bluuebunny/crossref_metadata_2025 huggingface dataset:
Transphosphorylation as the mechanism by which the high-affinity receptor for IgE is phosphorylated upon aggregation.
Authors: Pribluda V S , Pribluda C , Metzger H
Published: May 2006

When aggregated, the high-affinity receptors for IgE on mast cells (Fc epsilon RI) launch a series of phosphorylations, particularly of protein tyrosines. We have analyzed how aggregation initiates this cascade. We examined Fc epsilon RI from unstimulated cells and from cells exposed to a polyvalent hapten conjugate that aggregates the Fc epsilon RI via the receptor-bound anti-hapten IgE. We also examined the latter receptors after they had been disaggregated in vitro with monovalent hapten. By an in vitro kinase assay: (i) Unaggregated and disaggregated receptors are associated with a kinase that phosphorylates an exogenous (peptide) substrate but minimally, or not at all, the subunits of Fc epsilon RI or associated proteins (endogenous substrates). After aggregation, phosphorylation of the exogenous substrate is linear with time, but the modification of the endogenous substrates reaches a plateau, presumably because only those endogenous substrates that are adjacent to the kinase are phosphorylated. (ii) Aggregated receptors and disaggregated receptors have enhanced kinase activity toward exogenous substrate. The state of phosphorylation of the receptor correlates strongly with the yield of enhanced kinase activity. We propose that upon aggregation of Fc epsilon RI, a constitutively associated kinase phosphorylates endogenous substrates by transphosphorylation. As a result, additional kinase activity becomes manifest and this promotes further transphosphorylation. In view of the homology between Fc epsilon RI and other receptors central to the immune response, the latter receptors likely utilize a similar transphosphorylation mechanism.
Construction, expression, and immunogenicity of the Schistosoma mansoni P28 glutathione S-transferase as a genetic fusion to tetanus toxin fragment C in a live Aro attenuated vaccine strain of Salmonella.
Authors: Khan C M , Villarreal-Ramos B , Pierce R J , Riveau G , Demarco de Hormaeche R , McNeill H , Ali T , Fairweather N , Chatfield S , Capron A
Published: May 2006

A vector has been constructed to allow genetic fusions of guest antigens via a hinge domain to the C terminus of the highly immunogenic C fragment of tetanus toxin. A fusion has been constructed with the gene encoding the protective 28-kDa glutathione S-transferase (EC 2.5.1.18) from Schistosoma mansoni. The recombinant vector has been electroporated into the nonvirulent Salmonella typhimurium aroA live vaccine strain SL3261. The corresponding chimeric protein is stably expressed in a soluble form in Salmonella as evaluated by Western blotting with fragment C and glutathione S-transferase antisera. Mice immunized intravenously with a single dose of the live recombinant bacteria elicit antibodies to both fragment C and glutathione S-transferase as detected by enzyme-linked immunosorbent assays. Furthermore, all of the mice were solidly protected when challenged with lethal doses of either tetanus toxin or the virulent Salmonella typhimurium strain C5. Mice have also elicited antibodies to fragment C and glutathione S-transferase after oral immunization. It may be that a live trivalent vaccine against typhoid, tetanus, and schistosomiasis is feasible.
Characterization of calcium-binding sites in the kidney stone inhibitor glycoprotein nephrocalcin with vanadyl ions: electron paramagnetic resonance and electron nuclear double resonance spectroscopy.
Authors: Mustafi D , Nakagawa Y
Published: May 2006

Nephrocalcin (NC) is a calcium-binding glycoprotein of 14,000 molecular weight. It inhibits the growth of calcium oxalate monohydrate crystals in renal tubules. The NC used in this study was isolated from bovine kidney tissue and purified with the use of DEAE-cellulose chromatography into four isoforms, designated as fractions A-D. They differ primarily according to the content of phosphate and gamma-carboxy-glutamic acid. Fractions A and B are strong inhibitors of the growth of calcium oxalate monohydrate crystal, whereas fractions C and D inhibit crystal growth weakly. Fraction A, with the highest Ca(2+)-binding affinity, was characterized with respect to its metal-binding sites by using the vanadyl ion (VO2+) as a paramagnetic probe in electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopic studies. By EPR spectrometric titration, it was shown that fraction A of NC bound VO2+ with a stoichiometry of metal:protein binding of 4:1. Also, the binding of VO2+ to NC was shown to be competitive with Ca2+. Only protein residues were detected by proton ENDOR as ligands, and these ligands bound with complete exclusion of solvent from the inner coordination sphere of the metal ion. This type of metal-binding environment, as derived from VO(2+)-reconstituted NC, differs significantly from the binding sites in other Ca(2+)-binding proteins.
Polysialic acid engineering: synthesis of polysialylated neoglycosphingolipids by using the polysialyltransferase from neuroinvasive Escherichia coli K1.
Authors: Cho J W , Troy F A
Published: May 2006

The CMP-sialic acid:poly alpha 2,8sialosyl sialyltransferase (polyST) in neurotropic Escherichia coli K1 inner membranes catalyzes synthesis of the alpha 2,8-linked polysialic acid capsule. The capsule is a neurovirulent determinant associated with neonatal meningitis in humans. A functionally similar polyST in human neuroblastomas polysialylates neural cell adhesion molecules. While bacteria do not synthesize glycosphingolipids (GSLs), we report here that the E. coli K1 polyST can selectively polysialylate several structurally related GSLs, when added as exogenous sialyl acceptors. A structural feature common to the preferred sialyl acceptors (GD3 > GT1a > GQ1b = GT1b > GD2 = GD1b = GD1a > GM1) was the disialyl glycotope, Sia alpha 2,8Sia, alpha 2,3-linked to galactose (Sia is sialic acid). A linear tetrasaccharide with a terminal Sia residue (e.g., GD3) was the minimum length oligosaccharide recognized by the polyST. Endo-N-acylneuraminidase was used to confirm the alpha 2,8-specific polysialylation of GSL. Ceramide glycanase was used to release the polysialyllactose chains from the ceramide moiety. Size analysis of these chains showed that 60-80 Sia residues were transferred to the disialyllactose moiety of GD3. The significance of these findings is two-fold. (i) The E. coli K1 polyST can be used as a synthetic reagent to enzymatically engineer the glycosyl moiety of GSL, thus creating oligo- or polysialylated GSLs. Such "designer" GSLs may have potentially important biological and pharmacological properties. (ii) The use of GSLs as exogenous sialyl acceptors increases the sensitivity of detecting polyST activity. The practical advantage of this finding is that polyST activity can be identified and studied in those eukaryotic cells that express low levels of this developmentally regulated enzyme and/or its acceptor.
Cyclical behavior of genotype frequencies in a two-locus population under fluctuating haploid selection.
Authors: Kirzhner V M , Korol A B , Ronin Y I , Nevo E
Published: May 2006

Numerical iterations have shown that periodic haploid two-locus selection with nonoverlapping generations can produce large-scale cyclical behavior with a period differing significantly from the environmental period. Among other types, we observed slowly damping oscillations with very long periods, stable T-cycles with a length containing several environmental periods, and chaotic-like dynamics. Possible biological implications are discussed.
The peptide p2Ca is immunodominant in allorecognition of Ld by beta chain variable region V beta 8+ but not V beta 8- strains.
Authors: Connolly J M
Published: May 2006

An explanation for the vigorous allograft rejection that results from the recognition by CD8+ T cells of allogeneic major histocompatibility complex (MHC) molecules has long eluded immunologists. Recent evidence has demonstrated that alloreactivity involves recognition of both the allogeneic MHC molecule and its associated peptide ligand, suggesting the current theory that the strength of the allogeneic response results from the participation of numerous peptides. However, I report here that a single peptide, p2Ca, is immunodominant in allorecognition of the murine MHC class I molecule H-2Ld. The majority of Ld-alloreactive T-cell clones are specific for Ld-p2Ca and this immunodominance is not due to peptide cross-reactivity. Generation of Ld-alloreactive cytotoxic T lymphocytes in a strain tolerant to p2Ca did not affect the peptide immunodominance, demonstrating that tolerance to p2Ca is MHC-restricted. The p2Ca-specific clones express beta chain variable region V beta 8 T-cell receptors, however, Ld-alloreactive cytotoxic T lymphocytes generated in V beta 8- mice are not dominated by recognition of p2Ca, suggesting that the T-cell receptor repertoire is a factor in determining peptide immunodominance.
Molecular evolution of the MyoD family of transcription factors.
Authors: Atchley W R , Fitch W M , Bronner-Fraser M
Published: May 2006

Myogenesis in skeletal muscle is a cascade of developmental events whose initiation involves the MyoD family of transcription factors. Evolutionary analyses of amino acid sequences of this family of transcriptional activators suggest that the vertebrate genes MyoD1, myf-5, Myog (myogenin), and myf-6 were derived by gene duplications from a single ancestral gene. A common genetic origin predicts some functional redundancy between MyoD1 and myf-5 and between Myog and myf-6. Experimental studies have suggested that these pairs of genes can substitute for each other during myogenesis. Separate analyses of the conserved basic helix-loop-helix and nonconserved flanking elements yield similar branching sequences but show evolutionary change in the basic helix-loop-helix region has occurred at a much slower rate.
Interspecific transfer of mitochondrial genes in fungi and creation of a homologous hybrid gene.
Authors: Paquin B , Laforest M J , Lang B F
Published: May 2006

In eukaryotes, horizontal gene transfer is a rare event. Here we show that the mitochondrial genome of a lower fungus, Allomyces macrogynus, has an extra DNA segment not present in a close relative, Allomyces arbusculus. This insert consists of the C terminus of a foreign gene encoding a subunit of the ATP synthetase complex (atp6) plus an open reading frame encoding an endonuclease. The inserted atp6 portion is fused in phase to the resident gene, resulting in expression of a hybrid atp6 gene and the displacement of the original C-terminal atp6 region. We present evidence that this insertion may have been acquired by interspecific transfer and we discuss the possible role of the endonuclease in this process.
Targeted mutation in the neurotrophin-3 gene results in loss of muscle sensory neurons.
Authors: Tessarollo L , Vogel K S , Palko M E , Reid S W , Parada L F
Published: May 2006

Neurotrophin 3 (NT-3) is one of four related polypeptide growth factors that share structural and functional homology to nerve growth factor (NGF). NT-3 and its receptor, called neurotrophic tyrosine kinase receptor type 3 (Ntrk3; also called TrkC), are expressed early and throughout embryogenesis. We have inactivated the NT-3 gene in embryonic stem (ES) cells by homologous recombination. The mutated allele has been transmitted through the mouse germ line, and heterozygote intercrosses have yielded homozygous mutant newborn pups. The NT-3-deficient mutants fail to thrive and exhibit severe neurological dysfunction. Analysis of mutant embryos uncovers loss of Ntrk3/TrkC-expressing sensory neurons and abnormalities at early stages of sensory neuronal development. NT-3-deficient mice will permit further study of the role of this neurotrophin in neural development.
Expression of the allatostatin gene in endocrine cells of the cockroach midgut.
Authors: Reichwald K , Unnithan G C , Davis N T , Agricola H , Feyereisen R
Published: May 2006

Cockroach allatostatins are neuropeptides that have been isolated from the brain of Diploptera punctata and shown to inhibit juvenile hormone production by the corpora allata. Enzyme-linked immunoassay and immunocytochemistry with antisera to two allatostatins, ASB2 (AYSYVSEYKRLPVYNFGL-NH2) and ASAL (APSGAQRLYGFGL-NH2), revealed that allatostatins were located not only in the insect brain but also in several peripheral tissues including the cockroach midgut and hindgut. Allatostatin-like immunoreactivity was found in nerve fibers of the stomatogastric nervous system as well as in intrinsic endocrine cells of the midgut. Midgut extracts were shown to be biologically active in an allatostatin bioassay and to contain several allatostatin-like peptides, including the octadecapeptide ASB2, which was identified by mass spectrometry following HPLC purification. Reverse transcription of brain mRNA followed by PCR with degenerate oligonucleotides for ASB2 and ASAL yielded a 338-bp fragment of the allatostatin gene that encoded six allatostatins. In situ hybridization with this probe confirmed that an allatostatin gene is expressed in intrinsic endocrine cells of the midgut. Reverse transcription of midgut mRNA followed by PCR and sequencing of the product revealed that the same gene is expressed in the midgut and in the brain. Allatostatins are thus an example of insect "brain-gut peptides" and we suggest that their function may not be restricted to the regulation of juvenile hormone production.
Isolation of a DEAD-family protein gene that encodes a murine homolog of Drosophila vasa and its specific expression in germ cell lineage.
Authors: Fujiwara Y , Komiya T , Kawabata H , Sato M , Fujimoto H , Furusawa M , Noce T
Published: May 2006

In an effort to study the molecular basis of the determination processes of the mammalian germ cell lineage, we have tried to isolate a mouse gene homolog to vasa, which plays an essential role as a maternal determining factor for the formation of Drosophila germ cell precursors. By reverse transcriptase PCRs of mouse primordial germ cell cDNAs using family-specific primers, we obtained a gene (Mvh) encoding a DEAD-family protein that showed a much higher degree of similarity with the product of the Drosophila vasa gene (vas) than previously reported mouse genes. In adult tissues, Mvh transcripts were exclusively detected in testicular germ cells, in which Mvh protein was found to be localized in cytoplasm of spermatocytes and round spermatids including a perinuclear granule. The protein was also expressed in germ cells colonized in embryonic gonads but was not detected in pluripotential embryonic cells such as stem cells and germ cells. These results suggest the possibility that the Mvh protein may play an important role in the determination events of mouse germ cells as in the case of Drosophila vasa.
New Cretaceous marsupial from Mongolia and the early radiation of Metatheria.
Authors: Trofimov B A , Szalay F S
Published: May 2006

A marsupial mammal from Mongolia representing a previously unreported group, Asiadelphia, offers unequivocal evidence that metatherians were represented on the continent of Asia during the Late Cretaceous (Campanian), even in the northern arid interior. Asiatherium is known by skull, left and right mandibles, and most of its postcranial skeleton. Comparisons of the dentition, ear region, and aspects of the postcranium not only allow its diagnosis as an Asian group of metatherians but also permit a diagnosis of the Metatheria based on the unequivocally apomorphous condition of the dental formula and probable replacement pattern of the protometatherian, in contrast to the more primitive therian dental formula, represented by the few known Aegialodontidae and the first dental eutherians. It appears an inescapable conclusion that the first metatherians had a more derived dental formula (and probably replacement pattern) than the earliest dental eutherians, regardless of what specific derivation from a therian ancestry is contemplated. Such a diagnosis also supports the metatherian status of other Cretaceous Asian and American taxa such as the Deltatheroida and Kokopellia. The hypothesis that Metatheria originated in North America is largely dependent on the preponderance of Cretaceous forms from North America and negative evidence from Asia (i.e., on the lack of lower latitude Early Cretaceous tropical faunas). Conversely, the relative Cretaceous paucity of placentals from North America and their greater abundance in Asia suggests the earliest flowering of the Eutheria in an unspecified region of the Old World. The concept of a holophyletic Theria (= Tribosphenida) employed here, based on the biologically significant apomorphy of the molar form-function of the first therian, entails only the tribosphenic mammals (infraclasses Tribotheria, Metatheria, and Eutheria) but not the sundry quasi-triangular-toothed mammals and their derivatives in the Mesozoic (Eupantotheria, Peramura, Monotremata, etc.). It is suggested that the Asiadelphia and Deltatheroida, the protospecies of both, with the apomorphic therian dental formula, are ancient lineages of Metatheria independent from their North American relatives probably since the early part of the Late Cretaceous.
Binding of Vav to Grb2 through dimerization of Src homology 3 domains.
Authors: Ye Z S , Baltimore D
Published: May 2006

The protooncogenic protein Vav has the structure of an intracellular signal transducer. It is exclusively expressed in cells of hematopoietic lineage and plays a crucial role in hematopoietic cell differentiation. Here we report that both in cell extracts and within intact mammalian cells Vav binds to Grb2 (Sem-5/ASH/Drk), an adaptor molecule which plays a key role in Ras activation. The interaction became evident from a yeast two-hybrid screen and its specificity was demonstrated by in vitro binding assays. It is mediated by an unusual protein-protein binding reaction: dimerization of specific intact Src homology 3 domains of each of the partners. Signaling during hematopoietic lineage differentiation may therefore involve the tissue-specific signal transducer Vav linking into the ubiquitous pathway involving Grb2 and ultimately Ras.
Exposure of human ovarian carcinoma to cisplatin transiently sensitizes the tumor cells for liposome-mediated gene transfer.
Authors: Son K , Huang L
Published: May 2006

Human ovarian carcinoma cells (line 2008) grown as subcutaneous solid tumor in the severe combined immunodeficient mouse can be transfected by directly injecting a plasmid DNA-liposome complex into the tumor (in situ lipofection). The level of reporter gene expression in the tumor cells was significantly elevated if the animal received a single i.p. injection of cisplatin 1 week before the lipofection. Sensitization of the tumor for lipofection peaked 1 week after cisplatin injection and declined thereafter. Cells exposed to low concentration of cisplatin in vitro for four to five doubling times also showed elevated sensitivity for lipofection in vitro. Cisplatin was the only anticancer drug tested that exhibited this activity. These results suggest a sequential combinational gene therapy protocol with cisplatin for the ovarian carcinoma.
A second class I ribonucleotide reductase in Enterobacteriaceae: characterization of the Salmonella typhimurium enzyme.
Authors: Jordan A , Pontis E , Atta M , Krook M , Gibert I , Barbé J , Reichard P
Published: May 2006

The nrdA and nrdB genes of Escherichia coli and Salmonella typhimurium encode the R1 and R2 proteins that together form an active class I ribonucleotide reductase. Both organisms contain two additional chromosomal genes, nrdE and nrdF, whose corresponding protein sequences show some homology to the products of the genes nrdA and nrdB. When present on a plasmid, nrdE and nrdF together complement mutations in nrdA or nrdB. We have now obtained in nearly homogeneous form the two proteins encoded by the S. typhimurium nrdE and nrdF genes (R1E and R2F). They correspond to the R1 and R2 proteins. Each protein is a homodimer. Together they catalyze the reduction of CDP to dCDP, using dithiothreitol or reduced glutaredoxin, but not thioredoxin, as an electron donor. CDP reduction is strongly stimulated by low concentrations of dATP, presumably acting as an allosteric effector. Protein R2F contains an antiferromagnetically coupled dinuclear iron center and a tyrosyl free radical. The E. coli and S. typhimurium chromosome thus have maintained the information for a potentially active additional class I ribonucleotide reductase, whose role in vivo is as yet unknown. The allosteric regulation of this enzyme differs from that of the normally expressed reductase.
Fourier-transform infrared spectroscopy and gas chromatography-mass spectrometry reveal a remarkable degree of structural damage in the DNA of wild fish exposed to toxic chemicals.
Authors: Malins D C , Gunselman S J
Published: May 2006

The use of gas chromatography-mass spectrometry with selected ion monitoring (GC-MS/SIM) and Fourier-transform infrared (FT-IR) spectroscopy revealed a remarkable degree of damage in the hepatic DNA of fish exposed to toxic environmental chemicals, compared with controls. The exposed fish, which were neoplasm-free, were part of a population with a high incidence of liver cancer. GC-MS/SIM showed markedly high concentrations of hydroxyl radical-induced ring-opening products (e.g., 2,6-diamino-4-hydroxy-5-formamidopyrimidine) and 8-hydroxy adducts of adenine and guanine (e.g., 8-hydroxyguanine) in the DNA. FT-IR spectroscopy revealed substantial changes in spectral areas, such as those assigned to NH vibrations of nucleotide bases and CO vibrations of deoxyribose. This diverse and extensive damage to DNA provides a perspective of premalignant changes resulting from xenobiotic exposure and a promising basis for predicting cancer risk in animals and humans.
Stimulation of endogenous ADP-ribosylation by brefeldin A.
Authors: De Matteis M A , Di Girolamo M , Colanzi A , Pallas M , Di Tullio G , McDonald L J , Moss J , Santini G , Bannykh S , Corda D
Published: May 2006

Brefeldin A (BFA) is a fungal metabolite that exerts profound and generally inhibitory actions on membrane transport. At least some of the BFA effects are due to inhibition of the GDP-GTP exchange on the ADP-ribosylation factor (ARF) catalyzed by membrane protein(s). ARF activation is likely to be a key event in the association of non-clathrin coat components, including ARF itself, onto transport organelles. ARF, in addition to participating in membrane transport, is known to function as a cofactor in the enzymatic activity of cholera toxin, a bacterial ADP-ribosyltransferase. In this study we have examined whether BFA, in addition to inhibiting membrane transport, might affect endogenous ADP-ribosylation in eukaryotic cells. Two cytosolic proteins of 38 and 50 kDa were enzymatically ADP-ribosylated in the presence of BFA in cellular extracts. The 38-kDa substrate was tentatively identified as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase. The BFA-binding components mediating inhibition of membrane traffic and stimulation of ADP-ribosylation appear to have the same ligand specificity. These data demonstrate the existence of a BFA-sensitive mono(ADP-ribosyl)transferase that may play a role in membrane movements.
Chimeric restriction endonuclease.
Authors: Kim Y G , Chandrasegaran S
Published: May 2006

Fok I restriction endonuclease recognizes the nonpalindromic pentadeoxyribonucleotide 5'-GGATG-3'.5'-CATCC-3' in duplex DNA and cleaves 9 and 13 nt away from the recognition site. Recently, we reported the presence of two distinct and separable domains within this enzyme: one for the sequence-specific recognition of DNA (the DNA-binding domain) and the other for the endonuclease activity (the cleavage domain). Here, we report the construction of a chimeric restriction endonuclease by linking the Drosophila Ultrabithorax homeodomain to the cleavage domain (FN) of Fok I restriction endonuclease. The hybrid enzyme, Ubx-FN, was purified, and its cleavage properties were characterized. The hybrid enzyme shows the same DNA sequence-binding preference as that of Ubx; as expected, it cleaves the DNA away from the recognition site. On the 5'-TTAATGGTT-3' strand the hybrid enzyme cleaves 3 nt away from the recognition site, whereas it cuts the complementary 5'-AACCATTAA-3' strand 8, 9, or 10 nt away from the binding site. Similarly engineered hybrid enzymes could be valuable tools in physical mapping and sequencing of large eukaryotic genomes.
CP-96,345, a substance P antagonist, inhibits rat intestinal responses to Clostridium difficile toxin A but not cholera toxin.
Authors: Pothoulakis C , Castagliuolo I , LaMont J T , Jaffer A , O'Keane J C , Snider R M , Leeman S E
Published: May 2006

Toxin A from Clostridium difficile mediates acute inflammatory enterocolitis in experimental animals, while cholera toxin causes noninflammatory secretory diarrhea. The purpose of this study was to investigate whether an antagonist to the peptide substance P, a constituent of primary sensory neurons known to participate in inflammatory responses, would inhibit toxin A-mediated enteritis in the rat ileum. Pretreatment of rats with CP-96,345 (2.5 mg per kg of body weight), a substance P antagonist, dramatically inhibited fluid secretion (P < 0.01) and mannitol permeability (P < 0.01) in ileal loops exposed to toxin A. The protective effects, which were dose dependent, caused a significant reduction of inflammation in the lamina propria, reduction of the necrosis of intestinal epithelial cells, and complete inhibition of toxin A-mediated release of rat mast cell protease II, a specific product of rat mucosal mast cells. An inactive enantiomer of the substance P antagonist, CP-96,344, had no effect. In contrast, pretreatment with CP-96,345 had no inhibitory effect on the intestinal effects caused by administration of cholera toxin into the ileal loops. From these data, we conclude that the peptide substance P is involved in the secretory and inflammatory effects of toxin A but not of cholera toxin.
Convergence of quantum electrodynamics in a curved modification of Minkowski space.
Authors: Segal I E , Zhou Z
Published: May 2006

The interaction and total hamiltonians for quantum electrodynamics, in the interaction representation, are entirely regular self-adjoint operators in Hilbert space, in the universal covering manifold M of the conformal compactification of Minkowski space Mo. (M is conformally equivalent to the Einstein universe E, in which Mo may be canonically imbedded.) In a fixed Lorentz frame this may be expressed as convergence in a spherical space with suitable periodic boundary conditions in time. The traditional relativistic theory is the formal limit of the present variant as the space curvature vanishes.
In vivo transcription from the adenovirus E2 earlypromoter by RNA polymerase III.
Authors: Huang W , Pruzan R , Flint S J
Published: May 2006

We have previously reported that the subgroup C
adenovirus E2 early (E2E) RNA polymerase II promoter can specify efficient in
vitro transcription by RNA polymerase III. We now show that promoter proximal
sequences of the E2E transcription unit are also transcribed by RNA polymerase
III in nuclei isolated from adenovirus-infected cells. Small E2E RNA species
that possessed the same properties as in vitro synthesized RNA polymerase III
E2E transcripts were detected in cytoplasmic RNA populations from infected cells
by using blotting, primer extension, and RNase protection assays. The 3'
termini of these RNAs were mapped to thymidine-rich sequences typical of RNA
polymerase III termination sites. These results demonstrate that a single gene
can be transcribed by both RNA polymerase II and RNA polymerase III in
vivo.
Pseudomonas aeruginosa possesses homologues ofmammalian phenylalanine hydroxylase and 4 alpha-carbinolamine dehydratase/DCoHas part of a three-component gene cluster.
Authors: Zhao G , Xia T , Song J , Jensen R A
Published: May 2006

Pseudomonas aeruginosa possesses a multigene
operon that includes phenylalanine hydroxylase (PhhA; phenylalanine
4-monooxygenase, EC 1.14.16.1). phhA encodes PhhA (M(r) = 30,288), phhB (M(r) =
13,333) encodes a homologue of mammalian 4 alpha-carbinolamine
dehydratase/homeodomain protein transregulator, and phhC encodes an aromatic
aminotransferase (M(r) = 43,237). The reading frames specifying phhB and phhC
overlap by 2 bases. The P. aeruginosa PhhA appears to contain iron and is pterin
dependent. Unlike the multimeric mammalian hydroxylase, the native P. aeruginosa
enzyme is a monomer. The P. aeruginosa PhhA is homologous with mammalian PhhA,
tryptophan hydroxylase, and tyrosine hydroxylase. Expression of PhhA from its
native promoter required phhB. This may suggest a positive regulatory role for
phhB, consistent with the dual catalytic and regulatory roles of the
corresponding mammalian homologue.
Vanishing properties of cuspidal localsystems.
Authors: Lusztig G
Published: May 2006

Prehomogeneous vector spaces arising from
semisimple groups are considered, and it is shown that certain local systems on
the open orbit have the property that their middle extension is zero on the
complement of the open orbit.
KAP: a dual specificity phosphatase that interactswith cyclin-dependent kinases.
Authors: Hannon G J , Casso D , Beach D
Published: May 2006

The cyclin-dependent kinases are key cell cycle
regulators whose activation is required for passage from one cell cycle phase to
the next. In mammalian cells, CDK2 has been implicated in control of the G1 and
S phases. We have used a two-hybrid protein interaction screen to identify cDNAs
encoding proteins that can interact with CDK2. Among those identified was a
protein (KAP), which contained the HCXX-XXGR motif characteristic of protein
tyrosine phosphatases. KAP showed phosphatase activity toward substrates
containing either phosphotyrosine or phosphoserine residues. Since KAP is not
significantly similar to known phosphatases beyond the catalytic core motif, it
represents an additional class of dual specificity phosphatase. KAP interacted
with cdc2 and CDK2 in yeast. In mammalian cells, KAP also associated with cdc2
and CDK2 but showed a preference for cdc2. The ability of KAP to bind multiple
cyclin-dependent kinases suggests that it may play a role in cell cycle
regulation.
Utilization of modified surfactant-associated protein B for delivery of DNA to airway cells in culture.
Authors: Baatz J E , Bruno M D , Ciraolo P J , Glasser S W , Stripp B R , Smyth K L , Korfhagen T R
Published: May 2006

Pulmonary surfactant lines the airway epithelium and creates a potential barrier to successful transfection of the epithelium in vivo. Based on the functional properties of pulmonary surfactant protein B (SP-B) and the fact that this protein is neither toxic nor immunogenic in the airway, we hypothesized that SP-B could be modified to deliver DNA to airway cells. We have modified native bovine SP-B by the covalent linkage of poly(lysine) (average molecular mass of 3.3 or 10 kDa) to the N terminus of SP-B and formed complexes between a test plasmid and the modified SP-B. Transfection efficiency was determined by transfection of pulmonary adenocarcinoma cells (H441) in culture with the test plasmid pCPA-RSV followed by measurement of activity of the reporter gene encoding chloramphenicol acetyltransferase (CAT). Transfections were performed with DNA.protein complexes using poly(lysine)10kDa-SP-B ([Lys]10kDa-SP-B) or poly(lysine)3.3kDa-SP-B ([Lys]3.3kDa-SP-B), and results were compared with transfections using unmodified poly(lysine).DNA, unmodified SP-B.DNA, or DNA only. For [Lys]10kDa-SP-B.pCPA-RSV preparations, CAT activity was readily detectable above the background of [Lys]3.3kDa-SP-B or unmodified SP-B. The SP-B-poly(lysine) conjugates were effective over a broad range of protein-to-DNA molar ratios, although they were optimal at approximately 500:1-1000:1. Transfection efficiency varied with the tested cell line but was not specific to airway cells. Addition of replication-defective adenovirus to the [Lys]10kDa-SP-B.pCPA-RSV complex enhanced CAT activity about 30-fold with respect to that produced by the [Lys]10kDa-SP-B.pCPA-RSV complex alone. This increase suggests routing of the adenoviral.[Lys]10kDa-SP-B.pCPA-RSV complex through an endosomal pathway. Effects of covalent modification on the secondary structure of SP-B were examined by Fourier transform infrared spectrometry (FTIR). Results of FTIR indicated that the conformation of [Lys]10kDa-SP-B was comprised primarily of alpha-helical structure compared with a predominantly aggregated structure of unmodified poly(lysine). We conclude that poly(lysine) conjugates of SP-B effectively deliver DNA in vitro and may have utility as DNA delivery vehicles to the airway in vivo.
Interactions between p53 and MDM2 in a mammalian cell cycle checkpoint pathway.
Authors: Chen C Y , Oliner J D , Zhan Q , Fornace A J , Vogelstein B , Kastan M B
Published: May 2006

Normal p53 function is required for optimal arrest of cells in the G1 phase of the cell cycle following certain types of DNA damage. Loss of this cell cycle checkpoint may contribute to tumor development by increasing the number of genetic abnormalities in daughter cells following DNA damage. The MDM2 protein is an endogenous gene product that binds to the p53 protein and is able to block p53-mediated transactivation of cotransfected reporter constructs; thus, interactions between MDM2 and p53 in this checkpoint pathway following ionizing irradiation were examined. Though increases in p53 protein by DNA damage were not abrogated by MDM2 overexpression, increased levels of MDM2, resulting either from endogenous gene amplification or from transfection of an exogenous expression vector, were associated with a reduction in the ability of cells to arrest in G1 following irradiation. In addition, expression of endogenous MDM2 was enhanced by ionizing irradiation at the level of transcription in a p53-dependent fashion. These observations demonstrate that MDM2 overexpression can inhibit p53 function in a known physiologic pathway and are consistent with the hypothesis that MDM2 may function in a "feedback loop" mechanism with p53, possibly acting to limit the length or severity of the p53-mediated arrest following DNA damage.
An evolutionarily conserved palindrome in the Drosophila Gld promoter directs tissue-specific expression.
Authors: Gunaratne P , Ross J L , Zhang Q , Organ E L , Cavener D R
Published: May 2006

A conserved palindromic sequence (Gpal) in the promoter region of the Drosophila Gld directs expression of a heterologous reporter gene in transgenic flies to the anterior spiracular glands of third instar larvae and to the ejaculatory bulb of adult males. The Gld gene is normally expressed at high levels in the anterior spiracular glands but is not expressed in the ejaculatory bulb of Drosophila melanogaster. However, Gld promoters from other Drosophila species contain the Gpal element and express glucose dehydrogenase (GLD) in the adult male ejaculatory bulb. A gene fusion composed of the D. melanogaster Gld promoter and the lacZ gene is expressed in the anterior spiracular glands of transgenic larvae. Mutations of the Gpal sequence element in this gene fusion block expression of beta-galactosidase in the anterior spiracular gland. Together these experiments demonstrate that Gpal is necessary and sufficient for tissue-specific expression in the anterior spiracular glands. Based upon the tissue distribution and function of GLD, it is speculated that expression of GLD in the anterior spiracular glands represents the ancestral state and that GLD expression in other tissues arose as a fortuitous consequence of a shared combinatorial regulatory network.
T-cell receptor usage by melanoma-specific clonal and highly oligoclonal tumor-infiltrating lymphocyte lines.
Authors: Shilyansky J , Nishimura M I , Yannelli J R , Kawakami Y , Jacknin L S , Charmley P , Rosenberg S A
Published: May 2006

Tumor-infiltrating lymphocytes (TIL) obtained from human melanomas can specifically lyse autologous tumor in vitro and mediate tumor regression in vivo. To develop more effective therapeutic reagents and to further understand the T-cell response to tumors, the diversity of T-cell receptors (TCRs) involved in melanoma antigen recognition has been studied. The TCR variable (V) genes, joining (J) segments, and N diversity regions used by five clonal lines and one highly oligoclonal, melanoma-specific, CD8+ TIL line were examined utilizing PCR amplification with V gene subfamily-specific primers and anchor PCR. The TIL lysed multiple allogeneic melanomas expressing matched surface major histocompatibility complex class I molecules. TCR analysis confirmed the clonal nature of the TIL lines; however, the TCR repertoire was diverse. Even among the three HLA-A2 restricted TIL (TIL 1200, TIL F2-2, and TIL-5), no common V gene usage was found. Comparison of the third complementarity-determining regions of the TCRs from the HLA-A2 restricted TIL revealed no homology. Results presented here identify T-cell clonotypes that recognize epitopes on highly prevalent, shared melanoma tumor-associated antigens presented in the context of HLA-B55, HLA-A1, and HLA-A2. These T cells and the antigens they recognize represent important components for the design of new immunotherapies for patients with advanced melanoma.
Constraints of thermal noise on the effects of weak 60-Hz magnetic fields acting on biological magnetite.
Authors: Adair R K
Published: May 2006

Previous calculations of limits imposed by thermal noise on the effects of weak 60-Hz magnetic fields on biological magnetite are generalized and extended to consider multiple signals, the possibility of anomalously large magnetosome structures, and the possibility of anomalously small cytoplasm viscosities. The results indicate that the energies transmitted to the magnetite elements by fields less than 5 microT, characteristic of the electric power distribution system, will be much less than thermal noise energies. Hence, the effects of such weak fields will be masked by that noise and cannot be expected to affect biology or, therefore, the health of populations.
Chromosomal localization of the callipyge gene in sheep (Ovis aries) using bovine DNA markers.
Authors: Cockett N E , Jackson S P , Shay T L , Nielsen D , Moore S S , Steele M R , Barendse W , Green R D , Georges M
Published: May 2006

A mutation causing muscular hypertrophy, with associated leanness and improved feed efficiency, has been recently identified in domestic sheep (Ovis aries). Preliminary results indicate that an autosomal dominant gene may be responsible for this economically advantageous trait. We have exploited the conservation in sequence and chromosomal location of DNA markers across Bovidae to map the corresponding callipyge locus to ovine chromosome 18 using a battery of bovine chromosome 21 markers. Chromosomal localization of the ovine callipyge locus is the first step toward positional cloning of the corresponding gene.
Cell-adhesion-disrupting action of interleukin 6 in human ductal breast carcinoma cells.
Authors: Tamm I , Kikuchi T , Cardinale I , Krueger J G
Published: May 2006

Recombinant baculovirus-derived interleukin 6 (IL-6) disrupts the attachment of human ductal breast carcinoma subline ZR-75-1-Tx cells to neighbors and the substratum in culture without inhibiting the proliferation of the cells. The nonadherent cells lack pseudopodia and do not translocate directionally. These findings stand in contrast to the earlier observations in the Ro subline of ZR-75-1 cells in which IL-6 induces cell-cell separation without detachment of the cells from the substratum, with the cells displaying pseudopodia, increased motility, and decreased proliferation. The IL-6-induced ZR-75-1-Tx cell detachment and rounding are reversible by incubation of the treated cells in IL-6-free medium for several days. The distinctive changes induced by IL-6 in ZR-75-1-Ro cells are similarly reversible. Either acidic fibroblast growth factor or phorbol 12-myristate 13-acetate can replace serum as a cofactor in IL-6-induced ZR-75-1-Tx cell detachment. Our findings indicate that genetic changes can occur in breast carcinoma cells that through cytokine action markedly affect cell structure, adhesiveness, and motility.
Insertional mutagenesis of Drosophila heterochromatin with single P elements.
Authors: Zhang P , Spradling A C
Published: May 2006

Insertional mutagenesis with transposable P elements has greatly facilitated the identification and analysis of genes located throughout the 70% of the Drosophila melanogaster genome classified as euchromatin. In contrast, genetically marked P elements have only rarely been shown to transpose into heterochromatin. By carrying out single P element insertional mutagenesis under conditions where position-effect variegation was suppressed, we efficiently generated strains containing insertions at diverse sites within centromeric and Y-chromosome heterochromatin. The tendency of P elements to transpose locally was shown to operate within heterochromatin, and it further enhanced the recovery of heterochromatic insertions. Three of the insertions disrupted vital genes known to be present at low density in heterochromatin. Strains containing single P element insertions will greatly facilitate the structural and functional analysis of this poorly understood genomic component.
The human homologue of the retroviral oncogene qin maps to chromosome 14q13.
Authors: Kastury K , Li J , Druck T , Su H , Vogt P K , Croce C M , Huebner K
Published: May 2006

Chromosomal mapping of the human QIN gene (renamed FKH2 by the Human Genome Organization Nomenclature Committee) was initially accomplished by correlation of the presence of the QIN locus with specific chromosome regions in a rodent-human hybrid panel. This analysis revealed that the human QIN gene maps to chromosome region 14q11.2-->14q32, between the TCR and IGH loci. Further analysis by fluorescence in situ hybridization techniques with a human QIN genomic clone refined the human QIN gene localization to 14q13.
Molecular analysis of antigen-independent adhesion forces between T and B lymphocytes.
Authors: Amblard F , Auffray C , Sekaly R , Fischer A
Published: May 2006

The low-affinity interactions underlying antigen recognition by T-cell receptors (TCRs) are thought to involve antigen-independent adhesion mechanisms. Using a hydrodynamic approach, we found that antigen-independent adhesion occurred between human B cells and resting T cells in a transient and temperature-dependent fashion. The mean cell-cell adhesion force was 0.32 x 10(-9) N and was generated by similar contributions (0.16 x 10(-9) N) of the LFA-1- and CD2-dependent adhesion pathways. After T-cell stimulation with a phorbol ester, the force contributed by LFA-1 was drastically increased, while that of CD2 was unaffected. We propose that weak receptor-mediated adhesion initiates antigen-independent intercellular contacts required for antigen recognition by the TCR and is upregulated following TCR engagement. The method used permits adhesion forces between living cells to be resolved at the molecular level and should prove valuable for the rapid assessment of interaction forces between various types of cells and cell-sized particles.
A Rev-inducible mutant gag gene stably transferred into T lymphocytes: an approach to gene therapy against human immunodeficiency virus type 1 infection.
Authors: Smythe J A , Sun D , Thomson M , Markham P D , Reitz M S , Gallo R C , Lisziewicz J
Published: May 2006

One strategy for somatic gene therapy for human immunodeficiency virus type 1 (HIV-1) infection is based on the regulated expression of dominant negative mutants of the HIV-1 gag gene. To limit expression of the mutant Gag polypeptide to HIV-1-infected cells, we have constructed a replication-defective retroviral vector that contains a Rev-responsive element. By using this construct we have obviated problems that can be associated with constitutive expression of an exogenous gene, an important step toward developing a human therapy. In uncloned T lymphocytes infected (transduced) with this retroviral construct, HIV-1 replication was inhibited by 94% with a concomitant decrease in the cytopathic effects of the virus. In addition, simian immunodeficiency virus (SIV) replication was also shown to be significantly inhibited, suggesting that this mutant Gag protein may have antiviral efficacy against a broad range of primate lentiviruses and that an SIV/macaque model can be used for further in vivo studies. These results have important implications in assessing the potential of somatic gene therapy in the treatment of HIV-1 infection.
Amplification of trpEG: adaptation of Buchnera aphidicola to an endosymbiotic association with aphids.
Authors: Lai C Y , Baumann L , Baumann P
Published: May 2006

Survival of aphids is dependent on an association with a prokaryotic endosymbiont (Buchnera aphidicola) found in specialized cells within the aphid body cavity. Recent nutritional and physiological studies have indicated that one of the functions of the endosymbionts is the synthesis of tryptophan [Douglas, A. E. & Prosser, W. A. (1992) J. Insect Physiol. 38, 565-568]. B. aphidicola resembles in many of its properties free-living prokaryotes. An adaptation to an endosymbiosis involving the overproduction of tryptophan would necessitate alterations that modify the effect of regulatory systems that in free-living organisms function to reduce enzyme activity under conditions of excess tryptophan. We have cloned and sequenced the genes for B. aphidicola trpEG encoding anthranilate synthase, the first enzyme of the tryptophan biosynthetic pathway, which in free-living bacteria is feedback-inhibited by tryptophan. Amino acid sequence comparisons indicate that the B. aphidicola enzyme has all of the key residues involved in allosteric feedback inhibition. Evidence is presented indicating that trpEG is present as four tandem repeats on a circular plasmid. Relative to B. aphidicola trpDC(F)BA (the chromosomal genes coding for the remaining enzymes of the tryptophan biosynthetic pathway) trpEG is amplified 14- to 15-fold. These findings suggest that the effect of inhibition by accumulated tryptophan may be overcome by overproduction of anthranilate synthase. Our results demonstrate the acquisition of a new property (gene amplification) as an adaptation to an endosymbiotic association in which B. aphidicola overproduces tryptophan for the aphid host.
Expression of trk in MAH cells lacking the p75 low-affinity nerve growth factor receptor is sufficient to permit nerve growth factor-induced differentiation to postmitotic neurons.
Authors: Verdi J M , Ip N , Yancopoulos G D , Anderson D J
Published: May 2006

We have transfected MAH cells, an immortalized sympathoadrenal progenitor cell line, with a plasmid encoding the 140-kDa Trk protein, a nerve growth factor (NGF) receptor with protein-tyrosine kinase activity. NGF promotes neurite outgrowth and proliferation from such cells, indicating that Trk is sufficient to mediate such responses in the absence of significant levels of the endogenous 75-kDa low-affinity NGF receptor (p75). These initial NGF responses are indistinguishable from those evoked by basic fibroblast growth factor (bFGF). However, NGF is sufficient to promote terminal differentiation of a approximately 8% of trk-transfected MAH cells to postmitotic, NGF-dependent neurons, whereas all cells eventually die in medium with bFGF. Other environmental signals (such as depolarization or ciliary neurotrophic factor) can cooperate with NGF to enhance production of postmitotic NGF-dependent neurons in trk-transfected MAH cells. The terminal differentiation of sympathetic neurons thus involves sequential and cooperative actions of different growth and neurotrophic factors, as well as cell-intrinsic changes in the response to these factors.