bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of the alpha v beta 3 integrin in human melanoma cell invasion.
Authors: Seftor R E , Seftor E A , Gehlsen K R , Stetler-Stevenson W G , Brown P D , Ruoslahti E , Hendrix M J
Published: May 2006

The human melanoma cell line A375M expresses the vitronectin receptor (alpha v beta 3 integrin) on its cell surface. Treatment of A375M cells with either polyclonal or monoclonal anti-alpha v beta 3 antibodies resulted in stimulation of invasion through basement membrane matrices in vitro. Similar treatment of these cells with a monoclonal anti-alpha v antibody, which does not inhibit the adhesive function of the alpha v beta 3 antigen, also stimulated invasion; however, anti-beta 3 antibody treatment had no effect. Furthermore, pretreatment of the cells with vitronectin or addition of vitronectin to the basement membrane matrix also resulted in stimulation of invasion. Similar treatments with fibronectin receptor antibody or fibronectin had no effect on invasion. Analysis of type IV collagenase expression in cells treated with anti-alpha v beta 3 antibody showed higher levels of both the secreted 72-kDa enzyme and its mRNA. Signal transduction through the alpha v beta 3 integrin could underlie the elevated expression of metalloproteinase and the enhanced invasion of A375M cells through basement membrane matrices.
Synergistic and coordinate expression of the genes encoding ribonucleotide reductase subunits in Swiss 3T3 cells: effect of multiple signal-transduction pathways.
Authors: Albert D A , Rozengurt E
Published: May 2006

Ribonucleoside-diphosphate reductase (ribonucleotide reductase, EC 1.17.4.1) is the enzyme responsible for the in vivo production of deoxyribonucleotides for DNA synthesis and is essential for cell proliferation. We examined the signal transduction pathways leading to expression of the M1 and M2 subunits of this enzyme in Swiss 3T3 mouse fibroblasts by Northern blot analysis. Stimulation of quiescent cells resulted in coordinate expression of both subunits, beginning at 8 hr after serum addition, in late G1 phase, and peaking at 18-24 hr. Serum increased M2 message to 30 to 50 times that of quiescent cells, in contrast with M1 message, which was increased 10 times. Agents that elevated cAMP, including forskolin, and the cAMP analogue 8-bromo-cAMP modestly stimulated gene expression. Each of these agents was synergistic with insulin, and these combinations induced expression equivalent to that induced by serum stimulation. Likewise, agents that activate protein kinase C such as phorbol 12,13-dibutyrate, bombesin, and vasopressin were also synergistic with insulin with respect to ribonucleotide reductase gene expression, as was epidermal growth factor, which stimulates receptor tyrosine kinase activity. The time course for induction of mRNA expression by each of these agents alone or in combination was identical to that for induction stimulated by serum. Finally, the synergistic effects apparent in Northern analysis of ribonucleotide reductase gene expression were mirrored in parallel determinations of DNA synthesis. Thus, the combinatorial nature of signal transduction pathways resulting in proliferation of Swiss 3T3 cells is expressed at the level of ribonucleotide reductase gene expression.
Calcium entry-dependent oscillations of cytoplasmic calcium concentration in cultured endothelial cell monolayers.
Authors: Laskey R E , Adams D J , Cannell M , van Breemen C
Published: May 2006

Bovine endothelial cell monolayers grown to confluence and stimulated with bradykinin responded with periodic fluctuations in intracellular Ca2+ concentration ([Ca2+]i) when exposed to K(+)-free Hepes-buffered saline. The fluctuations in [Ca2+]i measured with fura-2 were synchronized among the population of cells observed and were sensitive to extracellular Ca2+ concentration ([Ca2+]o). Thapsigargin, which inhibits the endoplasmic reticular Ca2(+)-ATPase, did not inhibit the [Ca2+]i oscillations. Removal of extracellular Ca2+ or inhibition of Ca2+ entry by using La3+ or 1-(beta- [3-(4-methoxyphenyl)proproxy]-4-methoxyphenethyl)-1H-imidazole hydrochloride (SKF 96365) abolished the [Ca2+]i oscillations in endothelial cell monolayers. The fluctuations in [Ca2+]i were therefore dependent on Ca2+ influx rather than Ca2+ mobilization from intracellular stores. Simultaneous measurements of membrane potential (Em) using the potential-sensitive bisoxonol dye bis(1,3-dibutylbarbituric acid)trimethine oxonol [Di-BAC4(3)] and [Ca2+]i using fura-2 showed that Em oscillated at the same frequency as the fluctuations in [Ca2+]i. The peak depolarization signal coincided with the maximum rate of increase in the [Ca2+]i signal. Oscillations in the Em signal were inhibited by removal of Ca2+ or by addition of 1 mM Ni2+ to the external solution. Taken together, these observations suggest that the change in Em is the consequence of oscillatory changes in a membrane conductance that also allows Ca2+ to enter the cell. Oscillations in the DiBAC4(3) signal may reflect a rhythmic entry of Ca2+ through nonselective cation channels.
The c-rel protooncogene product c-Rel but not NF-kappa B binds to the intronic region of the human interferon-gamma gene at a site related to an interferon-stimulable response element.
Authors: Sica A , Tan T H , Rice N , Kretzschmar M , Ghosh P , Young H A
Published: May 2006

Interferon-gamma (IFN-gamma) is an important immunoregulatory protein that is expressed usually only in large granular lymphocytes and T cells. The gene encoding IFN-gamma was previously found to contain an intronic enhancer element that was not tissue-specific in its activity, despite the restricted expression of the intact IFN-gamma-encoding gene. Using nuclear extracts from the human T-cell line Jurkat, we have now identified two protein-binding regions in this intronic enhancer element. One of the protected regions has strong partial identify to the NF-kappa B site present in the promoter region of the human interleukin 2-encoding gene. Based on this observation and recent reports of the interaction of the c-rel protooncogene product (c-Rel) with NF-kappa B sites, we determined whether c-Rel could interact with the intronic enhancer element in the human IFN-gamma genomic DNA. Most surprisingly, gel-shift analysis, using c-Rel expressed in Escherichia coli established that c-Rel binds specifically to the IFN-gamma intronic DNA but not to the interleukin 2-like NF-kappa B site. Additional studies with antibodies prepared against c-Rel peptides verified specificity of the interaction of c-Rel with this binding site. In addition, using an affinity-purified p50 subunit of the NF-kappa B complex, we observed that the p50 protein did not bind to this additional c-Rel-binding site. Furthermore, nucleotide sequence analysis of this DNA region revealed a strong similarity of the additional c-Rel-binding site to a previously identified IFN-stimulable response element. These data show that c-Rel can interact with DNA regions distinct from that recognized by NF-kappa B and may, in fact, be involved in transcriptional regulation of the IFN-stimulable genes via the IFN-stimulable response element.
A peptide from the Drosophila Shaker K+ channel inhibits a voltage-gated K+ channel in basolateral membranes of Necturus enterocytes.
Authors: Dubinsky W P , Mayorga-Wark O , Schultz S G
Published: May 2006

A synthetic peptide composed of the first 22 amino acid residues of the Drosophila Shaker K+ channel inhibits a voltage-gated K+ channel in basolateral membrane vesicles from Necturus enterocytes reconstituted in planar phospholipid bilayers when added to the solution bathing the inner surface of this channel but not when added to the solution bathing its outer surface. A modified peptide in which the leucine in the 7 position is replaced with phenylalanine is also an effective inhibitor, but replacement of the leucine-7 with lysine or glutamate, or digestion with trypsin, renders the peptide ineffective; replacement of the leucine-7 with glycine markedly reduces but does not abolish the effectiveness of the peptide as an inhibitor. These results are analogous to those reported for the Shaker K+ channel +ADHoshi, T., Zagotta, W.N. & Aldrich, R.W. (1990) Science 250, 533-538; and Zagotta, W.N., Hoshi, T. & Aldrich, R.W. (1990) Science 250, 568-571.+BD and suggest that the molecular anatomy of the receptor at the inner face of the Necturus K+ channel with which the peptide interacts to bring about inhibition of that channel may be similar to that of the Shaker K+ channel.
Purification and characterization of the higher plant enzyme L-canaline reductase.
Authors: Rosenthal G A
Published: May 2006

A newly discovered enzyme, L-canaline reductase (NADPH:L-canaline oxidoreductase, EC 1.6.6-), has been isolated and purified from 10-day-old leaves of the jack bean Canavalia ensiformis (Leguminosae). This higher plant is representative of a large number of legumes that synthesize L-canavanine, an important nitrogen-storing nonprotein amino acid. Canavanine-storing legumes contain arginase, which hydrolyzes L-canavanine to form the toxic metabolite L-canaline. Canaline reductase, having a mass of approximately 167 kDa and composed of 82-kDa dimers, catalyzes a NADPH-dependent reductive cleavage of L-canaline to L-homoserine and ammonia. This is the only enzyme known to use reduced NADP to cleave an O-N bond. Canaline reductase performs at least three important functions for canavanine-synthesizing legumes. First, it detoxifies canaline. Second, it increases by one-half the overall yield of ammoniacal nitrogen released from canavanine. Third, it permits the carbon skeleton of canavanine, a secondary plant metabolite, to support vital primary metabolic reactions.
A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands.
Authors: Frommer M , McDonald L E , Millar D S , Collis C M , Watt F , Grigg G W , Molloy P L , Paul C L
Published: May 2006

The modulation of DNA-protein interactions by methylation of protein-binding sites in DNA and the occurrence in genomic imprinting, X chromosome inactivation, and fragile X syndrome of different methylation patterns in DNA of different chromosomal origin have underlined the need to establish methylation patterns in individual strands of particular genomic sequences. We report a genomic sequencing method that provides positive identification of 5-methylcytosine residues and yields strand-specific sequences of individual molecules in genomic DNA. The method utilizes bisulfite-induced modification of genomic DNA, under conditions whereby cytosine is converted to uracil, but 5-methylcytosine remains nonreactive. The sequence under investigation is then amplified by PCR with two sets of strand-specific primers to yield a pair of fragments, one from each strand, in which all uracil and thymine residues have been amplified as thymine and only 5-methylcytosine residues have been amplified as cytosine. The PCR products can be sequenced directly to provide a strand-specific average sequence for the population of molecules or can be cloned and sequenced to provide methylation maps of single DNA molecules. We tested the method by defining the methylation status within single DNA strands of two closely spaced CpG dinucleotides in the promoter of the human kininogen gene. During the analysis, we encountered in sperm DNA an unusual methylation pattern, which suggests that the high methylation level of single-copy sequences in sperm may be locally modulated by binding of protein factors in germ-line cells.
Cloning of a big tau microtubule-associated protein characteristic of the peripheral nervous system.
Authors: Goedert M , Spillantini M G , Crowther R A
Published: May 2006

Microtubule-associated protein tau consists in brain of a series of isoforms of 48- to 67-kDa apparent molecular mass that are encoded by mRNAs of approximately 6 kilobases (kb) and that are generated from a single gene by alternative splicing. Previously, a tau-like protein of 110-kDa apparent molecular mass was described in peripheral ganglia and in peripheral neuronlike cell lines. We now report the cloning and sequencing of a rat cDNA encoding this big tau. The corresponding protein contains sequence identical to the longest of the previously cloned small tau isoforms but with an additional 254 amino acid insert in the amino-terminal half. Big tau is produced from an 8-kb mRNA generated by alternative splicing from the same gene that encodes small tau. Production of big tau from the cloned sequence gives a protein of 110-kDa apparent molecular mass that aligns on SDS/PAGE with big tau protein extracted from peripheral ganglia. RNA blots show that in peripheral ganglia from adult rats only the 8-kb mRNA band corresponding to big tau is found, whereas in ganglia from newborn rats both 6- and 8-kb tau mRNA bands are found. In tissues from the central nervous system only the 6-kb mRNA band can be detected. Big tau protein is therefore produced specifically in the peripheral nervous system, and it will be interesting to see whether further molecular differences between the two major divisions of the vertebrate nervous system will be discovered.
Invariant chain can function as a chaperone protein for class II major histocompatibility complex molecules.
Authors: Anderson M S , Miller J
Published: May 2006

During biosynthesis, class II major histocompatibility complex molecules are intimately associated with invariant chain (Ii). The Ii-class II association has been shown to block peptide-class II binding and to affect the ultimate conformation of class II expressed on the cell surface. To assess the biochemical basis for the effects of Ii on class II, we have analyzed the biosynthesis of class II in EL4 cells transfected with I-Ad with and without Ii. In these studies, we found that Ii had a profound effect on the biosynthesis of I-Ad. In the absence of Ii, class II could form dimers efficiently, but these dimers appeared to be misfolded and this altered conformation resulted in the loss of some monoclonal antibody epitopes and inefficient transport from the endoplasmic reticulum to the Golgi. In addition, class II that was transported through the Golgi accumulated an abnormally increased molecular mass associated with N-linked glycosylation. Subsequent transfection of Ii into these cells resulted in recovery of normal class II conformation, causing a restoration of monoclonal antibody epitopes, efficient intracellular transport, and normal glycosylation. Together, these data indicate that Ii can have a profound effect on the folding, transport, and modification of class II molecules and suggest that one function of Ii may be to act as a class II-specific chaperone.
Absolute requirement for GTP in activation of human neutrophil NADPH oxidase in a cell-free system: role of ATP in regenerating GTP.
Authors: Peveri P , Heyworth P G , Curnutte J T
Published: May 2006

Guanine and/or adenine nucleotides appear to be involved in the activation of the superoxide-generating NADPH oxidase of phagocytic cells. Their precise roles, however, are unclear, as much of the evidence for their involvement comes from experiments in which nucleotides have been added to complex systems already rich in both endogenous nucleotides and enzymes capable of interconverting them. To circumvent this problem we have examined the role of nucleotides in neutrophil NADPH oxidase activation by using a cell-free system in which adenine and guanine nucleotide concentrations were carefully controlled and monitored by (i) depletion of endogenous nucleotides by extensive dialysis and charcoal treatment; (ii) reconstitution of the depleted system with reagents analyzed for purity; and (iii) measurement of nucleotide levels in cytosol preparations and in oxidase reaction mixtures by HPLC analysis. In contrast to previous reports that have demonstrated only a several-fold enhancement of oxidase activity by GTP or its analogs, we have shown that oxidase activation was absolutely dependent upon GTP in reactions containing dialyzed cytosol in which the total endogenous nucleotide levels were reduced by greater than 99.5%. Kinetic studies revealed that GTP is required at or before the rate-limiting step in oxidase activation. Two nonhydrolyzable analogs of GTP, guanosine 5'-(gamma-thio)triphosphate and guanylyl imidodiphosphate, were even more active than GTP, suggesting the involvement of one or more GTP-binding proteins. In contrast, ATP was neither necessary nor sufficient for oxidase activation. If reaction mixtures were contaminated with GDP and/or GMP, however, ATP (but not its nonhydrolyzable analog adenylyl imidodiphosphate) could indirectly support oxidase activation by means of endogenous enzymes that catalyze the ATP-dependent conversion of GMP and GDP to GTP.
Modification of Brassica seed oil by antisense expression of a stearoyl-acyl carrier protein desaturase gene.
Authors: Knutzon D S , Thompson G A , Radke S E , Johnson W B , Knauf V C , Kridl J C
Published: May 2006

Molecular gene transfer techniques have been used to engineer the fatty acid composition of Brassica rapa and Brassica napus (canola) oil. Stearoyl-acyl carrier protein (stearoyl-ACP) desaturase (EC 1.14.99.6) catalyzes the first desaturation step in seed oil biosynthesis, converting stearoyl-ACP to oleoyl-ACP. Seed-specific antisense gene constructs of B. rapa stearoyl-ACP desaturase were used to reduce the protein concentration and enzyme activity of stearoyl-ACP desaturase in developing rapeseed embryos during storage lipid biosynthesis. The resulting transgenic plants showed dramatically increased stearate levels in the seeds. A continuous distribution of stearate levels from 2% to 40% was observed in seeds of a transgenic B. napus plant, illustrating the potential to engineer specialized seed oil compositions.
DNA helical stability accounts for mutational defects in a yeast replication origin.
Authors: Natale D A , Schubert A E , Kowalski D
Published: May 2006

Earlier studies on the H4 autonomously replicating sequence (ARS) identified a DNA unwinding element (DUE), a required sequence that is hypersensitive to single-strand-specific nucleases and serves to facilitate origin unwinding. Here we demonstrate that a DUE can be identified in the C2G1 ARS, a chromosomal replication origin, by using a computer program that calculates DNA helical stability from the base sequence. The helical stability minima correctly predict the location and hierarchy of the nuclease-hypersensitive sites in a C2G1 ARS plasmid. Nucleotide-level mapping shows that the nuclease-hypersensitive site at the ARS spans a 100-base-pair sequence in the required 3'-flanking region. Mutations that stabilize the DNA helix in the broad 3'-flanking region reduce or abolish ARS-mediated plasmid replication, indicating that helical instability is required for origin function. The level of helical instability is quantitatively related to the replication efficiency of the ARS mutants. Multiple copies of either a consensus-related sequence present in the C2G1 ARS or the consensus sequence itself in synthetic ARS elements contribute to DNA helical instability. Our findings indicate that a DUE is a conserved component of the C2G1 ARS and is a major determinant of replication origin activity.
Gene expression in Leishmania: analysis of essential 5' DNA sequences.
Authors: Curotto de Lafaille M A , Laban A , Wirth D F
Published: May 2006

A major unanswered question in Kinetoplastida parasites is the mechanism of regulating gene expression. Using a transfection system, we have previously shown that the intergenic region of the alpha-tubulin gene of Leishmania enriettii contained sequences required for gene expression. The goal of the work reported here was to determine whether the Leishmania-derived sequences were providing transcriptional control signals or functioning at a post-transcriptional level, most likely in trans-splicing. The chloramphenicol acetyltransferase (cat) gene was used as the reporter gene and was stably introduced into L. enriettii as part of an extrachromosomal element by transfection. We show here that the production of cat mRNA was dramatically dependent on the presence of the intergenic region 5' to the cat gene. The intergenic region could be substituted by a smaller fragment (222 base pairs) that contained the trans-splice acceptor site and an adjacent polypyrimidine tract. This native fragment could be replaced by a synthetic polypyrimidine tract containing an AG site. The native and the synthetic fragments had unidirectional activity. No effect on transcription of the cat gene by the wild-type fragment or the synthetic polypyrimidine was detected. The results indicate that both regions contain signals that affect RNA stability, probably sequences involved in trans-splicing.
Functional characterization of a hybrid human-mouse interferon gamma receptor: evidence for species-specific interaction of the extracellular receptor domain with a putative signal transducer.
Authors: Hemmi S , Merlin G , Aguet M
Published: May 2006

The human interferon gamma (IFN-gamma) receptor expressed in mouse cells displays binding properties indistinguishable from those of the resident receptor on human cells. Still, mouse cells expressing the human IFN-gamma receptor remain insensitive to human IFN-gamma. It is widely accepted that at least one species-specific cofactor encoded within human chromosome 21 is required for signal transduction. To define structural domains of the human IFN-gamma receptor responsible for this species-specific interaction, a hybrid between the human and the murine receptor was constructed and expressed in mouse L929 cells or in mouse L cell-derived SCC16-5 cells, which contain human chromosome 21. This hybrid receptor, which consisted of the extracellular domain of the human IFN-gamma receptor and the transmembrane and cytoplasmic domains of the murine IFN-gamma receptor, was found to bind human IFN-gamma with high affinity. However, only SCC16-5 cells expressing the human/mouse hybrid receptor were responsive to human IFN-gamma as revealed by enhanced expression of major histocompatibility complex class I antigens, induction of the transcription factor IRF-1, and induction of a partial antiviral state. These findings strongly suggest that IFN-gamma-mediated signal transduction requires a species-specific interaction of the extracellular portion of the known ligand-binding IFN-gamma receptor chain with an additional, presumably membrane-anchored receptor subunit.
Synergistic suppression: anomalous inhibition of the proliferation of factor-dependent hemopoietic cells by combination of two colony-stimulating factors.
Authors: Metcalf D , Nicola N A , Gough N M , Elliott M , McArthur G , Li M
Published: May 2006

Cells of the continuous murine hemopoietic cell line FDC-P1 expressing macrophage-colony-stimulating factor (M-CSF) receptors following retroviral insertion of murine c-fms cDNA proliferated clonally when stimulated by granulocyte/macrophage (GM)-CSF, multipotential CSF, or M-CSF. However, M-CSF combined with either GM-CSF or multi-CSF, even at low CSF concentrations, strongly inhibited colony formation, with loss of clonogenicity in affected cells accompanied by increased macrophage differentiation. Stimulation by these CSF combinations did not induce short-term changes in CSF receptor expression or internalization. FDC-P1 cells expressing another inserted tyrosine kinase receptor, basic fibroblast growth factor receptor, did not exhibit suppression when GM-CSF was combined with fibroblast growth factor. This phenomenon of synergistic suppression may have relevance for the future clinical use of combinations of CSFs, because a potentially similar suppression is also observable with some normal macrophage progenitor cells.
Synthesis of 5-methylaminomethyl-2-selenouridine in tRNAs: 31P NMR studies show the labile selenium donor synthesized by the selD gene product contains selenium bonded to phosphorus.
Authors: Veres Z , Tsai L , Scholz T D , Politino M , Balaban R S , Stadtman T C
Published: May 2006

An enzyme preparation from Salmonella typhimurium catalyzes the conversion of 5-methylaminomethyl-2-thiouridine in tRNAs to 5-methylaminomethyl-2-selenouridine when supplemented with selenide and ATP. Similar preparations from a Salmonella mutant strain carrying a defective selD gene fail to catalyze this selenium substitution reaction. However, supplementation of the deficient enzyme preparation with the purified selD gene product (SELD protein) restored synthesis of seleno-tRNAs. In the absence of the complementary enzyme(s), the SELD protein catalyzes the synthesis of a labile selenium donor compound from selenide and ATP. 31P NMR studies show that among the products of this reaction are AMP and a compound containing selenium bonded to phosphorus. The reaction is completely dependent on the addition of both selenide and magnesium. The dependence of reaction velocity on ATP concentration shows sigmoidal kinetics, whereas dependence on selenide concentration obeys Michaelis-Menten kinetics indicating a Km value of 46 microM for selenide.
Suramin is an inhibitor of DNA topoisomerase II in vitro and in Chinese hamster fibrosarcoma cells.
Authors: Bojanowski K , Lelievre S , Markovits J , Couprie J , Jacquemin-Sablon A , Larsen A K
Published: May 2006

The antitrypanosomal and antifiliarial drug suramin is currently under investigation for treatment of advanced malignancies including prostatic cancer, adrenocortical cancer, and some lymphomas and sarcomas. Here we show that suramin is a potent inhibitor of the nuclear enzyme DNA topoisomerase II. Suramin inhibited purified yeast topoisomerase II with an IC50 of about 5 microM, as measured by decatenation or relaxation assays. Suramin did not stabilize the covalent DNA-topoisomerase II reaction intermediate ("cleavable complex"), whereas other inhibitors of this enzyme, such as amsacrine, etoposide, and the ellipticines, are known to stabilize the intermediate. In contrast, the presence of suramin strongly inhibited the cleavable-complex formation induced by amsacrine or etoposide. Accumulation of the endogenous cleavable complex was also inhibited. Suramin entered the nucleus of DC-3F Chinese hamster fibrosarcoma cells exposed to radiolabeled suramin for 24 hr as shown by both optic and electron microscopy. The suramin present in the nucleus seemed to interact with topoisomerase II, since suramin reduced the number of amsacrine-induced protein-associated DNA strand breaks in DC-3F cells and protected these cells from the cytotoxic action of amsacrine. Cells resistant to 9-hydroxyellipticine, which have been shown to have an altered topoisomerase II activity, are about 7-fold more resistant to suramin than the sensitive parental cells as shown by 72-hr growth inhibition assay. Our results suggest that DNA topoisomerase II is a target of suramin action and that this action may play a role in the cytotoxic activity of suramin.
Heparin- and sulfatide-binding peptides from the type I repeats of human thrombospondin promote melanoma cell adhesion.
Authors: Guo N H , Krutzsch H C , Nègre E , Vogel T , Blake D A , Roberts D D
Published: May 2006

Peptides from the three type I repeats of human endothelial cell thrombospondin, containing the consensus sequence-Trp-Ser-Xaa-Trp-, bind to sulfated glycoconjugates including heparin and sulfatide. The peptides are potent inhibitors for the binding of thrombospondin, laminin, or apolipoprotein E to these ligands. The thrombospondin peptides that inhibit heparin binding, but not adjacent peptides from the thrombospondin sequence containing the previously identified adhesive motif Val-Thr-Cys-Gly, promote melanoma cell adhesion when immobilized on plastic. Melanoma cell adhesion to the immobilized peptides is inhibited by soluble recombinant heparin-binding fragment of thrombospondin. The peptides also inhibit heparin-dependent binding of thrombospondin or laminin to human melanoma cells. The active peptides lack any previously identified heparin-binding consensus sequences and most do not contain any basic amino acids. Studies with homologous peptides showed that the tryptophan residues are required for binding. Adjacent basic residues in the second type I repeat enhance binding to heparin but not to sulfatide. Thus the type I peptides of thrombospondin define a distinct class of heparin-binding peptides.
Isolation of the mitochondrial benzodiazepine receptor: association with the voltage-dependent anion channel and the adenine nucleotide carrier.
Authors: McEnery M W , Snowman A M , Trifiletti R R , Snyder S H
Published: May 2006

The mitochondrial benzodiazepine receptor (mBzR) has been solubilized with retention of reversible ligand binding, and the associated subunits were characterized. mBzR comprises immunologically distinct protein subunits of 18-, 30-, and 32-kDa. The 18-kDa protein is labeled by the isoquinoline carboxamide mBzR ligand [3H]PK14105, whereas the 30- and 32-kDa subunits are labeled by the benzodiazepine (Bz) ligands [3H]flunitrazepam and [3H]AHN-086. Selective antibodies and reagents identify the 32- and 30-kDa proteins as the voltage-dependent anion channel (VDAC) and the adenine nucleotide carrier (ADC), respectively. While isoquinoline carboxamide and Bz ligands target different subunits, they interact allosterically, as the binding of Bz and isoquinoline carboxamide ligands is mutually competitive at low nanomolar concentrations. Moreover, eosin-5-maleimide and mercuric chloride inhibit [3H]PK11195 binding to the intact receptor via sulfhydryl groups that are present in ADC. VDAC and ADC, outer and inner mitochondrial membrane channel proteins, respectively, together with the 18-kDa subunit, may comprise mBzR at functionally important transport sites at the junction of two mitochondrial membranes.
Regulatory roles of Fnr, Fur, and Arc in expression of manganese-containing superoxide dismutase in Escherichia coli.
Authors: Hassan H M , Sun H C
Published: May 2006

Transcriptional regulation of the sodA gene, encoding the manganese superoxide dismutase (superoxide: superoxide oxidoreductase, EC 1.15.1.1) of Escherichia coli, was studied by monitoring expression of sodA-lacZ in different genetic backgrounds and under different growth conditions. Mutations in the fnr gene were found to affect aerobic and anaerobic expression of sodA-lacZ. Potential Fnr-binding sites were identified in the promoter region of sodA. Strains harboring simultaneous mutations in arcA/B and fur expressed sodA-lacZ under anaerobic growth conditions but were still inducible by iron chelators. However, in the triple mutants (fnr fur arcA/B) sodA-lacZ was fully expressed under anaerobiosis and was not further induced by the presence of 2,2'-dipyridyl, nitrate, or oxidants. On the other hand, aerobic expression of sodA-lacZ from a Fur-strain was approximately 3.8-fold higher than that from the wild-type strain but was diminished by introducing mutations in fnr or arcA/B. In conclusion, Fnr, Arc, and Fur act as anaerobic repressors of sodA. Furthermore, the regulation of sodA by Fur (ferric uptake regulation protein), Arc (aerobic respiratory control), and Fnr (fumarate nitrate reduction/regulator of anaerobic respiration) is independent of the superoxide response regulon SoxRS.
Initiator methionine tRNA is essential for Ty1 transposition in yeast.
Authors: Chapman K B , Byström A S , Boeke J D
Published: May 2006

The yeast retrotransposon Ty1 transposes through an RNA intermediate by a mechanism similar to that of retroviral reverse transcription and integration. Ty1 RNA contains a putative minus strand primer binding site (-PBS) that is complementary to the 3' acceptor stem of the initiator methionine tRNA (tRNA(iMet)). Here we demonstrate that the tRNA(iMet) is used as a primer for Ty1 reverse transcription. Mutations in the Ty1 element that alter 5 of 10 nucleotides that are complementary to the tRNA(iMet) abolish Ty1 transposition, even though they are silent with regard to Ty1 protein coding. We have constructed a yeast strain lacking wild-type tRNA(iMet) that is dependent on a mutant derivative of tRNA(iMet) that has an altered acceptor stem sequence, engineered to restore homology with the Ty1 -PBS mutant. The compensatory mutations made in the tRNA(iMet) alleviate the transposition defect of the Ty1 -PBS mutant. The mutant and wild-type tRNA(iMet) are enriched within Ty1 virus-like particles irrespective of complementarity to the Ty1 -PBS. Thus, complementarity between the Ty1 -PBS and tRNA(iMet) is essential for transposition but is not necessary for packaging of the tRNA inside virus-like particles.
Immature thymocytes are protected from deletion early in ontogeny.
Authors: Finkel T H , Kappler J W , Marrack P C
Published: May 2006

Self/non-self discrimination occurs within the thymus, where T cells undergo positive selection to produce a repertoire that recognizes foreign antigen in the context of self major histocompatibility complex proteins and negative selection to eliminate from the repertoire those T cells with self-reactive specificities. We have previously shown that two subpopulations of immature antigen receptor-bearing thymocytes exist, one that is susceptible to negative selection induced by ligation of the T-cell receptor for antigen and a second that is resistant to T-cell receptor-mediated negative selection. In the current work, we show that all antigen receptor-bearing thymocytes in early fetal thymuses are resistant to negative selection and that thymocytes susceptible to deletion do not develop until later gestational ages. Thus, deletion is a relatively late event in T-cell ontogeny. In addition, these data suggest that a thymocyte population can be isolated early in ontogeny that is capable of transducing selective signals through the antigen receptor, which do not lead to deletion but may result in positive selection.
Long-term abrogation of autoimmune diabetes in nonobese diabetic mice by immunotherapy with anti-lymphocyte serum.
Authors: Maki T , Ichikawa T , Blanco R , Porter J
Published: May 2006

We investigated the therapeutic effect of anti-lymphocyte serum (ALS) on clinically overt diabetes by using a nonobese diabetic (NOD) mouse model of type I diabetes mellitus. ALS given within 14 days of disease onset gradually reversed hyperglycemia with a 76% cumulative incidence of remission. Combined use of anti-CD4 and anti-CD8 monoclonal antibodies, but not anti-CD4 or anti-CD8 antibody alone, was also effective with overall 64% remission. Diabetic NOD mice that failed to respond to ALS treatment accepted subsequent islet isografts for a prolonged period (mostly greater than 100 days), whereas islet isografts in diabetic NOD mice previously treated with normal rabbit serum were all destroyed as acutely as isografts in untreated diabetic NOD mice. These results suggest that persistence of diabetes was due to irreversible beta-cell destruction and that ALS has indeed abrogated autoimmunity. In addition, ALS treatment at the time of islet isografting achieved significant prolongation of graft survival with 8 of 13 mice maintaining euglycemia for greater than 100 days. Although ALS prolonged islet allograft survival in diabetic NOD mice, the degree of prolongation was much less for allografts than for isografts, suggesting that ALS is capable of suppressing autoimmunity more effectively than allograft responses.
Abnormal fibrinogens IJmuiden (B beta Arg14----Cys) and Nijmegen (B beta Arg44----Cys) form disulfide-linked fibrinogen-albumin complexes.
Authors: Koopman J , Haverkate F , Grimbergen J , Engesser L , Nováková I , Kerst A F , Lord S T
Published: May 2006

The molecular defects in two congenital abnormal fibrinogens, IJmuiden and Nijmegen, were determined by sequence analysis of genomic DNA amplified by the polymerase chain reaction. Both fibrinogens were heterozygous, IJmuiden having a B beta Arg14----Cys substitution and Nijmegen having a B beta Arg44----Cys substitution. Clotting induced by thrombin or Reptilase was impaired in both fibrinogens, indicating defective fibrin polymerization. Immunoblot analysis of both purified fibrinogens demonstrated that some of the abnormal molecules were linked by disulfide bonds to albumin. In addition, abnormal high molecular weight fibrinogen complexes with Mrs between 600,000 and 700,000 were present. Fibrinogen-albumin and high molecular weight complexes were also detected in the patients' plasmas. Quantitative analysis demonstrated that of the total plasma fibrinogen in the IJmuiden patient, 20% was linked to albumin and 10% was present as high molecular weight complexes. In plasma Nijmegen, 13% was linked to albumin and 15% was present as high molecular weight complexes. These results demonstrate that the additional abnormal cysteine in fibrinogens IJmuiden and Nijmegen resulted in the formation of disulfide-linked complexes with other proteins, predominantly albumin. We also found that a significant fraction of the abnormal fibrinogen molecules contained free sulfhydryl groups. These findings complicate interpretation of functional studies of these altered fibrinogens.
Bacterial resistance to complement killing mediated by the Ail protein of Yersinia enterocolitica.
Authors: Bliska J B , Falkow S
Published: May 2006

Ail is a 17-kDa outer membrane Yersinia protein that mediates bacterial attachment to, and invasion of, cultured epithelial cells. We report here an alternative role for Ail in the pathogenesis of Yersinia infection. We found that Escherichia coli HB101 harboring the 4-kilobase recombinant ail clone pVM102 were highly resistant to killing in up to 50% normal human serum. A 674-base-pair fragment of DNA from pVM102, which encodes the ail gene, was inserted into pUC18 and shown to promote full resistance to complement killing in E. coli HB101. Cellular attachment and resistance to complement killing in a plasmid-cured inv- strain of Yersinia enterocolitica (0:8) was correlated with the thermoinduced expression of Ail at 37 degrees C. Insertional inactivation of ail in Y. enterocolitica resulted in loss of both thermoinduced bacterial properties. Cellular attachment and serum resistance were restored by complementation of the defect by plasmid-encoded ail. Complementation of cell attachment activity required bacterial growth at 37 degrees C, indicating that an additional thermoinduced factor is required for this Ail function. In addition, these studies reveal that functional homology exists between Ail and the structurally related protein Rck, which promotes resistance to complement killing in Salmonella typhimurium.
Parallel pathways in macaque monkey striate cortex: anatomically defined columns in layer III.
Authors: Lachica E A , Beck P D , Casagrande V A
Published: May 2006

Visual information reaching striate cortex comes from parallel pathways, and the information is organized, or processed, by the layers and columns of striate cortex. To better understand how this is accomplished anatomically, we asked whether parallel pathways originating in the lateral geniculate nucleus (LGN), and terminating separately in layer IV, remain separate in layer III of macaque monkeys. Layer III is of interest since it may play a special role in color and form vision but not in analysis of visual motion. The chief finding was that cells in "blobs" of layer III that stain densely for cytochrome oxidase receive indirect input, via layer IVC, from both LGN magnocellular (M) and parvocellular (P) cells. This is important because the P and M pathways may represent color/form and motion-processing channels, respectively. Interblob cells receive indirect input, via layers IVC and IVA, from the LGN P cells. Also, as suggested by others, our data demonstrate that layer III can be subdivided. The bottom tier, layer IIIB, receives direct projections from all cortical layers. Output from layer IIIB appears to remain intrinsic to striate cortex. In contrast, the top tier, layer IIIA, receives projections from layer IIIB as well as from layers IVA, IVB (blobs only), and V, but it receives no direct projections from LGN recipient layers IVC and VI. Unlike layer IIIB, the output of layer IIIA reaches extrastriate areas. Thus, impulses arriving from parallel LGN pathways may be recombined through serial stages in striate cortex to produce a set of parallel pathways that are qualitatively different from the original LGN set.
The synaptic vesicle protein synaptotagmin associates with calcium channels and is a putative Lambert-Eaton myasthenic syndrome antigen.
Authors: Leveque C , Hoshino T , David P , Shoji-Kasai Y , Leys K , Omori A , Lang B , el Far O , Sato K , Martin-Moutot N
Published: May 2006

Immunoglobulin G fractions from patients with Lambert-Eaton myasthenic syndrome (LEMS), an autoimmune disease of neuromuscular transmission, immunoprecipitate 125I-labeled omega-conotoxin GVIA-labeled calcium channels solubilized from rat brain. A 58-kDa antigen was detected by probing Western blots of partially purified calcium channels with LEMS plasma and IgG and was shown to be the relevant antigen in omega-conotoxin receptor immunoprecipitation. Monoclonal antibody 1D12, produced by immunizing mice with synaptic membranes, has properties similar to these autoimmune IgGs in both immunoprecipitation and Western blotting assays. 1D12 antigen was purified by immunoaffinity chromatography and shown to bind LEMS IgG. The antigen was identified by screening a rat brain cDNA library with 1D12 and was found to have strong homology to the synaptic vesicle membrane protein synaptotagmin. Our results indicate therefore that these antibodies immunoprecipitate omega-conotoxin receptors by binding to synaptotagmin that is associated with calcium channels. We suggest that the interaction between synaptotagmin and the voltage-gated calcium channel plays a role in docking synaptic vesicles at the plasma membrane prior to rapid neurotransmitter release and that autoantibody binding to a synaptotagmin-calcium-channel complex may be involved in the etiology of LEMS.
Molecular cloning and expression of human myocardial cGMP-inhibited cAMP phosphodiesterase.
Authors: Meacci E , Taira M , Moos M , Smith C J , Movsesian M A , Degerman E , Belfrage P , Manganiello V
Published: May 2006

We have cloned a cDNA for a myocardial cGMP-inhibited cAMP phosphodiesterase (cGI PDE) from a human heart cDNA library in lambda Zap II. The open reading frame [3.5 kilobases (kb)] of cDNA clone n.13.2 (7.7 kb) encodes a protein of 125 kDa. In Northern blots of total human ventricle RNA, a single mRNA species (8.3 kb) hybridized with a 4-kb EcoRI restriction fragment of clone n.13.2 cDNA (containing the entire open reading frame). The carboxyl-terminal region of the deduced amino acid sequence of the cGI PDE contains the putative catalytic domain conserved among mammalian PDE families. A partial cDNA clone, n.2, encoding a truncated, 54-kDa cGI PDE containing the conserved domain was expressed as a catalytically active fusion protein in Escherichia coli. cAMP hydrolytic activity was inhibited by cGMP and OPC 3911 but not by rolipram. Thus, this report provides direct proof that the conserved domain contains the catalytic core of cGI PDEs.
Phage display of ricin B chain and its single binding domains: system for screening galactose-binding mutants.
Authors: Swimmer C , Lehar S M , McCafferty J , Chiswell D J , Blättler W A , Guild B C
Published: May 2006

We demonstrate that the B chain of ricin toxin preserves its lectin activity when expressed as a fusion protein on the surface of fd phage. Moreover, B chain, which folds into two topologically similar globular domains, can be dissected into amino-terminal and carboxyl-terminal domains to form single binding domains (SBDs) of B chain, each of which displays specificity for complex galactosides. The specific binding exhibited by the fusion protein of these SBDs was eliminated when amino acid substitutions Gly-46 in SBD1 or Gly-255 in SBD2 for native asparagine were introduced to alter key residues implicated in hydrogen bonding with substrate. These data demonstrate that it is possible to use a prokaryotic expression system to stably express and screen ricin B chain and its SBDs for sugar-binding mutants. Expression of ricin B chain on the surface of fd phage provides a method that can be used to efficiently select mutants with altered binding activities from a randomly generated library.
Identification and characterization of "injurin," an inducer of expression of the gene for hepatocyte growth factor.
Authors: Matsumoto K , Tajima H , Hamanoue M , Kohno S , Kinoshita T , Nakamura T
Published: May 2006

The marked and rapid increase of hepatocyte growth factor (HGF) mRNA in the intact lung of rats after partial hepatectomy or unilateral nephrectomy suggests the existence of a humoral factor mediating a signal of injury to distal organs and may induce the expression of HGF gene in these organs. We have now identified a proteinous factor in the sera of rats with injury of liver or kidney that increases HGF mRNA in the intact lung. When the serum of rats with liver insult caused by partial hepatectomy or ischemic treatment was injected i.p. into normal noninjured rats, it induced a marked HGF mRNA expression in the lung of the recipient rats. The addition of serum from rats with various hepatic or renal injuries to MRC-5 human embryonic lung fibroblasts in culture also led to the induction of HGF mRNA expression, so that the production of HGF by MRC-5 cells after treatment with the sera was remarkably increased in the culture medium. However, serum from the normal intact rat induced no HGF production and no HGF mRNA in the lung in vivo and lung fibroblasts in vitro. This factor, which increases HGF production, was purified greater than 200-fold from sera of CCl4-treated rats. The factor proved to be an acid- and heat-stable protein with an apparent molecular mass of 10-20 kDa in SDS/PAGE. Its activity markedly increased within 3-6 hr in the plasma of rats after various treatments that injured the liver or kidney. These results suggest that the factor specifically appears in the blood of rats with organ injury and may be involved in organ regeneration through the potential to increase the synthesis of HGF. Since the factor seems to mediate various organ injuries, we named it "injurin."
Guanyl nucleotides modulate binding to steroid receptors in neuronal membranes.
Authors: Orchinik M , Murray T F , Franklin P H , Moore F L
Published: May 2006

The recently characterized corticosteroid receptor on amphibian neuronal membranes appears to mediate rapid, stress-induced changes in male reproductive behaviors. Because the transduction mechanisms associated with this receptor are unknown, we performed radioligand binding studies to determine whether this steroid receptor is negatively modulated by guanyl nucleotides. The binding of [3H]corticosterone to neuronal membranes was inhibited by nonhydrolyzable guanyl nucleotides in both equilibrium saturation binding and titration studies. The addition of guanyl nucleotide plus unlabeled corticosterone induced a rapid phase of [3H]corticosterone dissociation from membranes that was not induced by addition of unlabeled ligand alone. Furthermore, the equilibrium binding of [3H]corticosterone and the sensitivity of the receptor to modulation by guanyl nucleotides were both enhanced by Mg2+. These results are consistent with the formation of a ternary complex of steroid, receptor, and guanine nucleotide-binding protein that is subject to regulation by guanyl nucleotides. Therefore, rapid signal transduction through corticosteroid receptors on neuronal membranes appears to be mediated by guanine nucleotide-binding proteins.