bluuebunny/crossref_metadata_2025 huggingface dataset:
Nerve growth factor prevents the amblyopic effects of monocular deprivation.
Authors: Domenici L , Berardi N , Carmignoto G , Vantini G , Maffei L
Published: May 2006

Monocular deprivation early in life causes dramatic changes in the functional organization of mammalian visual cortex and severe reduction in visual acuity and contrast sensitivity of the deprived eye. We tested whether or not these changes could be from competition between the afferents from the two eyes for a target-derived neurotrophic factor. Rats monocularly deprived during early postnatal development were treated with repetitive intraventricular injections or topical administration of nerve growth factor. The effects of monocular deprivation were then assessed electrophysiologically. In untreated animals visual acuity and contrast sensitivity of the deprived eye were strongly reduced, whereas in nerve growth factor-treated animals these parameters were normal.
RNA-mediated ligation of self-cleavage products of a Neurospora mitochondrial plasmid transcript.
Authors: Saville B J , Collins R A
Published: May 2006

Neurospora VS RNA is a mitochondrial single-stranded RNA that combines certain features of catalytic RNAs and group I introns. We report here that monomeric VS RNA synthesized in vitro by self-cleavage of a multimeric transcript can perform an RNA-mediated self-ligation reaction producing circular RNAs indistinguishable from those isolated from mitochondria. We conclude that the active site for the ligation reaction is present in the RNA itself. Also, the mechanism for aligning the termini to be ligated may be different from mechanisms previously described. The lack of sequence similarity between VS RNA and previously characterized catalytic RNAs suggests that VS RNA is an independently evolved ribozyme capable of both cleavage and ligation.
Substrate channeling in glycolysis: a phantom phenomenon.
Authors: Wu X M , Gutfreund H , Lakatos S , Chock P B
Published: May 2006

It has been proposed that glycolytic enzymes form multienzyme complexes for direct transfer of metabolites from the producing enzyme to the utilizing one. Reexamination of the supporting evidence, which involves the transfer of NADH between its complexes with glycerol-3-phosphate dehydrogenase (alpha-glycerol phosphate dehydrogenase, GPDH; EC 1.1.1.8) and with L-lactate dehydrogenase (LDH; EC 1.1.1.27), has shown that the supporting evidence is based on misinterpretation of the kinetics of ligand exchange. Srivastava et al. have responded with a revision of their own and criticism of our data. To clarify this problem, we have carried out detailed kinetic studies on NADH binding to GPHD and LDH and on the displacement of enzyme-bound NADH by LDH or GPDH. The experiments were conducted at 10 degrees C in 50 mM Hepes, pH 7.5/100 mM KCl/1 mM EDTA/1 mM 2-mercaptoethanol, using rabbit muscle GPDH and LDH. The results show that the kinetic patterns exhibited by the displacement of NADH-bound enzyme by either GPDH or LDH are consistent with a dissociative mechanism but not with a direct transfer mechanism. Theoretical analysis shows that a combined dissociative and direct transfer mechanism can explain the transient kinetic data reported by Srivastava et al. if, and only if, a majority (approximately 90%) of the enzyme present in lower concentration exists as a complex with the second enzyme. However, data from tracer and traditional sedimentation equilibrium and from gel filtration experiments show that LDH and GPDH do not form complexes in the presence of saturating NADH concentration when the enzyme concentrations are ranged between 4 and 50 microM, a concentration equal to or greater than that used by Srivastava et al. Our results demonstrate that GPDH and LDH do not form multienzyme complex and the transfer of NADH between these enzymes proceeds via a dissociative mechanism.
Crystal structure of interleukin 8: symbiosis of NMR and crystallography.
Authors: Baldwin E T , Weber I T , St Charles R , Xuan J C , Appella E , Yamada M , Matsushima K , Edwards B F , Clore G M , Gronenborn A M
Published: May 2006

The crystal structure of a host defense system chemotactic factor, interleukin 8, has been solved by molecular replacement using as a model the solution structure derived from nuclear magnetic resonance experiments. The structure was refined with 2 A x-ray data to an R factor of 0.187 (0.217 at 1.6 A). A comparison indicates some potential differences between the structure in solution and in the crystalline state. Our analysis also predicts that residues 4 through 9 on the amino terminus and the beta-bend, which includes His-33, may be important for receptor binding.
Cell-autonomous role of Notch, an epidermal growth factor homologue, in sensory organ differentiation in Drosophila.
Authors: de Celis J F , Marí-Beffa M , García-Bellido A
Published: May 2006

The gene Notch (N) codes for a transmembrane protein with an extracellular domain that has homologies to epidermal growth factors and an intracellular domain that could be involved in signal transduction. N null alleles cause the transformation of most epidermal cells into neuroblasts in central and peripheral nervous systems. Alleles of the same gene, called Abruptex (Ax), that map to the extracellular domain of N protein cause the absence of adult sensory organs. Both types of alleles show cell autonomy in mosaic analysis carried out in the last stages of the formation of adult sensory organs. The phenotypes are different: cells lacking N gene products differentiate as sensory organ mother cells early and as its neural sublineage later, whereas in the homozygous Ax condition epidermal cells do not enter the sensory organ mother cell pathway. The results indicate that N gene products act internally in the cell, probably as receptors of intercellular signals both in sensory organ mother cell singularization and in fate specification of its daughter cells. Ax mutations behave as an excess of N+ function in this signal transduction process. N proteins modified by these mutations act as constitutively activated.
Heterogeneity of thymic epithelial cells in promoting T-lymphocyte differentiation in vivo.
Authors: Gutierrez J C , Palacios R
Published: May 2006

To study in vivo the contribution of different thymic epithelial cells to T-lymphocyte differentiation, we have established several nontransformed thymic epithelial cell lines and developed an in vivo assay, not involving exposure to drugs or radiation, that permitted us to study the capacity of these epithelial lines to support T-cell differentiation. We found that cell lines EA2 and ET, which express markers of cortical epithelial cells, produce interleukin 7 mRNA and after being injected into the spleens of young athymic nude mice support in vivo generation of CD4+CD8- T-cell receptor alpha beta+ T lymphocytes (ET line) or both CD4+CD8- and CD4-CD8+ T-cell receptor alpha beta+ T cells (EA2 line). Both cell lines also supported generation of T-cell receptor gamma delta+ T cells but appear not to support development of double-positive (CD4+CD8+) cells. One cell line, EB3, which expresses markers of medullary epithelial cells, produces interleukin 1 alpha RNA transcripts but does not support T-lymphocyte differentiation. The results provide direct evidence for functional heterogeneity of thymic epithelial cells in vivo and show the involvement of different cortical epithelial cells in the differentiation of T-cell progenitors into distinct thymocyte subsets.
Hairpin formation within the enhancer region of the human enkephalin gene.
Authors: McMurray C T , Wilson W D , Douglass J O
Published: May 2006

The 3',5'-cyclic adenosine monophosphate (cAMP)-inducible enhancer of the human enkephalin gene is located within an imperfect palindrome of 23 base pairs. We have found that a 23-base-pair oligonucleotide duplex containing the enhancer undergoes a reversible conformational transition from the duplex to two individual hairpin structures each formed from one strand of the duplex. Each individual hairpin forms with mismatched base pairs, one containing two GT pairs and the other containing two AC pairs. The conformational transition is stabilized by proton transfer to the hairpin containing AC mismatched pairs. The unique physical and thermodynamic properties of the enkephalin enhancer DNA suggest a model in which DNA secondary structure within the enhancer region plays an active role in cAMP-inducible activation of the human enkephalin gene via formation of cruciform structures.
Overexpression of c-jun, junB, or junD affects cell growth differently.
Authors: Castellazzi M , Spyrou G , La Vista N , Dangy J P , Piu F , Yaniv M , Brun G
Published: May 2006

The coding sequences of murine c-jun, junB, or junD, which code for proteins with practically identical dimerization and DNA binding properties, were introduced into a nondefective retroviral vector, and the phenotype of primary avian fibroblasts chronically infected with each of these viruses was studied. Cells expressing c-jun grew in low-serum medium and developed into colonies in agar, two properties characteristic of in vitro transformation. Cells expressing junB grew in agar, with a reduced efficiency as compared to c-jun, but did not grow in low-serum medium. Finally, no effect of junD expression on cell growth was observed. These different phenotypes suggest that these three closely related transcription factors play distinct roles during normal cell growth. Analysis of c-jun deletion mutants and of c-jun/junB and c-jun/junD chimeric genes showed that the N-terminal portion (amino acids 2-168) of the c-Jun protein that is involved in transcriptional activation is required for efficient transformation. On the contrary, cells expressing a truncated mouse c-Jun lacking this N-terminal domain grew slower than normal embryo fibroblasts. The reduced growth rate may be related to the finding that expression of the intact or the truncated mouse c-jun repressed the endogenous avian c-Jun homologue, suggesting that functional c-Jun product is required for normal cell growth.
X/Y translocations resulting from recombination between homologous sequences on Xp and Yq.
Authors: Yen P H , Tsai S P , Wenger S L , Steele M W , Mohandas T K , Shapiro L J
Published: May 2006

Several regions of sequence homology between the human X and Y chromosomes have been identified. These segments are thought to represent areas of these chromosomes that have engaged in meiotic recombination in relatively recent evolutionary times. Normally, the X and Y chromosomes pair during meiosis and exchange DNA only within the pseudoautosomal region at the distal short arms of both chromosomes. However, it has been suggested that aberrant recombination involving other segments of high homology could be responsible for the production of X/Y translocations. We have studied four X/Y translocation patients using molecular probes detecting homologous sequences on X and Y chromosomes. In one translocation the breakpoints have been isolated and sequenced. The mapping data are consistent with the hypothesis that X/Y translocations arise by homologous recombination. The sequencing data from one translocation demonstrate this directly.
Ca(2+)-channel blockers modulate expression of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and low density lipoprotein receptor genes stimulated by platelet-derived growth factor.
Authors: Block L H , Matthys H , Emmons L R , Perruchoud A , Erne P , Roth M
Published: May 2006

The effects of Ca(2+)-channel blockers (amlodipine, nifedipine, nitrendipine, and verapamil) on expression of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (EC 1.1.1.88) and low density lipoprotein receptor (LDL-R) genes stimulated by recombinant platelet-derived growth factor BB isomer (PDGF-BB) were evaluated in human skin fibroblasts. The drugs enhanced expression of the LDL-R protein on the plasma membrane of the cells; in contrast, they inhibited expression of the HMG-CoA reductase gene. In addition, PDGF-BB-dependent stimulation of transcription of c-fos mRNA was inhibited also by the Ca(2+)-channel blockers. We conclude that PDGF-BB-dependent activation of the two genes is inhibited effectively by the Ca(2+)-channel blockers, at therapeutic concentrations, although they are unable to lower systemic cholesterol levels at these concentrations; however, they do modify responses of the two genes that are involved crucially in regulation of cellular cholesterol homeostasis.
Antioncogenic effect of adenovirus E1A in human tumor cells.
Authors: Frisch S M
Published: May 2006

Stable expression of the adenovirus 5 E1A gene reduced anchorage-independent growth and tumorigenic potential, caused cytoskeletal reorganization, induced flat morphology, and restored contact inhibition in three human tumor cell lines. By these criteria, E1A appears to be functionally indistinguishable from a tumor suppressor gene in this context. The apparent paradox accorded by the observations of the ability of E1A to transform rodent cells in cooperation with other oncogenes suggests that E1A may be the prototype of a class of growth-regulatory proteins having context-specific transforming and antioncogenic activities.
Cloning and characterization of a 3-methyladenine DNA glycosylase cDNA from human cells whose gene maps to chromosome 16.
Authors: Samson L , Derfler B , Boosalis M , Call K
Published: May 2006

We described previously the isolation of a Saccharomyces cerevisiae 3-methyladenine (3-MeAde) DNA glycosylase repair gene (MAG) by its expression in glycosylase-deficient Escherichia coli alkA tag mutant cells and its ability to rescue these cells from the toxic effects of alkylating agents. Here we extend this cross-species functional complementation approach to the isolation of a full-length human 3-MeAde DNA glycosylase cDNA that rescues alkA tag E. coli from killing by methyl methanesulfonate, and we have mapped the gene to human chromosome 16. The cloned cDNA, expressed from the pBR322 beta-lactamase promoter, contains an 894-base-pair open reading frame encoding a 32,894-Da protein able to release 3-MeAde, but not 7-methylguanine, from alkylated DNA. Surprisingly, the predicted human protein does not share significant amino acid sequence homology with the bacterial AlkA and Tag glycosylases or the yeast MAG glycosylase, but it does share extensive amino acid sequence homology with a rat 3-MeAde DNA glycosylase and significant DNA sequence homology with genes from several mammalian species. The cloning of a human 3-MeAde DNA glycosylase cDNA represents a key step in generating 3-MeAde repair-deficient cells and the determination of the in vivo role of this DNA repair enzyme in protecting against the toxic and carcinogenic effects of alkylating agents.
Antigenic variation in Plasmodium falciparum.
Authors: Biggs B A , Goozé L , Wycherley K , Wollish W , Southwell B , Leech J H , Brown G V
Published: May 2006

Antigenic variation of infectious organisms is a major factor in evasion of the host immune response. However, there has been no definitive demonstration of this phenomenon in the malaria parasite Plasmodium falciparum. In this study, cloned parasites were examined serologically and biochemically for the expression of erythrocyte surface antigens. A cloned line of P. falciparum gave rise to progeny that expressed antigenically distinct forms of an erythrocyte surface antigen but were otherwise identical. This demonstrates that antigenic differences on the surface of P. falciparum-infected erythrocytes can arise by antigenic variation of clonal parasite populations. The antigenic differences were shown to result from antigenic variation of the parasite-encoded protein, the P. falciparum erythrocyte membrane protein 1.
Widespread expression of MyoD genes in Xenopus embryos is amplified in presumptive muscle as a delayed response to mesoderm induction.
Authors: Harvey R P
Published: May 2006

The MyoD gene codes for an important regulatory factor in skeletal myogenesis. To explore the relationship between mesoderm induction in Xenopus embryos and expression of MyoD, I have monitored MyoD mRNA levels in normal embryos and cultured explants by RNase protection. Transcription from the two Xenopus MyoD gene copies is activated weakly across the whole embryo at the midblastula transition, and this activation occurs in the absence of mesoderm induction. In response to induction this basal expression is amplified 50- to 100-fold, but in animal-pole explants 6-10 hr elapse before induced mRNAs appear, and this induction requires prior protein synthesis. The promiscuous transcripts disappear from animal explants at a time when induction "competence" is lost, suggesting a link between these events. The data highlight a broad, but transient, permissiveness for MyoD expression in embryos, which is propagated and amplified only in presumptive muscle in response to induction. Moreover, muscle-specific MyoD expression is a relatively late (postgastrulation) event in the mesoderm-induction cascade.
Suppression of H-2b-associated resistance to Friend erythroleukemia virus by a class I gene from the H-2d major histocompatibility complex haplotype.
Authors: Polsky D , Lilly F
Published: May 2006

Mice homozygous for the H-2d haplotype at the major histocompatibility complex are markedly more susceptible to erythroleukemia induction by the Friend isolate of murine leukemia retrovirus (FV) than are congenic mice homozygous for the H-2b haplotype. The resistance conferred by the H-2b haplotype is recessive in this cross, since heterozygous F1 mice are as susceptible as parental strain H-2d homozygotes. However, H-2b-associated resistance is not an intrinsically recessive trait, since H-2b/H-2dm1 heterozygotes resemble H-2b homozygotes in their relative resistance to FV; the mutant H-2dm1 haplotype lacks the entire D region of the parental haplotype except for a single class I gene formed by the fusion of its terminal D-region genes to produce a class I gene differing from both parental genes, and thus this finding indicates that one or more D-region genes of the H-2d haplotype can actively suppress H-2b-associated resistance. Unlike H-2dm1, the mutant H-2dm2 haplotype, which retains only the class IDd gene in the D region of the H-2d haplotype, strongly suppresses resistance in H-2b/H-2dm2 heterozygotes, and the presence of Dd as a transgene significantly reduces the resistance of H-2b homozygotes. Since H-2b-associated resistance to FV appears to be due mainly to the capacity of Lb (also called Db), the only class I molecule encoded in the D region of the H-2b haplotype, to present viral epitopes for recognition by FV-specific cytotoxic T lymphocytes, suppression of resistance to FV by the Dd molecule implies that the presence of one class I molecule of the major histocompatibility complex can interfere with either the presentation of viral epitopes by another class I molecule or the generation of T cells that recognize viral epitopes so presented.
Inhibition of cAMP accumulation by intracellular calcium mobilization in C6-2B cells stably transfected with substance K receptor cDNA.
Authors: DeBernardi M A , Seki T , Brooker G
Published: May 2006

C6-2B rat glioma cells were stably transfected with substance K receptor cDNA and used to study interactions between cAMP and Ca2+ signaling pathways. Activation of the newly expressed receptors by substance K increased the intracellular free Ca2+ concentration, as monitored by single-cell fura-2 imaging, and markedly inhibited agonist-stimulated cAMP accumulation. Blockade of intracellular Ca2+ mobilization abolished the substance K receptor-mediated inhibition of isoproterenol-induced cAMP production. Phosphodiesterase inhibitors, down-regulation or inhibition of protein kinase C, and pertussis toxin failed to prevent substance K-induced inhibition of agonist-stimulated cAMP accumulation. An increased intracellular Ca2+ concentration caused by either calcium ionophores or activation of endogenous bradykinin receptors was found to markedly reduce cAMP production in wild-type cells. These results demonstrate that elevated intracellular Ca2+ concentration can negatively modulate agonist-stimulated adenylate cyclase activity in C6-2B glioma cells.
Hydrophobic ion transfer between membranes of adjacent hepatocytes: a possible probe of tight junction structure.
Authors: Turin L , Béhé P , Plonsky I , Dunina-Barkovskaya A
Published: May 2006

The topology of the tight junction is probed by introducing dipicrylamine (dpa-), a lipid-soluble anion, into the membranes of hepatocyte pairs in culture. Once partitioned into the membrane, dpa- ions are free to move in the hydrophobic core of the membrane, where their mobile charges greatly increase membrane capacitance. If tight junctions are lines of membrane fusion, dpa- will cross the tight junction without traversing a polar headgroup layer. Furthermore, the electric potential across the tight junction will be equal to the difference in membrane potentials of the two cells. dpa- can therefore be expected to move electrophoretically from cell membrane to cell membrane across the junction in response to an intercellular voltage difference. Experiments performed under double whole-cell clamp show that this transfer occurs as follows: First, dpa- causes an intercellular current unrelated to gap junctions to flow in response to an intercellular voltage difference. Second, this electrophoretic removal or addition of dpa- from a cell's membrane through the tight junction must reduce or increase its dpa- content and thus its capacitance. Experiments confirm this prediction: We detect rapid, symmetric, and reversible changes in membrane capacitance in response to changes in the membrane potential of the neighboring cell. Finally, we find that hepatocyte membranes contain a negatively charged endogenous molecule that contain a negatively charged endogenous molecule that can move from cell to cell like dpa- under the influence of an intercellular potential difference. We conclude that membrane fusion occurs at tight junctions and that this hydrophobic intercellular pathway can play a role in intercellular communication.
Cross-reactions and specificities of monoclonal antibodies against myelin basic protein and against the synthetic copolymer 1.
Authors: Teitelbaum D , Aharoni R , Sela M , Arnon R
Published: May 2006

Antibody cross-reactivity is here demonstrated between basic protein (BP), the encephalitogenic molecule of myelin, and copolymer 1 (Cop 1), the synthetic amino acid copolymer, which has a suppressive effect on experimental allergic encephalomyelitis and is effective in reducing the number of relapses in exacerbating-remitting multiple sclerosis. This cross-reactivity is conclusively established using mouse monoclonal antibodies (mAbs). About a third of anti-rat BP mAbs and most of anti-mouse BP mAbs cross-reacted with Cop 1. This cross-reactivity could be demonstrated with anti-BP mAbs of different specificities. In addition, several anti-Cop 1 hybridomas cross-reacted with BP. This cross-reactivity was verified in several assay systems, including competitive inhibition experiments. Moreover, some anti-BP mAbs and anti-Cop 1 mAbs reacted in a heteroclitic manner and favored the cross-reactive antigen over the immunogen. In contrast to the mAbs, no cross-reactivity could be demonstrated with the antisera of immunized mice. This observation may reflect the different B-cell populations expressed in the mAb response as compared to the polyclonal response. Thus, the use of mAbs has uncovered specificities that are not evident in antisera and has revealed pronounced cross-reactivity between BP and Cop 1 at the B-cell level. These results further establish the immunological interrelationships between Cop 1 and BP, demonstrated earlier at the T-cell level.
Fc receptors for IgG (Fc gamma Rs) on human monocytes and macrophages are not infectivity receptors for human immunodeficiency virus type 1 (HIV-1): studies using bispecific antibodies to target HIV-1 to various myeloid cell surface molecules, including the Fc gamma R.
Authors: Connor R I , Dinces N B , Howell A L , Romet-Lemonne J L , Pasquali J L , Fanger M W
Published: May 2006

Fc gamma Rs (Fc gamma RI, Fc gamma RII, and Fc gamma RIII) are highly expressed on human mononuclear phagocytes and function in the clearance of immune complexes and opsonized pathogens. We have examined the role of Fc gamma R in mediating antibody-dependent clearance of HIV-1 by human monocytes and monocyte-derived macrophages by using bispecific antibodies (BsAbs) to independently target the virus to Fc gamma RI, Fc gamma RII, or Fc gamma RIII. Virus production was markedly reduced in monocytes cultured with strain HIV-1IIIB opsonized with BsAbs that target the virus to either Fc gamma RI or Fc gamma RII compared to monocytes cultured with virus in the absence of BsAbs or in the presence of BsAbs that target the virus to non-Fc gamma R surface antigens (CD33 and HLA-A,B,C). These results were confirmed using the monotropic isolate HIV-1JRFL. Interaction of HIV-1JRFL with Fc gamma RI or Fc gamma RII on human monocytes and Fc gamma RI, Fc gamma RII, or Fc gamma RIII on monocyte-derived macrophages resulted in markedly reduced levels of virus production in these cultures. Moreover, HIV-1 infection of monocytes and monocyte-derived macrophages was completely blocked by anti-CD4 monoclonal antibodies, indicating that interaction with CD4 is required for infectivity even under conditions of antibody-mediated binding of HIV-1 to Fc gamma R. Thus, we propose that highly opsonized HIV-1 initiates high-affinity multivalent interactions with Fc gamma R that trigger endocytosis and intracellular degradation of the antibody-virus complex. At lower levels of antibody opsonization, there are two few interactions with Fc gamma R to initiate endocytosis and intracellular degradation of the antibody-virus complex, but there are enough interactions to stabilize the virus at the cell surface, allowing antibody-dependent enhancement of HIV-1 infection through high-affinity CD4 interactions. However, our results suggest that interaction of highly opsonized HIV-1 with Fc gamma Rs through BsAbs may reduce viral infectivity through Fc gamma R-mediated cytotoxic mechanisms and, therefore, that BsAbs offer promise as therapeutic reagents in HIV-1 infections.
On the use of combinatorial antibody libraries to clone the "fossil record" of an individual's immune response.
Authors: Lerner R A , Barbas C F , Kang A S , Burton D R
Published: May 2006

For about the last century the record of immunological events could only be obtained from serum proteins. We suggest that in the future this will change and the far more detailed nucleic acid record will provide new insights into immunological processes as well as selective access to the complete repertoire.
Transforming growth factor alpha contributes to the mechanism by which hypothalamic injury induces precocious puberty.
Authors: Junier M P , Ma Y J , Costa M E , Hoffman G , Hill D F , Ojeda S R
Published: May 2006

It has long been known that lesions of the hypothalamus lead to female sexual precocity. While an increased production of luteinizing hormone-releasing hormone (LHRH), the neurohormone that controls sexual development, appears to mediate the advancement of puberty induced by these lesions, little is known about the mechanism(s) by which hypothalamic injury activates LHRH secretion. Since brain lesions result in accumulation of neurotrophic/mitogenic activities in the injured area, we tested the hypothesis that transforming growth factor alpha (TGF-alpha), a mitogenic polypeptide recently shown to stimulate LHRH release, is produced in response to hypothalamic injury and mediates the effect of the lesion on puberty. Radiofrequency lesions of the preoptic area-anterior hypothalamic area (POA-AHA) of 22-day-old female rats resulted in precocious puberty within 7 days after the operation. RNA blot hybridization revealed that lesion-induced puberty was preceded by an increase in TGF-alpha mRNA levels in the POA-AHA. Epidermal growth factor (EGF) mRNA was undetectable in both intact and lesioned hypothalami. TGF-alpha mRNA levels, quantitated by RNase protection assays, were 3.5-fold greater in lesioned animals approaching puberty than in age-matched controls. Immunohistochemical studies, utilizing single- and double-staining procedures, demonstrated the presence of TGF-alpha precursor-like immunoreactivity in reactive astrocytes surrounding the lesion site. Hybridization histochemistry showed increased TGF-alpha mRNA expression in cells of the same area, further implicating reactive astrocytes as a site of TGF-alpha synthesis. The actions of TGF-alpha are mediated by its interaction with EGF receptors. Continuous infusion of RG-50864, an inhibitor of EGF receptor kinase activity, at the site of injury prevented the advancement of puberty induced by the lesion. These results suggest that TGF-alpha acting via EGF-like receptors contributes to the acceleration of puberty induced by anterior hypothalamic lesions. They also indicate that activation of TGF-alpha gene expression in glial cells is a component of the hypothalamic response to injury.
Spectral kinetics of glutamate-1-semialdehyde aminomutase of Synechococcus.
Authors: Smith M A , Grimm B , Kannangara C G , von Wettstein D
Published: May 2006

Purified Synechococcus glutamate-1-semialdehyde aminotransferase (GSA-AT; EC 5.4.3.8) has absorption maxima characteristic of vitamin B6-containing enzymes and can be converted to the pyridoxamine 5'-phosphate or pyridoxal 5'-phosphate form by reaction with diaminovalerate or dioxovalerate, respectively, suggesting that these two analogues are intermediates in the conversion of glutamate 1-semialdehyde (GSA) to 5-aminolevulinate (ALA). Values for Km and kmax were calculated for GSA, diaminovalerate, ALA, and gabaculine from absorption change rates during conversion of one coenzyme form of GSA-AT to the other, upon addition of one of these compounds. The substrate specificity (kmax/Km) of diaminovalerate is about 3 orders of magnitude larger than that of dioxovalerate, making the latter an unlikely intermediate in the enzymic conversion of GSA to ALA. GSA reacts with both coenzyme forms, whereas ALA only reacts with the pyridoxamine 5'-phosphate form of the enzyme. However, ALA does form a complex with the pyridoxal 5'-phosphate form of GSA-AT and inhibits reactions between gabaculine and GSA-AT. This relatively stable complex (Ki = 8 M) may have significance in enzyme inhibition. Both L and D enantiomers of GSA react with GSA-AT. Spectral changes observed upon addition of DL-GSA are apparently due to reaction with the less reactive D-isomer. L-GSA is converted to ALA prior to major spectral changes induced by the racemic mixture.
Clone pAT 133 identifies a gene that encodes another human member of a class of growth factor-induced genes with almost identical zinc-finger domains.
Authors: Müller H J , Skerka C , Bialonski A , Zipfel P F
Published: May 2006

We report the structure and regulation of a gene represented by clone pAT 133, which is induced upon transition from a resting state (G0) through the early phase of the cell cycle (G1). The pAT 133 gene is immediately induced, with FOS-like kinetics, in human T cells and in fibroblasts. Primary structure analysis showed that the encoded protein contains three tandem zinc-finger sequences of the type Cys2-Xaa12-His2. This zinc-finger region, which is thought to bind DNA in a sequence-specific manner, is similar (greater than 80% on the amino acid level) to two previously described transcription factors pAT 225/EGR1 and pAT 591/EGR2. Except for the conserved zinc-finger domains, the amino acid sequences of the three proteins are distinct. This structural similarity suggests that the pAT 133 gene encodes a transcription factor with a specific biological function. Comparing the regulation of these related zinc-finger-encoding genes showed coordinate induction upon mitogenic stimulation of resting T lymphocytes and of resting fibroblasts. However, upon transition from a proliferating (G1) to a resting state of the cell cycle the three genes were differently regulated. In human histiocytic U937 cells mRNA of clone pAT 133 was constitutively expressed, whereas mRNA of pAT 225/EGR1 was induced upon induction of terminal differentiation. In contrast mRNA representing pAT 591/EGR2 was not expressed in these cells. This difference in gene regulation suggests distinct biological roles in the control of cell proliferation for the respective proteins.
Relationship between alternatively spliced exons and functional domains in tropomyosin.
Authors: Cho Y J , Hitchcock-DeGregori S E
Published: May 2006

Smooth and striated muscle alpha-tropomyosins differ as a consequence of alternative splicing of exons 2 and 9 encoding amino acid residues 39-80 and 258-284, respectively [Ruiz-Opazo, N., Weinberger, J. & Nadal-Ginard, B. (1985) Nature (London) 315, 67-70]. To understand the relationship between alternatively spliced exons and functional domains in tropomyosin, recombinant unacetylated striated muscle, smooth muscle, and chimeric rat alpha-tropomyosins (+H3N-tropomyosins) expressed in and purified from Escherichia coli were analyzed. The functional differences between the isoforms can be primarily ascribed to exon 9. +H3N-Tropomyosins with the smooth muscle exon 9 bound to skeletal muscle filamentous actin with at least a 5-fold higher affinity than +H3N-tropomyosins with the striated muscle exon 9. On the other hand, in the presence of Ca2+, troponin increased the affinity of +H3N-tropomyosins with the striated muscle exon 9 at least 50-fold, whereas it had little effect on +H3N-tropomyosins with the smooth muscle exon 9. The unique striated muscle alpha-tropomyosin exon 9 seems to be specialized for Ca(2+)-insensitive interaction with troponin on the thin filament. The unique smooth muscle alpha-tropomyosin exon 2 was associated with a slightly lower actin affinity than the striated muscle exon 2. Although the regions encoded by exons 2 and 9 correspond to functional domains, they are not recognizable as independent units or structural domains in the extended coiled-coil structure of this fibrous actin binding protein.
Evolution of gene position: chromosomal arrangement and sequence comparison of the Drosophila melanogaster and Drosophila virilis sina and Rh4 genes.
Authors: Neufeld T P , Carthew R W , Rubin G M
Published: May 2006

The seven in absentia (sina) gene of Drosophila encodes a nuclear protein required for normal eye development. In Drosophila melanogaster, the sina gene is located within an intron of the Rh4 opsin gene. We examine here the nucleotide sequences and chromosomal arrangements of these genes in Drosophila virilis. An interspecies comparison between D. melanogaster and D. virilis reveals that the protein-coding sequences of the sina and Rh4 genes are highly conserved, but the relative chromosomal position and structural arrangement of these genes differ between the two species. In particular, the sina and Rh4 genes are widely separated in D. virilis, and there is no intron in the Rh4 gene. Our results suggest that the Rh4 gene was translocated to another chromosomal location by a retrotransposition event.
Specific lysis of human immunodeficiency virus type 1-infected cells by a HLA-A3.1-restricted CD8+ cytotoxic T-lymphocyte clone that recognizes a conserved peptide sequence within the gp41 subunit of the envelope protein.
Authors: Takahashi K , Dai L C , Fuerst T R , Biddison W E , Earl P L , Moss B , Ennis F A
Published: May 2006

A HLA-A3.1-restricted CD8+ cytotoxic T-cell clone, E7.20, that lyses cells infected with human immunodeficiency virus type 1 was isolated from an infected individual. The epitope was localized to amino acids 768-778 (RLRDLLLIVTR, NL43 env sequence) of the cytoplasmic domain of gp41 by successive use of a panel of recombinant vaccinia viruses that express truncated env genes and synthetic peptides. The epitope is conserved on 7 (NL43, BRU, HXB2, BRVA, SC, JH3, and JFL) of 13 human immunodeficiency virus type 1 isolates from North America. Synthetic peptides of this region of strains RF and CDC4 are also recognized by E7.20 despite a nonconservative Thr----Val or Thr----Ala change at amino acid 777; however, an MN peptide, which has four amino acid substitutions, was not reactive. The epitope recognized by E7.20 has a predicted hydrophobic alpha-helical structure, with three contiguous Leu residues followed by Ile and Val at amino acids 772-776. Cytotoxicity was restricted by HLA-A3.1 using allogeneic target cells that shared HLA class I antigens with the donor and an HLA-A and -B negative human plasma cell line transfected with the HLA-A3.1 gene. The transfected cells were infectable by human immunodeficiency virus type 1 strains IIIB and MN but only the former virus sensitized them to killing by E7.20. The ability of E7.20 to specifically lyse a human lymphocyte line infected with a human immunodeficiency virus type 1 strain carrying the conserved epitope is consistent with an important role for cytotoxic T cells in controlling infection.
The predominant form of the amyloid beta-protein precursor in human brain is protease nexin 2.
Authors: Van Nostrand W E , Farrow J S , Wagner S L , Bhasin R , Goldgaber D , Cotman C W , Cunningham D D
Published: May 2006

The amyloid beta protein and the amyloid beta-protein precursor (APP) are major constituents of senile plaques and cerebrovascular deposits in patients with Alzheimer disease and Down syndrome. Most human tissues contain mRNA that encodes forms of APP that contain the Kunitz protease inhibitor (KPI+) domain. A major 120-kDa protein corresponding to this KPI+ mRNA is also found in these tissues. This protein is identical to the protease inhibitor protease nexin 2. Brain contains an additional mRNA species that encodes a form of APP that lacks the KPI domain (KPI-). This latter mRNA has been suggested to encode a 105-kDa KPI- form of APP protein also found in brain. Using protease inhibitory functional assays, we show that both the 105-kDa and 120-kDa APP proteins in normal and Alzheimer disease brain contain the KPI domain. Moreover, KPI domain-specific precipitation assays reveal that KPI- forms of APP protein represent less than 14% of total brain APP. Lastly, an enriched fraction from total brain homogenate contains proteolytic activity that can process the purified 120-kDa KPI+ form of APP into a 105-kDa form, resulting in a high-molecular-mass doublet identical to that seen in brain. These findings indicate that although KPI- APP mRNA is abundant in brain, little corresponding protein is present. Thus, KPI+ APP protein (equivalent to protease nexin 2) is the predominant form of APP in human brain.
Identification of the staphylococcal enterotoxin A superantigen binding site in the beta 1 domain of the human histocompatibility antigen HLA-DR.
Authors: Herman A , Labrecque N , Thibodeau J , Marrack P , Kappler J W , Sekaly R P
Published: May 2006

The staphylococcal enterotoxin A (SEA) is a superantigen that must bind to class II molecules of the major histocompatibility complex to be recognized by T cells. In humans, most HLA-DR class II allelic and isotypic forms, such as DR1, bind SEA well. DRw53 is an exception, binding SEA very poorly. We have localized this difference to a single residue (amino acid 81) in the beta 1 domain. A highly conserved histidine at residue 81 allows SEA binding, but a tyrosine does not. Residue 81 is predicted to lie in an alpha-helix on the surface of the molecule, with its side chain pointing up out of the pocket associated with binding of conventional peptide antigens. This finding supports the hypothesis that superantigens and conventional antigens bind to different sites on the class II molecule.
Phosphorylation controls binding of acidic proteins to the ribosome.
Authors: Naranda T , Ballesta J P
Published: May 2006

The replacement of each one of the eight serine residues present in the amino acid sequence of the Saccharomyces cerevisiae acidic ribosomal phosphoprotein YP2 beta (L45) by different amino acids has been performed by heteroduplex site-directed mutagenesis in the cloned gene. The mutated DNA was used to transform a yeast strain previously deprived of the original protein YP2 beta (L45) by gene disruption. The replacement of serine in position 19 by either alanine, aspartic acid, or threonine prevents in vivo phosphorylation of the protein and its interaction with the ribosome. The serine-19 mutated gene is unable to rescue the negative effect on the growth rate caused by elimination of the original protein in YP2 beta (L45) gene disrupted strains. The mutation of any one of the other seven serine residues has no effect on either the phosphorylation or the ribosome binding capacity of the protein, although replacement of serine-72 seems to increase the sensitivity of the polypeptide to degradation. These results provide strong evidence indicating that ribosomal protein phosphorylation plays an important part in the activity of the particle and that it supports the existence of a control mechanism of protein synthesis, which would regulate the level of phosphorylation of acidic proteins.
Transfer of oxygen from an artificial protease to peptide carbon during proteolysis.
Authors: Rana T M , Meares C F
Published: May 2006

Site-specific cleavage of proteins with metal chelates is an approach for designing artificial proteolytic reagents that are directed by proximity to a peptide bond rather than by an amino acid residue type. In the presence of ascorbate and H2O2, an iron chelate attached to Cys-212 of the enzyme human carbonic anhydrase I quickly cleaved the protein between residues Leu-189 and Asp-190 to produce two discrete fragments. The transfer of an 18O atom from [18O]H2O2 (or [18O]O2) to the carboxyl group of Leu-189 was demonstrated by mass spectrometry. Quantitative experiments revealed that one molecule of H2O2 and one molecule of ascorbate afforded the hydrolysis of one peptide bond (1:1:1 stoichiometry) and that the reaction required ascorbate and H2O2. The process is catalytic, since related experiments on the protein bovine serum albumin revealed two cleavage events for each polypeptide chain cleaved. Hydroxyl radical scavengers had no significant effect. These results may be explained by generation of a highly nucleophilic oxygen species, such as peroxide coordinated to the iron chelate, that attacks a carbonyl carbon nearby.
Evidence for common ancestry of a chestnut blight hypovirulence-associated double-stranded RNA and a group of positive-strand RNA plant viruses.
Authors: Koonin E V , Choi G H , Nuss D L , Shapira R , Carrington J C
Published: May 2006

Computer-assisted analysis of the putative polypeptide products encoded by the two open reading frames present in a large virus-like double-stranded RNA, L-dsRNA, associated with hypovirulence of the chestnut blight fungus, Cryphonectria parasitica, revealed five distinct domains with significant sequence similarity to previously described conserved domains within plant potyvirus-encoded polyproteins. These included the putative RNA-dependent RNA polymerase, RNA helicase, two papain-like cysteine proteases related to the potyvirus helper-component protease, and a cysteine-rich domain of unknown function similar to the N-terminal portion of the potyvirus helper-component protein. Phylogenetic trees derived from the alignment of the polymerase domains of L-dsRNA, a subset of positive-stranded RNA viruses, and double-stranded RNA viruses, using three independent algorithms, suggested that the hypovirulence-associated dsRNA and potyvirus genomes share a common ancestry. However, comparison of the organization of the conserved domains within the encoded polyproteins of the respective viruses indicated that the proposed subsequent evolution involved extensive genome rearrangement.
Mouse kappa light-chain recombination signal sequences mediate recombination more frequently than do those of lambda light chain.
Authors: Ramsden D A , Wu G E
Published: May 2006

Immunoglobulin and T-cell receptor genes are somatically rearranged by site-specific recombination. Recombination signal sequences (RSS) have been identified as the major targeting element of this process. Recent reports demonstrate that differences in RSS affect the frequency of recombination, suggesting a role for RSS in the development of the B-cell repertoire. Examination of mouse light-chain RSS indicates that kappa light-chain RSS consistently show a greater degree of similarity to a consensus sequence than do those of lambda light chain. To determine whether this difference in natural RSS could affect the patterns of light-chain gene rearrangement and expression, we have constructed recombination substrates containing both a typical mouse kappa RSS pair and a typical mouse lambda RSS pair. Experiments using these substrates demonstrate that the kappa RSS pair mediates recombination at a vastly higher frequency than does the lambda RSS pair. This result argues that RSS differences may contribute significantly to the patterns of mouse immunoglobulin light-chain rearrangement, ultimately resulting in a high proportion of kappa light chain relative to lambda.
Inhibition of the activation of Hageman factor (factor XII) by human vascular endothelial cell culture supernates.
Authors: Ratnoff O D , Everson B , Embury P , Ziats N P , Anderson J M , Emanuelson M M , Malemud C J
Published: May 2006

The supernatant fluid (conditioned medium) of cultured human vascular endothelial cells inhibits activation of Hageman factor (factor XII), whether by ellagic acid, bovine brain sulfatides, or bismuth subgallate; inhibition appears to be a property of one or more proteins in the culture supernates. This phenomenon may contribute to maintaining the fluidity of circulating blood by inhibiting surface activation of the intrinsic pathway of coagulation.
A mutation in a Rhodobacter capsulatus gene encoding an integration host factor-like protein impairs in vivo hydrogenase expression.
Authors: Toussaint B , Bosc C , Richaud P , Colbeau A , Vignais P M
Published: May 2006

A gene capable of encoding a protein sharing 45% identical amino acids with the alpha subunit of the integration host factor (IHF) of Escherichia coli was isolated from the photosynthetic bacterium Rhodobacter capsulatus strain B10 by complementation of a hydrogenase-deficient (Hup-) mutant, IR4. A DNA fragment of 274 base pairs containing an IHF binding consensus sequence, isolated from the promoter region of the hydrogenase structural genes (hupSL), was shown by gel retardation assays to bind the IHF protein from E. coli. The product of the R. capsulatus gene was shown to bind specifically to the 274-base-pair DNA fragment from the hupSL promoter. By analogy to the E. coli himA gene, which encodes the alpha subunit of IHF, the gene complementing the IR4 mutant was named himA of R. capsulatus. The wild-type himA gene, cloned in plasmid pBO2, was introduced into the IR4 strain and shown to restore, in trans, hydrogenase activity and autotrophic growth in the mutant. In IR4, a C----T transition mutation had replaced Arg-8 by Cys-8. Gel mobility shifts of the 274-base-pair DNA fragment, not observed with the himA gene product of IR4, were restored with extracts from IR4(pBO2) cells, containing the himA gene on the recombinant plasmid pBO2.