bluuebunny/crossref_metadata_2025 huggingface dataset:
Transforming growth factor beta 1 is present at sites of extracellular matrix gene expression in human pulmonary fibrosis.
Authors: Broekelmann T J , Limper A H , Colby T V , McDonald J A
Published: May 2006

Idiopathic pulmonary fibrosis is an inexorably fatal disorder characterized by connective tissue deposition within the terminal air spaces resulting in loss of lung function and eventual respiratory failure. Previously, we demonstrated that foci of activated fibroblasts expressing high levels of fibronectin, procollagen, and smooth muscle actin and thus resembling those found in healing wounds are responsible for the connective tissue deposition and scarring in idiopathic pulmonary fibrosis. Using in situ hybridization and immunohistochemistry, we now demonstrate the presence of transforming growth factor beta 1 (TGF-beta 1), a potent profibrotic cytokine, in the foci containing these activated fibroblasts. These results suggest that matrix-associated TGF-beta 1 may serve as a stimulus for the persistent expression of connective tissue genes. One potential source of the TGF-beta 1 is the alveolar macrophage, and we demonstrate the expression of abundant TGF-beta 1 mRNA in alveolar macrophages in lung tissue from patients with idiopathic pulmonary fibrosis.
Single amino acid substitutions dissociate fibrinogen-clotting and thrombomodulin-binding activities of human thrombin.
Authors: Wu Q Y , Sheehan J P , Tsiang M , Lentz S R , Birktoft J J , Sadler J E
Published: May 2006

Thrombin is a serine protease that acts as a procoagulant by clotting fibrinogen and activating platelets and as an anticoagulant by activating protein C in a thrombomodulin-dependent reaction. Fibrinogen and thrombomodulin bind competitively to an anion-binding exosite on thrombin. We prepared recombinant normal human thrombin and mutant thrombins with single amino acid substitutions in order to localize and distinguish the fibrinogen- and thrombomodulin-binding sites. Normal and mutant thrombins had similar amidolytic activity. Thrombin K52E had approximately 2.5-fold increased protein C-activating activity but only approximately 17% of normal fibrinogen-clotting activity. Thrombin R70E had normal fibrinogen-clotting activity but only approximately 7% of normal protein C-activating activity. Thrombin R68E had markedly reduced activity in both assays. Decreased activation of protein C correlated with decreased binding affinity for thrombomodulin, and ability to activate platelets correlated directly with fibrinogen-clotting activity. These results demonstrate that thrombins with predominantly anticoagulant or procoagulant activity can be created by mutagenesis and that thrombomodulin- and fibrinogen-binding sites on thrombin may overlap but are not identical.
Evidence that a downstream pseudoknot is required for translational read-through of the Moloney murine leukemia virus gag stop codon.
Authors: Wills N M , Gesteland R F , Atkins J F
Published: May 2006

Approximately 5% of the ribosomes translating the gag gene of murine leukemia viruses read through the UAG terminator and translate the in-frame pol gene to produce the gag-pol fusion polyprotein, the sole source of the pol gene products. We show that a pseudoknot located eight nucleotides 3' of the UAG codon in the Moloney murine leukemia virus is required for read-through. This requirement is markedly different from that known to be involved in other cases of read-through but surprisingly similar to some stimulatory sequences known to promote ribosomal frameshifting.
Elevation of membrane tyrosine phosphatase activity in density-dependent growth-arrested fibroblasts.
Authors: Pallen C J , Tong P H
Published: May 2006

Swiss 3T3 cells harvested at high density contain a membrane protein-tyrosine-phosphatase (EC 3.1.3.48) whose specific activity is on average 8-fold higher than that of cells harvested at low or medium densities. Investigation of the conditions affecting this elevation of specific activity suggests that it is associated with density-dependent growth arrest. Fibroblasts in the exponentially doubling phase have a relatively low level of membrane phosphatase specific activity, which rises only as the rate of cell proliferation decreases and is maximal when cell growth is contact inhibited. These observations have been extended to BALB/c 3T3 fibroblasts and normal human diploid fibroblasts. The increase in membrane tyrosine phosphatase activity is coupled to density arrest and not to cellular quiescence in general, as no increase in phosphatase specific activity is detected when non-contact-inhibited cells are induced to arrest their growth through serum deprivation. The observed alterations in specific activity are attributable to a tyrosine phosphatase of Mr 37,000 that was purified and characterized from solubilized membrane fractions of Swiss 3T3 cells. A regulatory mechanism controlling tyrosine phosphatase activity may play a role in cell proliferation and growth arrest caused by cell contact.
Double targeted gene replacement for creating null mutants.
Authors: Cruz A , Coburn C M , Beverley S M
Published: May 2006

We have used double gene targeting to create homozygous gene replacements in the protozoan parasite Leishmania major, an asexual diploid. This method uses two independent selectable markers in successive rounds of gene targeting to replace both alleles of an endogenous gene. We developed an improved hygromycin B-resistance cassette encoding hygromycin phosphotransferase (HYG) for use as a selectable marker for Leishmania. HYG-containing vectors functioned equivalently to those containing the neomycin phosphotransferase (NEO) cassette previously used for extrachromosomal transformation or gene targeting. Drug resistances conferred by the NEO and HYG markers were independent, allowing simultaneous selection for both markers. A HYG targeting vector was utilized to replace the single dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene remaining in a line heterozygous for a NEO replacement at the dhfr-ts locus (+/neo), with a targeting efficiency comparable to that seen with wild-type recipients. The resultant dhfr-ts- line (hyg/neo) was auxotrophic for thymidine. The double targeted replacement method will enable functional genetic testing in a variety of asexual diploids, including cultured mammalian cells and fungi such as Candida albicans. Additionally, it may be possible to use Leishmania bearing conditionally auxotrophic gene replacements as safe, improved live vaccines for leishmaniasis.
The human enhancer-binding protein Gata3 binds to several T-cell receptor regulatory elements.
Authors: Marine J , Winoto A
Published: May 2006

The tissue-specific developmental regulation of the alpha, beta, gamma and delta T-cell antigen receptor (TCR) genes is controlled by the corresponding distinct enhancers and their enhancer-binding proteins. To find a common TCR regulatory element, we have studied the ability of the newly described enhancer-binding protein Gata3 to bind to the sequence motif (A/T)GATA(G/A) shared between enhancer elements of all four TCR genes. Gata3 was shown in the chicken to be an enhancer-binding protein containing a zinc-finger domain. We have isolated a full-length human Gata3 cDNA and characterized its role in TCR gene regulation. Northern blot analysis showed that the Gata3 mRNA is present in T-cell, but not B-cell, macrophage or HeLa cell lines. The in vitro transcribed and translated Gata3 cDNA product bound specifically to the enhancer elements of at least three TCR genes containing the above consensus sequence. This binding specificity was consistent with the in vivo activity of the TCR enhancer-binding element in transient transfection assays. Hence, the Gata3 protein, together with other T-cell-specific enhancer-binding proteins, may form the basis of tissue-specific expression of the TCR genes.
Glu-192----Gln substitution in thrombin mimics the catalytic switch induced by thrombomodulin.
Authors: Le Bonniec B F , Esmon C T
Published: May 2006

In serine proteases, residue 192, three residues prior to the active site Ser-195, plays an important role in determining substrate specificity. In trypsin (EC 3.4.21.4) and most trypsin-like enzymes with relatively broad specificity, this position is occupied by Gln. In thrombin (EC 3.4.21.5), an enzyme with restricted specificity, position 192 is occupied by Glu. The potential importance of Glu-192 in restricting the specificity of thrombin was investigated by isosterically replacing Glu-192 with Gln. Unlike trypsin, thrombin cleavage of peptides with acidic residues in positions P3 and P'3 [where P3 and P'3 refer to three residues removed from the Arg (P1) cleavage site on the amino and carboxyl side, respectively] is inefficient. Protein C, an anticoagulant zymogen, has Asp residues in positions P3 and P'3. Thrombomodulin, an endothelial cell protein, complexes with thrombin to activate protein C rapidly thus altering the specificity of thrombin. Compared to thrombin, the Glu-192----Gln mutant thrombin activates protein C 22 times more rapidly and cleaves the P7-P'5 peptide from the protein C activation site 19 times faster. Enhanced protein C activation results primarily from an increase in the catalytic rate constant rather than an improved Michaelis constant, a property that is shared by the thrombin-thrombomodulin complex. The Glu-192----Gln mutation does not influence fibrinopeptide A release and only increases the rate of fibrinopeptide B release 2.7-fold. These results demonstrate that Glu-192 plays a critical role in restricting the specificity of thrombin and suggest that thrombomodulin may function in part by altering the enzyme-substrate interaction near residue 192 in thrombin.
Senile plaque neurites in Alzheimer disease accumulate amyloid precursor protein.
Authors: Cras P , Kawai M , Lowery D , Gonzalez-DeWhitt P , Greenberg B , Perry G
Published: May 2006

Senile plaques are polymorphous beta-amyloid protein deposits found in the brain in Alzheimer disease and normal aging. This beta-amyloid protein is derived from a larger precursor molecule of which neurons are the principal producers in brain. We found that amyloid precursor protein (APP)-immunoreactive neurites were involved in senile plaques and that only a subset of these neurites showed markers for the abnormal filaments characteristic of neurofibrillary pathology. In the neocortex of nondemented individuals with senile plaques but spared of neurofibrillary pathology, dystrophic neurites in senile plaques showed only APP accumulation. In contrast, in the brains of Alzheimer patients, virtually all APP-immunoreactive neurites also showed immunoreactivity with ubiquitin, tau, and phosphorylated neurofilaments. The presence of tau and neurofilament epitopes in dystrophic neurites in senile plaques was correlated with the extent of neurofibrillary pathology in the surrounding brain tissue. Accumulation of APP and the formation of neurofibrillary pathology in senile plaque neurites are therefore distinct phenomena. Our findings suggest that APP accumulation in senile plaque neurites occurs prior to tau accumulation and is therefore more closely related to appearance of neuritic dystrophy.
Molecular cloning and chromosomal localization of one of the human glutamate receptor genes.
Authors: Puckett C , Gomez C M , Korenberg J R , Tung H , Meier T J , Chen X N , Hood L
Published: May 2006

Glutamate receptors are the predominant excitatory neurotransmitter receptors in the mammalian brain and are classified on the basis of their activation by different agonists. The agonists kainate and alpha-amino-3-hydroxy-5-methyl-4-isoxasolepropionic acid define a class of glutamate receptors termed kainate receptors. We have isolated and sequenced a human glutamate receptor (GluHI) cDNA and determined the chromosomal localization of its gene. The DNA sequence of GluHI would encode a 907-amino acid protein that has a 97% identity to one of the rodent kainate receptor subunits. Many of the changes between the predicted amino acid sequence of GluHI and the most similar rodent kainate receptor (GluRI) occur in a region of the protein encoded in rodents by an alternatively spliced exon. The extreme conservation between the human and rat kainate receptor subunits suggests that a similar gene family will encode human kainate receptors. The GluHI mRNA is widely expressed in human brain. The human gene encoding the GluHI subunit is located at 5q33. While the GluHI gene is not located near a chromosomal region associated with any human neurogenetic disorders, the homologous region on mouse chromosome 11 contains the sites of five neurologic mutations.
Angiotensin II receptor subtypes are coupled with distinct signal-transduction mechanisms in neurons and astrocytes from rat brain.
Authors: Sumners C , Tang W , Zelezna B , Raizada M K
Published: May 2006

Both neurons and astrocytes contain specific receptors for angiotensin II (AII). We used selective ligands for the AT1 and AT2 types of AII receptors to investigate the expression of functional receptor subtypes in astrocyte cultures and neuron cultures from 1-day-old (neonatal) rat brain. In astrocyte cultures, competition of 125I-labeled AII (125I-AII) specific binding with AT1 (DuP753) or AT2 (PD123177, CGP42112A, [Phe(p-NH2)6]AII) selective receptor ligands revealed a potency series of AII greater than DuP753 much greater than CGP42112A greater than [Phe(p-NH2)6]AII greater than PD123177. These results suggest a predominance of the AT1 receptor subtype in neonatal astrocytes. Also, in astrocyte cultures, AII stimulated increases in inositolphospholipid hydrolysis that were significantly reduced by the AT1 receptor antagonist DuP753 but not altered by the AT2 receptor antagonist PD123177. In neonatal neuron cultures, competition of 125I-AII specific binding with the above ligands revealed a potency series of CGP42112A = AII greater than [Phe(p- NH2)6]AII greater than PD123177 much greater than DuP753. 125I-AII specific binding to neonate neuronal cultures was reduced 73-84% by 1 microM PD123177, and the residual 125I-AII specific binding was eliminated by DuP753. Also, in neuron cultures, AII induced decreases in basal cGMP that were completely blocked by PD123177 or CGP42112A but not by DuP753. Our results suggest that astrocyte cultures from neonatal rat brains contain predominantly AT1 receptors that are coupled to a stimulation of inositophospholipid hydrolysis. In contrast, neuron cultures from neonatal rat brain contain mostly AT2 receptors that are coupled to a reduction in basal cGMP levels, but a smaller population of AT1 receptors is also present in these neurons.
Leukotriene A4 hydrolase: determination of the three zinc-binding ligands by site-directed mutagenesis and zinc analysis.
Authors: Medina J F , Wetterholm A , Rådmark O , Shapiro R , Haeggström J Z , Vallee B L , Samuelsson B
Published: May 2006

Three mutants of recombinant mouse leukotriene A4 (LTA4) hydrolase (3.3.2.6) were produced by site-directed mutagenesis on cDNA. The codons corresponding to His-295, His-299, or Glu-318 were replaced by codons encoding tyrosine, tyrosine, and glutamine, respectively. The mutated cDNAs were expressed in Escherichia coli, and the three mutated proteins were purified to apparent homogeneity. None of these mutants contained significant amounts of zinc, as determined by atomic absorption spectrometry, and all of them were practically devoid of both LTA4 hydrolase and peptidase enzyme activities. Nevertheless, the mutated proteins could be positively identified by their immunoreactivities with an antiserum for human LTA4 hydrolase in immunoblot analysis. Site-directed mutagenesis was also carried out on human LTA4 hydrolase cDNA. Codons encoding His-295, His-299, and Glu-318 were replaced by ones encoding tyrosine, leucine, and alanine, respectively, and the three mutants were expressed in E. coli. The LTA4 hydrolase activities of the total soluble proteins produced in these expressions were less than 10% of that obtained for bacteria harboring nonmutated cDNA. In agreement with earlier predictions, our experimental data demonstrate that His-295, His-299, and Glu-318 constitute the three ligands of the intrinsic zinc atom in LTA4 hydrolase. Additionally, the combined loss of enzyme activities and zinc content in the purified mutated mouse proteins, emphasizes the critical role of the zinc atom for catalysis, whereas the virtually identical chromatographic behaviors of the mutated and nonmutated mouse LTA4 hydrolase proteins suggest that the metal is of limited importance for the maintenance of the enzyme tertiary structure.
Antisense oligodeoxynucleotide to the cystic fibrosis gene inhibits anion transport in normal cultured sweat duct cells.
Authors: Sorscher E J , Kirk K L , Weaver M L , Jilling T , Blalock J E , LeBoeuf R D
Published: May 2006

We have tested the hypothesis that the cystic fibrosis (CF) gene product, called the CF transmembrane conductance regulator (CFTR), mediates anion transport in normal human sweat duct cells. Sweat duct cells in primary culture were treated with oligodeoxynucleotides that were antisense to the CFTR gene transcript in order to block the expression of the wild-type CFTR. Anion transport in CFTR transcript antisense-treated cells was then assessed with a halide-specific dye, 6-methoxy-N-(3-sulfopropyl)quinolinium, and fluorescent digital imaging microscopy to monitor halide influx and efflux from single sweat duct cells. Antisense oligodeoxynucleotide treatment (3.9 or 1.3 microM) for 24 hr virtually abolished Cl- transport in sweat duct cells compared with untreated cells or control cells treated with sense oligodeoxynucleotides. Br- uptake into sweat duct cells was also blocked after a 24-hr CFTR transcript antisense treatment, but not after treatment for only 4 hr. Lower concentrations of antisense oligodeoxynucleotides were less effective at inhibiting Cl- transport. These results indicate that oligodeoxynucleotides that are antisense to CFTR transcript inhibit sweat duct Cl- permeability in both a time-dependent and dose-dependent manner. This approach provides evidence that inhibition of the expression of the wild-type CFTR gene in a normal, untransfected epithelial cell results in an inhibition of Cl- permeability.
Mapping of human microtubule-associated protein 1B in proximity to the spinal muscular atrophy locus at 5q13.
Authors: Lien L L , Boyce F M , Kleyn P , Brzustowicz L M , Menninger J , Ward D C , Gilliam T C , Kunkel L M
Published: May 2006

A polyclonal antiserum directed against the C-terminal domain of dystrophin was used to isolate a cDNA clone encoding an antigenically cross-reactive protein, microtubule-associated protein 1B (MAP-1B). Physical mapping of the human MAP-1B locus places its chromosomal location at 5q13, in proximity to the spinal muscular atrophy (SMA) locus. SMA is a degenerative disorder primarily affecting motor neurons. Genetic linkage analysis of SMA families using a human dinucleotide repeat polymorphism just 3' of the MAP-1B gene has shown tight linkage to SMA mutations. These mapping data together with the postulated role of MAP-1B in neuronal morphogenesis and its localization in anterior horn motor neurons suggest a possible association with SMA.
Amino acid substitutions in the coat protein result in loss of insect transmissibility of a plant virus.
Authors: Atreya P L , Atreya C D , Pirone T P
Published: May 2006

Amino acids near the N terminus of the coat protein of tobacco vein mottling virus were deleted or altered by site-directed mutagenesis to determine the effect on aphid transmissibility of the virus. Deletion of a three amino acid sequence Asp-Ala-Gly, which is conserved in aphid-transmissible potyvirus isolates, abolished transmission. The mutation Ala----Thr in this triplet drastically reduced transmission, whereas the mutation Asp----Asn had no effect, and the mutation Asp----Lys consistently reverted to the wild-type residue. The mutation Lys----Glu, in the residue adjacent to the glycine of the triplet, drastically reduced transmission, whereas the mutation Gln----Pro, seven residues downstream from the glycine had no effect. Comparison of the sequences of other potyviruses suggests that the presence of a glycine residue at the third position of the Asp-Ala-Gly triplet is critical for aphid transmissibility and that certain changes in the residues adjacent to this position abolish or greatly reduce aphid transmissibility.
In vitro activity of CD4+ and CD8+ T lymphocytes from mice immunized with a synthetic malaria peptide.
Authors: Rénia L , Marussig M S , Grillot D , Pied S , Corradin G , Miltgen F , Del Giudice G , Mazier D
Published: May 2006

In previous work, a T-helper epitope was mapped within the circumsporozoite protein of the murine malaria parasite Plasmodium yoelii. A 21-mer synthetic peptide corresponding to this epitope (amino acid positions 59-79; referred to as Py1) induced a specific T-cell proliferation in BALB/c and C57BL/6 mice and provided help for the production of antibodies to peptides from the repetitive region, (Gln-Gly-Pro-Gly-Ala-Pro)n, of the P. yoelii circumsporozoite protein when mice were immunized with the Py1 peptide conjugated to the repetitive peptide. Experiments were then designed to study the in vitro antiparasite efficacy of T cells elicited in vivo by peptide immunization. T-cell activity was evaluated on cultured hepatic stages of P. yoelii. Peptide immunizations led to the preferential activation of CD8+ T cells in BALB/c mice and of both CD4+ and CD8+ T cells in C57BL/6 mice. Parasite elimination was mediated directly by these cells and did not seem to be dependent on lymphokine secretion. These data suggest that peptide-primed CD4+ T cells as well as CD8+ T cells could be cytolytic for the hepatic phase of malaria parasites. The fact that the same peptide could activate different lymphocyte populations, depending on the strain of mouse, highlights the importance of a better understanding of the fine mechanisms behind the immune responses to synthetic peptides being tested for malaria vaccine development.
Transmembrane signaling by interferon alpha involves diacylglycerol production and activation of the epsilon isoform of protein kinase C in Daudi cells.
Authors: Pfeffer L M , Eisenkraft B L , Reich N C , Improta T , Baxter G , Daniel-Issakani S , Strulovici B
Published: May 2006

The early events that occur after treatment of the highly interferon alpha (IFN-alpha)-sensitive human lymphoblastoid Daudi cell line with human leukocyte IFN-alpha have been examined. IFN-alpha treatment of Daudi cells results in a rapid and transient increase in the cellular content of diacylglycerol, which occurs in the absence of inositol phospholipid turnover, or an increase in intracellular calcium concentration. Furthermore, IFN-alpha treatment results in a selective, time-dependent activation of the Ca(2+)-independent epsilon isoform of protein kinase C (PKC), while the alpha isoform is unaffected by IFN-alpha treatment. In contrast, IFN-alpha treatment of an IFN-resistant subclone of Daudi cells had no effect on the diacylglycerol content of cells and on the activation of PKC-epsilon. The selective PKC inhibitor staurosporine blocked the transcriptional activation of IFN-alpha-stimulated genes, the cytoplasmic accumulation of mRNAs for these genes, and the induction of antiviral activity by IFN-alpha against vesicular stomatitis virus in IFN-sensitive cells. These observations suggest that transmembrane signaling of IFN-alpha involves diacylglycerol production and activation of PKC-epsilon in Daudi cells.
Direction of transneuronal transport of herpes simplex virus 1 in the primate motor system is strain-dependent.
Authors: Zemanick M C , Strick P L , Dix R D
Published: May 2006

We examined the axonal transport of two strains of herpes simplex virus 1 (HSV-1) within the central nervous system of cebus monkeys. Each strain was injected into the "arm area" of the primary motor cortex. One strain, HSV-1(McIntyre-B), was transported transneuronally in the retrograde direction. It infected neurons at sites known to project to the arm area of the primary motor cortex (e.g., ventrolateral thalamus). In addition, "second-order" neurons were labeled in the deep cerebellar nuclei (dentate and interpositus) and in the globus pallidus (internal segment). This result supports the concept that the arm area of the primary motor cortex is a target of both cerebellar and basal ganglia output. In contrast, the other strain, HSV-1(H129), was transported transneuronally in the anterograde direction. It infected neurons at sites known to receive input from the arm area of the primary motor cortex (e.g., putamen, pontine nuclei). In addition, "third-order" neurons were labeled in the cerebellar cortex (granule and Golgi cells) and in the globus pallidus (largely the external segment). Our observations suggest that strain differences have an important impact on the direction of transneuronal transport of HSV-1. Furthermore, it should be possible to examine the organization of cerebellar and basal ganglia loops with cerebral cortex by exploiting transneuronal transport of HSV-1 and virus strain differences.
A first-order reaction controls the binding of antigenic peptides to major histocompatibility complex class II molecules.
Authors: Witt S N , McConnell H M
Published: May 2006

Major histocompatibility complex class II molecules have been reported to bind antigenic peptides very slowly in vitro. To investigate the molecular events that govern the slow binding reaction, we have determined the dependence of complex formation and dissociation on peptide concentration. The complex between the purified major histocompatibility complex class II protein I-Ek and a fluoresceinated peptide representing amino acids 89-104 of pigeon cytochrome c (FpCytc) was studied. Two important results emerge from this study. (i) At pH 5.4, the half-time for I-Ek-FpCytc complex formation is equal to approximately 7 hr for peptide concentrations that vary over a range of three orders of magnitude. There is in fact a small but significant decrease in the half-time for complex formation at low peptide concentrations. The small decrease in half-time is related to the release of endogenous peptides. (ii) At large ratios of peptide to protein [( FpCytc]/[I-Ek] greater than 40), the half-times for I-Ek-FpCytc complex formation and dissociation are equal to one another to within a factor of two between pH 7.5 and 4.5. The percent results demonstrate that a slow, first-order reaction precedes complex formation between I-Ek and FpCytc. This first-order reaction may involve a protein conformational change in addition to the release of endogenous peptides.
Identification of a soluble salicylic acid-binding protein that may function in signal transduction in the plant disease-resistance response.
Authors: Chen Z , Klessig D F
Published: May 2006

It has recently been demonstrated that salicylic acid (SA) may serve as an endogenous signal molecule in the induction of systemic acquired resistance in tobacco and cucumber. In addition, SA is an endogenous regulator of heat and odor production in the inflorescence of some thermogenic plants. No information, however, is currently available concerning the mode(s) of action of SA in plant signal transduction. In a search for possible cellular factors that directly interact with SA, we have detected and partially characterized a SA-binding protein in tobacco leaves. The SA-binding activity is both SDS and proteinase sensitive and behaves as a soluble protein with an apparent mass of 650 kDa. The protein has an apparent Kd of 14 microM for SA, which is consistent with the range of physiological concentrations of SA observed for the induction of plant resistance responses. Furthermore, the ability of SA analogues to compete with SA for binding to this soluble protein is strictly correlated with their biological activity to induce the expression of genes associated with disease resistance. Biologically active analogues effectively inhibit SA binding while biologically inactive analogues do not. These results collectively indicate that this SA-binding protein may play a role in perceiving and transducing the SA signal to appropriate response elements, which ultimately activate one or more of the plant disease-resistance responses.
Identification of a fungal cutinase promoter that is inducible by a plant signal via a phosphorylated trans-acting factor.
Authors: Bajar A , Podila G K , Kolattukudy P E
Published: May 2006

Plant cutin monomers trigger, and glucose suppresses, the expression of the cutinase gene of pathogenic fungi. To identify the cutinase promoter region responsible for induction by the unique plant components, a promoter analysis was done with transformants. Plasmids were constructed that contained (i) the 5' flanking region of the cutinase gene or its deletion mutants from Fusarium solani pisi fused with a chloramphenicol acetyltransferase (CAT) reporter gene and (ii) a constitutive promoter fused with a hygromycin phosphotransferase gene. Hygromycin-resistant transformants of F. solani pisi generated by electroporation were assayed for CAT activity inducible by cutin hydrolysate and for glucose repression of this induction. CAT was induced in a glucose-repressible manner when fused with a 360-base-pair (bp), or longer, segment of the 5' flanking region of the cutinase gene, and deletion of the next 135 bp abolished this induction. Gel retardation assays showed that a protein(s) in nuclear extract from the fungus bound to the 5' flanking region of cutinase gene, and this binding was also abolished when the same 135-bp segment was deleted. These results show that the -225 to -360 segment of the cutinase gene contains a cis-acting regulatory element that binds trans-acting factor(s) in the nuclei. Treatment of the nuclear extract with immobilized phosphatase abolished binding to the promoter, suggesting that binding required a phosphorylated form of the protein. With isolated nuclei, phosphorylation of a protein occurred only in the presence of both cutin monomer and the fungal protein factor. The presence of protein kinase inhibitor H7 during the preincubation of nuclei with the monomer and protein factor inhibited cutinase gene transcription. These results suggest that cutin monomer causes phosphorylation of a transcription factor that binds to the -225 to -360 segment of the cutinase gene and enhances transcription of this gene.
Gene amplification accompanied by the loss of a chromosome containing the native allele and the appearance of the amplified DNA at a new chromosomal location.
Authors: Heard E , Williams S V , Sheer D , Fried M
Published: May 2006

The organization of amplified DNA in mammalian cells in the form of inverted repeats rather than tandem repeats was first observed and studied in the 3B rat cell line. The structure and chromosomal location of the amplified inverted duplications in this cell line have been further analyzed by cloning, long-range mapping, and fluorescence in situ hybridization. The amplification unit is at least 450 kilobases in size and all of the amplicons are located in a single chromosomal location of approximately 10 or 11 megabases. No heterogeneity in either size or molecular structure is detected between the 3B amplicons, indicating that the 20- to 40-fold amplification occurred in a single event and not through a series of events, which would result in heterogeneity among the amplicons. Thus the amplification in 3B cells may reflect more closely the situation seen in tumors containing amplified oncogenes/protooncogenes than the amplifications present in cell lines after multiple selections with cytotoxic drugs. The progenitor Rat-2 cell line contains three alleles of the region of DNA that is amplified in 3B cells; two are located on the two normal homologues of rat chromosome 2 and the third is at the equivalent position on a marker chromosome, der(3)t(2;3). 3B cells contain only one of the two normal homologues of chromosome 2 in addition to chromosome der(3)t(2;3). All of the amplified DNA is located on a new marker chromosome, M2, whose amplified DNA region does not resemble chromosome 2. These results are consistent with the amplification model proposed by Passananti et al. [Passananti, C., Davies, B., Ford, M. & Fried, M. (1987) EMBO J. 6, 1697-1703], in which the excision from a chromosome of the DNA to be amplified results in the loss of rearrangement of that chromosome. In this model the excised DNA can be amplified extrachromosomally during a single S phase before becoming stabilized by integration into a chromosome, probably at a different location to that of its unamplified allele.
Identification of a nonsense mutation in the rod photoreceptor cGMP phosphodiesterase beta-subunit gene of the rd mouse.
Authors: Pittler S J , Baehr W
Published: May 2006

Retinal degeneration in the mouse mutant, rd, was previously shown to be a disorder of cyclic nucleotide metabolism involving a deficiency in the activity of the rod photoreceptor cGMP phosphodiesterase (PDE). We have characterized the normal and rd PDE beta-subunit gene, and their respective transcripts, by PCR and direct sequence analysis. We show that the gene consists of at least 22 exons ranging in size from 48 base pairs to several hundred base pairs, covering greater than 25 kilobases. Within a 67-base-pair exon of the rd PDE beta-subunit gene, we identified a nonsense ochre mutation (a C----A transversion in codon 347) that truncates the normal gene product, eliminating more than one-half of the peptide chain, including the putative catalytic domain. The consequences of the truncation are consistent with the observed phenotypes in rd mice heterozygous and homozygous for the disorder. The nonsense mutation was also found in another related and in six unrelated strains displaying the rd phenotype, indicating that the rd allele arose from a single genetic event. The results strongly argue for the nonsense mutation being responsible for retinal degeneration in the rd mouse.
Endocytosis by the asialoglycoprotein receptor is independent of cytoplasmic serine residues.
Authors: Geffen I , Fuhrer C , Spiess M
Published: May 2006

The human asialoglycoprotein (ASGP) receptor, like most other plasma membrane receptors, has previously been shown to be phosphorylated at serine residues within the cytoplasmic domain. Phorbol esters, which activate protein kinase C, cause hyperphosphorylation and down-regulation of the ASGP receptor in HepG2 cells. To test the importance of serine residues for receptor traffic and function, we have mutated all the cytoplasmic serines of the two receptor subunits H1 (at positions 16 and 37) and H2 (at positions 12, 13, and 55) to alanines or glycines. Stable transfected fibroblast cell lines expressing either mutant H1 alone or both mutant subunits together were created and compared to cell lines expressing the respective wild-type proteins. Mutant and wild-type subunits were found to have very similar distributions between the cell surface and intracellular compartments. Constitutive internalization of H1 alone and ligand uptake and degradation by cells expressing both receptor subunits were not affected by the mutations. Cytoplasmic serines and serine phosphorylation are thus not essential for receptor function and intracellular traffic. Analysis of individual serine mutations identified serine-12 of subunit H2 as the major site of phosphorylation in the ASGP receptor.
The large form of hepatitis delta antigen is crucial for assembly of hepatitis delta virus.
Authors: Chang F L , Chen P J , Tu S J , Wang C J , Chen D S
Published: May 2006

The virions of hepatitis delta virus (HDV) contain two species of HDV-specific protein, a large and a small form of hepatitis delta antigen (HDAg). We examined the role of individual HDAgs in virion assembly in cotransfection experiments. First, we constructed a replication-competent HDV mutant expressing only the small HDAg. When cotransfected with a plasmid expressing hepatitis B virus surface antigens to the HuH-7 cells, the mutant did not produce HDV virions, whereas the wild-type HDV clone did. Therefore, though the small HDAg is important for viral replication and is incorporated into the virus, the small-form delta antigen by itself is insufficient for virion formation. When the system was co-transfected with an additional plasmid providing the large HDAg, the HDV virion was then recovered. There was also evidence suggesting that the large HDAg could be copackaged into the HBsAg particles, without the presence of the HDV genome and the small HDAg. The results indicate a crucial role of the large HDAg in HDV assembly.
Ligand-binding and heterodimerization activities of a conserved region in the ligand-binding domain of the thyroid hormone receptor.
Authors: Spanjaard R A , Darling D S , Chin W W
Published: May 2006

The ligand-binding domain of the thyroid hormone (3,5,3'-triiodothyronine) receptor (TR) contains poorly characterized subdomains involved with ligand binding, transactivation, and protein-protein interactions. The region between residues 288-331 of rat TR alpha-1 was analyzed by modeling and site-directed mutagenesis. Our results suggest that part of this sequence adopts an amphipathic alpha-helical conformation. The integrity of the putative helix is important for 3,5,3'-triiodothyronine binding but not necessarily for heterodimerization with nuclear factor(s). Mutants defective for both activities were found clustered in a region overlapping the C-terminal portion of the helix and further downstream. The sequence conservation of this particular region among the entire superfamily suggests a similar role in dimerization in other receptors.
Metabolic rate of membrane-permeant diacylglycerol and its relation to human resting T-lymphocyte activation.
Authors: Asaoka Y , Oka M , Yoshida K , Nishizuka Y
Published: May 2006

DL-1,2-Dioctanoylglycerol (1,2-DiC8) added to human peripheral resting T lymphocytes was rapidly metabolized to produce octanoic acid and further to small molecules, probably by the action of diacylglycerol lipase and/or nonspecific esterase. Only a small portion was converted to the corresponding phosphatidic acid or was isomerized to 1,3-DiC8 before being metabolized. The uptake of 1,2-DiC8 by the cell was apparently fast, and the rate of disappearance of 1,2-DiC8 was dependent on the cell densities; at a higher density of T lymphocytes 1,2-DiC8 was removed quickly, whereas at a lower cell density 1,2-DiC8 remained for a longer period of time. With a fixed amount of 1,2-DiC8 added, the extent of interleukin 2 receptor alpha-subunit (IL-2R alpha) expression was inversely related to the cell density and proportional to the duration of exposure of the cells to 1,2-DiC8. Repeated doses of 1,2-DiC8 potentiated IL-2R alpha expression. In contrast, a single dose of phorbol 12-myristate 13-acetate caused T-lymphocyte activation to similar extents irrespective of the cell density, probably because the phorbol ester was not metabolized and remained in membranes. The available evidence supports a proposal made in a previous paper and indicates that the sustained activation of protein kinase C for at least the first 3-4 hr is essential for the activation of resting T lymphocytes.
Protective effect of medroxyprogesterone acetate plus testosterone against radiation-induced damage to the reproductive function of male rats and their offspring.
Authors: Jégou B , Velez de la Calle J F , Bauché F
Published: May 2006

This study attempted to protect spermatogenesis and the reproductive performance of rats against the effects of acute scrotal exposure to x-rays. Daily subcutaneous injections of medroxyprogesterone acetate (8 mg/kg) plus testosterone (1 mg/kg) (MT group) were administered for 55 days (experiment A) or 15 days (experiment B). The rats were irradiated (3 grays) on the last day of MT pretreatment (MTX group). In both experiments, on days 1 and 130 posttreatment, rats from each of the four groups (control, x-irradiated, MT, and MTX groups) were killed to measure the weight of the reproductive organs and the number of epididymal spermatozoa. Breeding was started 3 days posttreatment by housing all males from the four groups each with two virgin females for six successive periods of 19 days, separated by a period of 2 days. The percentage of fertile males, the litter size, postimplantation losses, and dominant lethal mutations were calculated. In experiment A, in the last fertility trial, animals of both sexes were selected at random from the progeny of each group (F1). When they were adults, their fertility was tested in a mating trial. A fertility trial was also performed with the F2 males. Our data essentially reveal that (i) in addition to their adverse quantitative effects on spermatogenesis, x-rays also produce a significant increase in dominant lethal mutations in all germ cell classes, including stem spermatogonia; (ii) the F1 and F2 male descendants of irradiated male rats provoked abnormal rates of postimplantation losses in their female mates; (iii) the short as well as the long MT pretreatment protects testicular function of irradiated rats; and (iv) in experiment A, MT pretreatment totally prevented qualitative damage to spermatozoa and protected the descendants of the irradiated animals against altered spermatogenesis as well as against genetic damage in germ cells. In conclusion, pretreatment with MT, even for a short period of time, offers a method for potentially reducing the toxic and genotoxic effects of irradiation on the male reproductive system.
Cell-type-specific and hypoxia-inducible expression of the human erythropoietin gene in transgenic mice.
Authors: Semenza G L , Koury S T , Nejfelt M K , Gearhart J D , Antonarakis S E
Published: May 2006

Synthesis of erythropoietin, the primary humoral regulator of erythropoiesis, in liver and kidney is inducible by anemia or hypoxia. Analysis of human erythropoietin gene expression in transgenic mice revealed that sequences located 6-14 kilobases 5' to the gene direct expression to the kidney, whereas sequences within the immediate 3'-flanking region control hepatocyte-specific expression. Human erythropoietin transcription initiation sites were differentially utilized in liver and kidney. Inducible transgene expression was precisely targeted to peritubular interstitial cells in the renal cortex that synthesize endogenous mouse erythropoietin. These studies demonstrate that multiple erythropoietin gene regulatory elements control cell-type-specific expression and inducibility by a fundamental physiologic stimulus, hypoxia.
Cloning, characterization, and autoimmune recognition of rat islet glutamic acid decarboxylase in insulin-dependent diabetes mellitus.
Authors: Michelsen B K , Petersen J S , Boel E , Møldrup A , Dyrberg T , Madsen O D
Published: May 2006

A 64-kDa islet protein is a major autoantigen in insulin-dependent diabetes mellitus (IDDM). Autoantibodies against the 64-kDa protein were recently shown to immunoprecipitate glutamic acid decarboxylase (GAD; L-glutamate 1-carboxy-lyase, EC 4.1.1.15) from brain and from islets. We present evidence that the autoantisera also recognize a hydrophilic islet protein of approximately 67 kDa in addition to the amphiphilic 64-kDa form. We have isolated a full-length rat islet GAD cDNA encoding a hydrophilic 67-kDa protein, which appears to be identical to rat brain 67-kDa GAD. A partial sequence of human insulinoma 67-kDa GAD was identical to human brain 67-kDa GAD. Allelic variations were observed in rat as well as in human 67-kDa GAD sequences. The expressed rat islet 67-kDa GAD protein is functional and is immunoprecipitated by IDDM sera; it comigrates electrophoretically with the 67-kDa islet autoantigen. The hydrophilic 67-kDa form of GAD in islets is an additional autoantigen in IDDM and is recognized by a different subset of autoantibodies than the 64-kDa autoantigen. Thus, mammalian cell lines expressing functionally active, recombinant GAD may become important tools to study the nature and the role of GAD autoreactivity in IDDM.
Functional heterogeneity of mutant rhodopsins responsible for autosomal dominant retinitis pigmentosa.
Authors: Sung C H , Schneider B G , Agarwal N , Papermaster D S , Nathans J
Published: May 2006

Thirteen mutant rhodopsins responsible for autosomal dominant retinitis pigmentosa (ADRP) have been produced by transfection of cloned cDNA into tissue culture cells. Three mutants [class I: Phe-45----Leu, Gln-344----termination (deletion of C-terminal positions 344-348), and Pro-347----Leu] resemble wild-type rhodopsin in yield, regenerability with 11-cis-retinal, and plasma membrane localization. Ten mutants [class II: Thr-17----Met, Pro-23----His, Thr-58----Arg, Val-87----Asp, Gly-89----Asp, Gly-106----Trp, Arg-135----Leu, Arg-135----Trp, Tyr-178----Cys, and Asp-190----Gly] accumulate to significantly lower levels, regenerate with 11-cis-retinal variably or not at all, and are transported inefficiently to the plasma membrane, remaining primarily in the endoplasmic reticulum. These data suggest that there are at least two distinct biochemical defects associated with different rhodopsin mutants in ADRP.
Expression of a unique 56-kDa polypeptide by neurons in the subplate zone of the developing cerebral cortex.
Authors: Naegele J R , Barnstable C J , Wahle P R
Published: May 2006

In the mammalian cerebral cortex, neurons destined for layers 2-6 are generated only after the period of genesis for a group of transient neurons that populate the subplate and marginal zones. Although a number of molecular markers for the subplate zone exist, most are also expressed by other cell populations in the cortical plate. To begin to study molecular properties of the subplate, we generated monoclonal antibodies against homogenates of cat cortical subplate zone. One monoclonal antibody, termed subplate 1 (SP1), recognized a polypeptide of 56 kDa. This antigen was strongly expressed within the subplate neurons only during a 3-week period beginning at birth and extending until 3 weeks after birth. From postnatal day 1, the number of SP1-immunoreactive neurons below the visual cortex increased until the end of second postnatal week and then declined thereafter. This period coincides with the period when a majority of the subplate neurons undergo naturally occurring cell death. The antigen was not expressed by subplate neurons surviving in the adult white matter. At the peak of antigen expression, 14% or less of the immunoreactive neurons also coexpressed gamma-aminobutyric acid, somatostatin, or neuropeptide Y. Biochemical and immunocytochemical properties of the SP1 antigen were also compared with the Alz-50 antigen (A68), a marker for dying neurons. On Western blots, SP1- and Alz-50-reactive polypeptides were selectively enriched in cytosolic fractions of kitten cerebral cortex, but each marker recognized different molecular weight polypeptides. In tissue sections many subplate, cortical plate, and layer 1 neurons were Alz-50 immunoreactive. In contrast, a rarer subpopulation of neurons restricted to the subplate was labeled by SP1. We propose that the SP1 antigen is a protein expressed within dying cortical subplate neurons, at the commencement of cell death.