-74 (HLA-A3, -Aw68, -Aw69, -B7) residue generates a mutant that provides C1R cell line transfectants an NK-resistant phenotype. As His-74 blocks access to a side pocket in the HLA-A2 antigen-binding cleft, these results support the critical involvement of residues within the peptide-binding groove of class I molecules in determining the NK susceptibility phenotype of class I+ target cells.
1H NMR study of renal trimethylamine responses to dehydration and acute volume loading in man.
Authors: Avison M J , Rothman D L , Nixon T W , Long W S , Siegel N J
Published: May 2006

We have used volume-localized 1H NMR spectroscopy to detect and measure changes in medullary trimethylamines (TMAs) in the human kidney in vivo. Localized water-suppressed 1H spectra were collected from a volume of interest located within the renal medulla by using a stimulated echo-based localization scheme. The principal resonances in the medullary 1H spectrum were residual water (4.7 ppm), lipid (0.9-1.4 ppm), and TMAs (3.25 ppm). The TMA line width was 7-15 Hz before filtering, and the signal-to-noise ratio was 40:1. In four normal volunteers, 15 hr of dehydration led to a significant increase in urine osmolality and decrease in body weight and an increase in medullary TMAs. A subsequent water load [20 ml.(kg of body weight)-1] caused a transient water diuresis, a return to euvolemic body weight, and a significant reduction in medullary TMAs within 4 hr. These results suggest that TMAs may play an osmoregulatory role in the medulla of the normal human kidney.
Aminoguanidine effects on nerve blood flow, vascular permeability, electrophysiology, and oxygen free radicals.
Authors: Kihara M , Schmelzer J D , Poduslo J F , Curran G L , Nickander K K , Low P A
Published: May 2006

Since advanced glycosylation end products have been suggested to mediate hyperglycemia-induced microvascular atherogenesis and because aminoguanidine (AG) prevents their generation, we examined whether AG could prevent or ameliorate the physiologic and biochemical indices of streptozotocin (STZ)-induced experimental diabetic neuropathy. Four groups of adult Sprague-Dawley rats were studied: group I received STZ plus AG (25 mg.kg-1.day-1), group II received STZ plus AG (50 mg.kg-1.day-1), group III received STZ alone, and group IV was a control. We monitored conduction and action potential amplitudes serially in sciatic-tibial and caudal nerves, nerve blood flow, oxygen free radical activity (conjugated dienes and hydroperoxides), and the product of the permeability coefficient and surface area to 125I-labeled albumin. STZ-induced diabetes (group III) caused a 57% reduction in nerve blood flow and in abnormal nerve conduction and amplitudes and a 60% increase in conjugated dienes. Nerve blood flow was normalized by 8 weeks with AG (groups I and II) and conduction was significantly improved, in a dose-dependent manner, by 16 and 24 weeks in sciatic-tibial and caudal nerves, respectively. The permeability coefficient was not impaired, suggesting a normal blood-nerve barrier function for albumin, and the oxygen free-radical indices were not ameliorated by AG. We suggest that AG reverses nerve ischemia and more gradually improves their electrophysiology by an action on nerve microvessels. AG may have potential in the treatment of diabetic neuropathy.
Islet-specific T-cell clones from nonobese diabetic mice express heterogeneous T-cell receptors.
Authors: Candéias S , Katz J , Benoist C , Mathis D , Haskins K
Published: May 2006

Nonobese diabetic (NOD) mice spontaneously develop a T-cell-mediated autoimmune disease that is similar in many respects to insulin-dependent diabetes mellitus in humans. T-cell clones that specifically recognize pancreatic islet cell antigens can be derived from NOD mice, and most of these have been diabetogenic upon transfer to healthy recipients. We report herein the sequences of the T-cell receptor alpha and beta chains from four NOD-derived, islet-specific clones. The sequences are quite heterogeneous--in the junctional regions, specifically--so there seems to be little hope for treating this disease with specific anti-T-cell receptor reagents. This result contrasts with the strikingly restricted junctional region sequences reported for the receptors on clones derived from mice with experimental allergic encephalomyelitis, another T-cell-mediated autoimmune disease. We discuss possible explanations for this difference.
Formation of the complex of bovine papillomavirus E1 and E2 proteins is modulated by E2 phosphorylation and depends upon sequences within the carboxyl terminus of E1.
Authors: Lusky M , Fontane E
Published: May 2006

The 68-kDa bovine papillomavirus (BPV) type 1 replication protein E1 and the 48-kDa transactivator protein E2 form a complex that specifically binds DNA [Mohr, I.J., Clark, R., Sun, S., Androphy, E.J., MacPherson, P. & Botchan, M.R. (1990) Science 250, 1694-1699]. We have confirmed this observation and shown that the E1-E2 complex binds to DNA fragments that contain the BPV plasmid maintenance sequence 1 and a site for the initiation of bidirectional BPV DNA synthesis. The E1 protein was found to bind preferentially to non- or underphosphorylated species of E2, suggesting that the phosphorylation state of E2 modulates the association of the two proteins. Replication-deficient E1 mutants with single amino acid substitutions and deletions in the carboxyl terminus failed to interact with E2, indicating that a region in the E1 carboxyl terminus is required for E1 to interact with E2. Our results suggest that the replication deficiency of some E1 mutants reflects their inability to associate with E2.
Nitric oxide mediates glutamate neurotoxicity in primary cortical cultures.
Authors: Dawson V L , Dawson T M , London E D , Bredt D S , Snyder S H
Published: May 2006

Nitric oxide (NO) mediates several biological actions, including relaxation of blood vessels, cytotoxicity of activated macrophages, and formation of cGMP by activation of glutamate receptors in cerebellar slices. Nitric oxide synthase (EC 1.14.23.-) immunoreactivity is colocalized with nicotinamide adenine di-nucleotide phosphate diaphorase in neurons that are uniquely resistant to toxic insults. We show that the nitric oxide synthase inhibitors, N omega-nitro-L-arginine (EC50 = 20 microM) and N omega-monomethyl-L-arginine (EC50 = 170 microM), prevent neurotoxicity elicited by N-methyl-D-aspartate and related excitatory amino acids. This effect is competitively reversed by L-arginine. Depletion of the culture medium of arginine by arginase or arginine-free growth medium completely attenuates N-methyl-D-aspartate toxicity. Sodium nitroprusside, which spontaneously releases NO, produces dose-dependent cell death that parallels cGMP formation. Hemoglobin, which complexes NO, prevents neurotoxic effects of both N-methyl-D-aspartate and sodium nitroprusside. These data establish that NO mediates the neurotoxicity of glutamate.
Molecular identification of a major palmitoylated erythrocyte membrane protein containing the src homology 3 motif.
Authors: Ruff P , Speicher D W , Husain-Chishti A
Published: May 2006

The complete amino acid sequence of a 55-kDa erythrocyte membrane protein was deduced from cDNA clones isolated from a human reticulocyte library. This protein, p55, is copurified during the isolation of dematin, an actin-bundling protein of the erythrocyte membrane cytoskeleton. Fractions enriched in p55 also contain protein kinase activity that completely abolishes the actin-bundling property of purified dematin in vitro. The predicted amino acid sequence of p55 does not contain any consensus sequence corresponding to the catalytic domains of protein kinases but does contain a conserved sequence found in the noncatalytic domains of oncogene-encoded tyrosine kinases. This conserved src homology 3 (SH-3) motif appears to suppress the tyrosine kinase activity of various oncoproteins and has also been found in several plasma membrane associated proteins involved in signal transduction. Northern blot analysis indicated that p55 mRNA was constitutively expressed during erythropoiesis and underwent 2-fold amplification after induction of K562 erythroleukemia cells toward the erythropoietic lineage. The abundant expression of p55 mRNA, along with protein 4.1 mRNA, was evident in terminally differentiated human reticulocytes. Although p55 has many features consistent with known peripheral membrane proteins, its tight association with the plasma membrane is reminiscent of an integral membrane protein. This fact may be partly explained by the observation that p55 is the most extensively palmitoylated protein of the erythrocyte membrane.
N coordination of FeMo cofactor requires His-195 of the MoFe protein alpha subunit and is essential for biological nitrogen fixation.
Authors: Thomann H , Bernardo M , Newton W E , Dean D R
Published: May 2006

Electron spin echo envelope modulation (ESEEM) spectroscopy, a pulsed electron spin resonance technique, was used to analyze the N coordination of the iron-molybdenum (FeMo) cofactor contained within the nitrogenase MoFe protein. Comparison of spectra obtained from whole cells and purified MoFe protein established that the N coordination of the FeMo cofactor provided by the MoFe-protein polypeptide matrix can be unambiguously recognized in whole cells. ESEEM spectra of altered MoFe proteins, which were produced in certain mutant strains of Azotobacter vinelandii, showed that the N coordination to FeMo cofactor requires His-195 of the MoFe protein alpha subunit. Moreover, this requirement for His-195 was shown to be essential for biological nitrogen fixation.
Enhancement of human immunodeficiency virus 1 replication in monocytes by 1,25-dihydroxycholecalciferol.
Authors: Skolnik P R , Jahn B , Wang M Z , Rota T R , Hirsch M S , Krane S M
Published: May 2006

Human immunodeficiency virus (HIV) expression and replication are under tight regulatory control. We demonstrate that 1,25-dihydroxycholecalciferol [1,25-(OH)2D3] enhances the replication of monocyte- and lymphocyte-tropic strains of HIV-1 up to 10,000-fold in monocyte cell lines, peripheral blood monocytes, and unfractionated peripheral blood mononuclear cells. 1,25(OH)2D3 is therefore one of the most potent regulators of HIV-1 replication described to date. Precursors of 1,25(OH)2D3 enhance HIV-1 replication in proportion to their affinity for the 1,25(OH)2D3 intracellular receptor, suggesting that 1,25(OH)2D3 influences HIV-1 replication by mechanisms involving this receptor. These studies may have important implications for the design of effective therapy of HIV-1 infection.
Protein kinase C redistribution within CA3 stratum oriens during acquisition of nictitating membrane conditioning in the rabbit.
Authors: Scharenberg A M , Olds J L , Schreurs B G , Craig A M , Alkon D L
Published: May 2006

This manuscript describes experiments designed to investigate protein kinase C redistribution occurring during acquisition of the rabbit nictitating membrane (NM) conditioned response (CR). The first experiment defined the acquisition phase of the NM response for our laboratory. A group of rabbits (n = 6) was given 2 days of paired NM training; a second group (n = 6) was given 2 days of unpaired NM training. The data document a variable level of responding on day 1 for rabbits given paired training (mean +/- SEM, 21 +/- 11% CRs) but show that on day 2 most rabbits reached the behavioral asymptote (five of six rabbits responding with greater than 85% CRs). Rabbits responding at the behavioral asymptote were defined as having acquired the NM conditioned response. These data were interpreted to indicate that 1 day of training initiated processes necessary for behavioral acquisition (i.e., responding at the behavioral asymptote). A quantitative film autoradiographic study of [3H]phorbol 12,13-dibutyrate binding was then used to determine the distribution of hippocampal protein kinase C in rabbits sacrificed after receiving either 1 day of paired stimuli (n = 10), 1 day of unpaired stimuli (n = 6), or no stimuli (n = 6). Autoradiograms were analyzed by measuring binding in strictly defined regions of interest and from transept profiles. A significant increase in binding of the phorbol ester was found in the CA3 stratum oriens in the paired group relative to unpaired and naive controls. No other significant differences were found.
Altered antigen receptor signaling in anergic T cells from self-tolerant T-cell receptor beta-chain transgenic mice.
Authors: Blackman M A , Finkel T H , Kappler J , Cambier J , Marrack P
Published: May 2006

T-cell tolerance to the minor lymphocyte-stimulating antigen Mls-1a in a T-cell receptor (TcR) V beta 8.1 transgenic line of mice is maintained by both clonal deletion and clonal anergy. Approximately 20-50% of peripheral CD4+ (but not CD8+) T cells isolated from these mice are anergic and fail to proliferate following TcR ligation. We have examined key events in T-cell signaling in peripheral T cells isolated from these mice. In this report, we show that the anergic CD4+ T cells did not mobilize calcium or express receptors for interleukin 2 (IL-2) following TcR ligation. However, the cells retained viability and functional potential because stimulation with phorbol 12-myristate 13-acetate and ionomycin bypassed the block in receptor-mediated signaling and induced IL-2 receptor expression and proliferation of the anergic cells.
Oxathiin carboxanilide, a potent inhibitor of human immunodeficiency virus reproduction.
Authors: Bader J P , McMahon J B , Schultz R J , Narayanan V L , Pierce J B , Harrison W A , Weislow O S , Midelfort C F , Stinson S F , Boyd M R
Published: May 2006

Oxathiin carboxanilide (OC), NSC 615985, a compound originally synthesized as a potential fungicide, was demonstrated to be highly active in preventing human immunodeficiency virus (HIV)-induced cell killing and in inhibiting HIV reproduction. Virus-infected CD4+ lymphocytes were completely protected by 0.5 microM OC, whereas no toxicity was observed at concentrations below 50 microM OC. Production of infectious virus, viral p24 antigen, and virion reverse transcriptase were reduced by OC at concentrations that prevented viral cell killing. A variety of CD4+ T-cell lines were protected by OC from HIV cytopathicity, and OC inhibited two distinct strains of HIV-1. However, HIV-2 infections were unaffected by OC. OC had no direct effect on virions of HIV or on the enzymatic activities of HIV reverse transcriptase or HIV protease. Time-limited treatments of cells with OC before, during, or after exposure of cells to virus failed to protect cells from the eventual cytopathic effects of HIV, and OC failed to inhibit the production of virus from cells in which infection was established or from chronically infected cells. We conclude that the highly active OC has a reversible effect on some early stage of HIV-1 reproduction and cytopathicity. Pilot in vivo experiments showed that circulating concentrations of OC exceeding 1 microM could be achieved and sustained in hamsters for at least a week with no remarkable toxicological sequelae. OC represents a new class of anti-HIV agents that are promising candidates for drug development.
Dosage compensation of the Drosophila pseudoobscura Hsp82 gene and the Drosophila melanogaster Adh gene at ectopic sites in D. melanogaster.
Authors: Sass H , Meselson M
Published: May 2006

Measurements were made of the amounts of larval RNA transcribed from the autosomal Adh gene of Drosophila melanogaster and the X chromosomal Hsp82 gene of Drosophila pseudoobscura carried on the same P-element transposon inserted at various sites in the D. melanogaster genome. Both genes were fully compensated at sites in euchromatic regions of the X chromosome but neither was compensated at a site in the centric beta-heterochromatin of the X chromosome. No compensation of the D. pseudoobscura Hsp82 gene was found at any of 10 autosomal insertion sites tested. The compensation behavior of the transposed genes was, therefore, not determined by closely linked sequences but instead was determined in each case by their new chromosomal environment.
Gamma-interferon promotes proliferation of adult human astrocytes in vitro and reactive gliosis in the adult mouse brain in vivo.
Authors: Yong V W , Moumdjian R , Yong F P , Ruijs T C , Freedman M S , Cashman N , Antel J P
Published: May 2006

Reactive gliosis is a characteristic response of astrocytes to inflammation and trauma of the central nervous system. To investigate whether soluble factors (cytokines) from inflammatory mononuclear cells that accumulate at lesion sites can provide the cellular signals to initiate gliosis and to identify such cytokines, we have tested and found that supernatants derived from subsets of activated human T lymphocytes (CD8+ or CD4+) are potent mitogens for cultured human adult astrocytes. This effect is blocked by a neutralizing antibody to gamma-interferon (IFN). Recombinant IFN alone can induce proliferation of human adult astrocytes in vitro and increase the extent of trauma-initiated gliosis in the adult mouse brain. The astrocyte proliferation-inducing activity of supernatants of glial cultures treated with IFN can be completely blocked with IFN-neutralizing antibody, suggesting that the proliferative effect does not require intermediary cytokines or cells. These results implicate IFN as an important mediator of the gliosis observed in pathologic conditions of the adult central nervous system associated with infiltrating lymphocytes.
A mutant alpha subunit of Gi2 induces neoplastic transformation of Rat-1 cells.
Authors: Pace A M , Wong Y H , Bourne H R
Published: May 2006

In a recently discovered class of oncogenes, GTPase-inhibiting mutations constitutively activate alpha subunits of signal-transducing guanine nucleotide-binding proteins (G proteins). Somatic mutations in a subclass of endocrine tumors are found in the arginine-179 codon of the alpha subunit of Gi2 (alpha i2), creating the putative gip2 oncogene. We have tested the ability of gip2 to mediate neoplastic transformation of Rat-1 and NIH 3T3 fibroblasts in tissue culture. Expression of a mutant alpha i2 cDNA encoding cysteine in place of arginine-179 (alpha i2-R179C) caused Rat-1 cells to grow to a higher density in monolayer culture, to lose anchorage dependence, and to form tumors when injected subcutaneously into nude mice. In contrast, expression of alpha i2-R179C failed to alter growth or tumorigenicity of NIH 3T3 cells. We conclude that gip2 is an oncogene, by the criterion that it induces neoplastic transformation of Rat-1 cells. Failure of gip2 to transform NIH 3T3 cells is in keeping with clinical indications that gip2 is a tissue-selective oncogene.
The Rex regulatory protein of human T-cell lymphotropic virus type I binds specifically to its target site within the viral RNA.
Authors: Unge T , Solomin L , Mellini M , Derse D , Felber B K , Pavlakis G N
Published: May 2006

The Rex protein of human T-cell leukemia virus type I (HTLV-I) was expressed in bacteria and partially purified. Rex was shown to bind in vitro specifically to an RNA sequence located in the 3' long terminal repeat of HTLV-I, named Rex-responsive element (RXRE). Rex also bound in vitro to the human immunodeficiency virus type 1 (HIV-1) Rev-responsive element (RRE), while purified HIV-1 Rev protein did not bind to the RXRE. The binding results obtained in vitro are therefore in agreement with the nonreciprocal function of Rev and Rex in vivo. Rex binds specifically to both RRE and RXRE and activates expression in both HIV-1 and HTLV-I, while Rev binds to RRE and activates only HIV-1. Binding of Rex to RRE deletion mutants previously shown to lack either the Rev-responsive or the Rex-responsive portion suggested preferential binding of Rex to a distinct target within the RRE. These results demonstrated that Rex, like Rev, acts by binding to a specific RNA target.
Glucagon induces disaggregation of polymer-like structures of the alpha subunit of the stimulatory G protein in liver membranes.
Authors: Nakamura S , Rodbell M
Published: May 2006

The hydrodynamic behavior of G alpha s, the alpha subunit of the stimulatory guanine nucleotide-binding regulatory protein (G protein), in octyl glucoside extracts of rat liver membranes was investigated. As was previously shown for G proteins similarly extracted from brain synaptoneurosomes, G alpha s behaved as polydisperse structures with S values higher than that of heterotrimeric G proteins. At concentrations of guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]) greater than 100 microM, incubation with membranes led to smaller structures having S values in the range of 4-5 S. Incubation of liver membranes with glucagon also caused a marked increase in structures having these S values; glucagon action required the presence of low concentrations of GTP[gamma S] (maximal, 10 microM), was rapid (within 10 sec), and was not observed with vasopressin, angiotensin II, or glucagon-(19-29). When G alpha s in its membrane-bound form was [32P]ADP-ribosylated by cholera toxin and the treated membranes were extracted with octyl glucoside, greater than 35% of the labeled G alpha s was found in material that sedimented through sucrose gradients and contained relatively low levels of immunoreactive G alpha s. Glucagon selectively converted the apparently large molecular weight structures to the 4-5 S structures in the presence of GTP[gamma S], even at 1 mM (the maximal effect of the nucleotide alone), when incubated with the toxin-treated membranes. These findings suggest that the glucagon receptor selectively interacts with polymer-like structures of G alpha s and that activation by GTP[gamma S] results in disaggregation. The role of the beta and gamma subunits of G proteins in the hormone-induced process is not clear since the polymer-like structures extracted with octyl glucoside are devoid of beta and gamma subunits.
T-cell receptor peptide immunization leads to enhanced and chronic experimental allergic encephalomyelitis.
Authors: Desquenne-Clark L , Esch T R , Otvos L , Heber-Katz E
Published: May 2006

It has previously been reported that synthetic peptides corresponding to sequences derived from T-cell receptor variable regions identified as dominant in the T-cell-mediated autoimmune disease experimental allergic encephalomyelitis in both the mouse and the rat can down-regulate disease in Lewis rats. In contrast to these results, we have found that immunization of Lewis rats with such peptides in complete Freund's adjuvant prior to induction of experimental allergic encephalomyelitis with myelin basic protein leads to responses ranging from profound disease enhancement to lack of disease. In some cases, enhanced disease was followed by a prolonged neurologic deficit that resembles multiple sclerosis more closely than does acute experimental allergic encephalomyelitis. These findings, on the one hand, support previous results showing T-cell receptor peptide-induced modulation of the disease experimental allergic encephalomyelitis and, on the other, indicate that such immunization is not a reliable method for inducing suppression of encephalitogenic effector cells.
Prevention of human immunodeficiency virus type 1 integrase expression in Escherichia coli by a ribozyme.
Authors: Sioud M , Drlica K
Published: May 2006

Ribozymes are potentially very powerful agents for perturbing intracellular gene expression. However, pilot experiments in eukaryotes have met with mixed success. We now report that a ribozyme designed to cleave the integrase gene of the human immunodeficiency virus (HIV), when transcribed from a plasmid in Escherichia coli, led to destruction of integrase RNA and complete blockage of integrase protein synthesis. These results indicate that ribozymes can be used to study intracellular gene expression in bacteria and that the HIV-1 integrase gene may be a useful target for therapeutic ribozymes.
Localization of heterogeneous nuclear ribonucleoprotein in the interphase nuclear matrix core filaments and on perichromosomal filaments at mitosis.
Authors: He D C , Martin T , Penman S
Published: May 2006

Although heterogeneous nuclear RNA (hnRNA) has been localized to the core filament substructure of the nuclear matrix, its precise location in the filament network has been unknown. The fA12 monoclonal antibody can localize, at high resolution, hn ribonucleoproteins (hnRNPs) and, presumably, hnRNA. Gold bead immunolabeling of resinless electron microscopy sections showed the fA12 antigens were in the fibrogranular material enmeshed in the filament network and not in the filaments themselves. At mitosis, hnRNP antigens became dispersed into a halo surrounding the chromosomes and spindle poles. Immunogold staining showed fA12 stained fibrogranular material associated with perichromosomal and pericentriolar filaments distinct from the mitotic spindle fibers. fA12 also labeled the midbody remaining after cytokinesis.
Repression of the interleukin 6 gene promoter by p53 and the retinoblastoma susceptibility gene product.
Authors: Santhanam U , Ray A , Sehgal P B
Published: May 2006

The aberrant overexpression of interleukin 6 (IL-6) is implicated as an autocrine mechanism in the enhanced proliferation of the neoplastic cell elements in various B- and T-cell malignancies and in some carcinomas and sarcomas; many of these neoplasms have been shown to be associated with a mutated p53 gene. The possibility that wild-type (wt) p53, a nuclear tumor-suppressor protein, but not its transforming mutants might serve to repress IL-6 gene expression was investigated in HeLa cells. We transiently cotransfected these cells with constitutive cytomegalovirus (CMV) enhancer/promoter expression plasmids overproducing wt or mutant human or murine p53 and with appropriate chloramphenicol acetyltransferase (CAT) reporter plasmids containing the promoter elements of human IL-6, c-fos, or beta-actin genes or of porcine major histocompatibility complex (MHC) class I gene in pN-38 to evaluate the effect of the various p53 species on these promoters. Murine and human wt p53 derived from pCMVNc9 and pC53-SN3, respectively, strongly repressed the IL-6 (promoter position -225 to +13), c-fos (-711 to +42), beta-actin (-3400 to +912), and MHC (-528 to -38) promoters in serum-induced HeLa cells; additionally, IL-6 promoter/CAT transcription unit constructs induced by IL-1, phorbol ester, or pseudorabies virus were also repressed by wt human and murine p53. The murine transforming mutant p53 (pCMVc5) was less active in repressing the IL-6, c-fos, beta-actin, and MHC promoter constructs. The human p53 mutant derived from pC53-SCX3 was also less active than the wt protein in repressing the IL-6, c-fos, beta-actin, and MHC promoters, except that serum-induced IL-6/CAT expression was equally repressed by both human wt and mutant p53. In similar transient transfection experiments in HeLa cells, overexpression of the wt human retinoblastoma susceptibility gene product, RB, was found to repress the serum-induced IL-6 (-225 to +13), c-fos (-711 to +42), and beta-actin (-3400 to +912) promoters but not the PRV-induced IL-6 (-110 to +13) or the serum-induced MHC (-528 to -38) promoters. These observations identify transcriptional repression as a property of p53 and suggest that p53 and RB may be involved as transcriptional repressors in modulating IL-6 gene expression during cellular differentiation and oncogenesis.
Recombination by sequence repeats with formation of suppressive or residual mitochondrial DNA in Neurospora.
Authors: Almasan A , Mishra N C
Published: May 2006

Recombination junctions of several Neurospora mitochondrial DNA (mtDNA) mutants and their revertants were identified. Their nucleotide sequences and putative secondary structures were determined in order to understand the nature of the elements involved in intramolecular recombination. Multiple deletions, involving the same portion of Neurospora mtDNA, were identified in six independently isolated mutants. A 9-nucleotide repeat element, CCCCNCCCC, was found to be involved in these and other Neurospora mitochondrial recombination events. The repeat elements were clustered as hot spots on the Neurospora mtDNA and were associated with palindromic DNA sequences. The palindromes have a potential to generate hairpin structures. A much lower free energy of the putative hairpins at the 5' end of the recombination site, and the possible formation of non-B-DNA structure by polypyrimidine tracks, may be important in the initiation of recombination. Using PCR, we found low levels of a specific mitochondrial deletion in certain Neurospora mutants. Their presence in low amounts in a population with a much larger number of normal mtDNA is unexpected. Contrary to earlier belief, this finding supports the view that deleted, smaller DNA molecules are not always suppressive relative to normal mtDNAs.