bluuebunny/crossref_metadata_2025 huggingface dataset:
Thymidine phosphorylase is angiogenic and promotes tumor growth.
Authors: Moghaddam A , Zhang H T , Fan T P , Hu D E , Lees V C , Turley H , Fox S B , Gatter K C , Harris A L , Bicknell R
Published: May 2006

Platelet-derived endothelial cell growth factor was previously identified as the sole angiogenic activity present in platelets; it is now known to be thymidine phosphorylase (TP). The effect of TP on [methyl-3H]thymidine uptake does not arise from de novo DNA synthesis and the molecule is not a growth factor. Despite this, TP is strongly angiogenic in a rat sponge and freeze-injured skin graft model. Neutralizing antibodies and site-directed mutagenesis confirmed that the enzyme activity of TP is a condition for its angiogenic activity. The level of TP was found to be elevated in human breast tumors compared to normal breast tissue (P < 0.001). Overexpression of TP in MCF-7 breast carcinoma cells had no effect on growth in vitro but markedly enhanced tumor growth in vivo. These data and the correlation of expression in tumors with malignancy identify TP as a target for antitumor strategies.
Biological and structural characterization of a Ras transforming mutation at the phenylalanine-156 residue, which is conserved in all members of the Ras superfamily.
Authors: Quilliam L A , Zhong S , Rabun K M , Carpenter J W , South T L , Der C J , Campbell-Burk S
Published: May 2006

Although Ras residue phenylalanine-156 (F156) is strictly conserved in all members of the Ras superfamily of proteins, it is located outside of the consensus GDP/GTP-binding pocket. Its location within the hydrophobic core of Ras suggests that its strict conservation reflects a crucial role in structural stability. However, mutation of the equivalent residue (F157L) in the Drosophila Ras-related protein Rap results in a gain-of-function phenotype, suggesting an alternative role for this residue. Therefore, we have introduced an F156L mutation into Ras to evaluate the role of this residue in Ras structure and function. Whereas introduction of this mutation activated the transforming potential of wild-type Ras, it did not impair that of oncogenic Ras. Further, Ras (156L) exhibited an extremely rapid off rate for bound GDP/GTP in vitro and showed increased levels of Ras.GTP in vivo. To determine the structural basis for these altered properties, we used high-resolution nuclear magnetic resonance spectroscopy. The F156L mutation caused loss of contact with residues 6, 23, 55, and 79, resulting in disruption of secondary structure in alpha-helix 1 and in beta-sheets 1-5. These major structural changes contrast with the isolated alterations induced by oncogenic mutation (residues 12 or 61) that perturb GTPase activity, and instead, weaken Ras contacts with Mg2+ and its guanine nucleotide substrate and result in increased rates of GDP/GTP dissociation. Altogether, these observations demonstrate the essential role of this conserved residue in Ras structure and its function as a regulated GDP/GTP switch.
Maintenance of an extrachromosomal plasmid vector in mouse embryonic stem cells.
Authors: Gassmann M , Donoho G , Berg P
Published: May 2006

We have constructed and characterized a polyoma virus-based plasmid that is maintained as an autonomously replicating extrachromosomal element (episome) in mouse embryonic stem (ES) cells. Plasmid pMGD20neo contains the polyoma origin of replication harboring a mutated enhancer (PyF101), a modified polyoma early region that encodes the large tumor (T) antigen only, and a gene that confers resistance to G418 (neo). After transfection, the plasmid replicates in ES cells and is maintained as an extrachromosomal element in 15% of G418-resistant clones. Integration of the plasmid DNA is undetectable for at least 28 cell generations. In one clone, the transfected DNA persists unaltered as an episome at 10-30 copies per cell for at least 74 cell generations in the presence of G418. Cells that maintain the autonomously replicating plasmid can efficiently replicate and maintain a second plasmid that carries the polyoma origin of replication. Independent vector-containing ES cell lines showed no significant alteration of the karyotype, and two cell lines yielded several chimeric animals when introduced into blastocysts, suggesting that the presence of an episomal element and expression of polyoma large T do not eliminate the ES cells' ability to populate an embryo. This system offers an efficient means for manipulating and analyzing various aspects of gene expression in ES cells.
Xenopus Gq alpha subunit activates the phosphatidylinositol pathway in Xenopus oocytes but does not consistently induce oocyte maturation.
Authors: Guttridge K L , Smith L D , Miledi R
Published: May 2006

We cloned the Xenopus laevis form of Gq alpha subunit to study its effects on oocyte maturation. Injection of Xenopus Gq alpha mRNA into stage 6 oocytes activated the phospholipase C/phosphatidylinositol pathway. The oocyte membrane became permeable to calcium ions and was able to generate transient inward currents (T(in)), due to the opening of Ca(2+)-dependent Cl- channels. The T(in) amplitude developed over several hours and disappeared by 24 hr. Diacylglycerol levels were found to parallel the appearance and disappearance of the T(in). The concurrent decline of T(in) values and diacylglycerol was not due to a failure in the synthesis of Gq alpha protein, which was produced continuously for > 24 hr. After Xenopus Gq alpha mRNA injection, germinal vesicle breakdown (GVBD) was variable (0-100%) in stage 6 oocytes, whereas none of the stage 4 oocytes underwent GVBD. In contrast, stage 6 oocytes injected with mRNA encoding the Go alpha G protein consistently underwent GVBD but did not acquire T(in). Our results show that activation of phospholipase C is not an absolute requisite for the induction of maturation, although in oocytes of some frogs phospholipase C activation can trigger a pathway to GVBD.
Multiple members of the E2F transcription factor family are the products of oncogenes.
Authors: Xu G , Livingston D M , Krek W
Published: May 2006

The retinoblastoma gene product (pRB) is a known tumor suppressor, capable of arresting growth in mid-to-late G1. Part of its growth suppression action arises from interaction(s) with one or more members of the E2F family of transcription factors. These proteins most likely contribute to progression from G0 to S phase in mammalian cells, and pRB binding most likely inhibits aspects of their suspected growth-promoting function. Given their growth-stimulating potential, we asked whether one or more E2F alleles can function as oncogenes. Uncloned pools of NIH 3T3 cells producing the pRB binding target E2F-1, E2F-2, or E2F-3 grew in semisolid medium. In addition, they grew to much higher saturation density than controls. From the study of cells producing selected E2F-1 mutant species, it appears that E2F DNA-binding function contributes to, and pRB/E2F binding suppresses, soft-agar growth. Thus, three E2F family members can act as oncogene products, suggesting that part of the normal role of pRB is to down-modulate this potential activity.
Protein kinase C epsilon is localized to the Golgi via its zinc-finger domain and modulates Golgi function.
Authors: Lehel C , Olah Z , Jakab G , Anderson W B
Published: May 2006

Protein kinase C (PKC) is a multigene family of serine/threonine kinases that are central to many signal transduction pathways. Among the PKC isozymes, only PKC epsilon has been reported to exhibit full oncogenic potential. PKC epsilon also displays unique substrate specificity and intracellular localization. To examine the interrelationship between the biological effects and domain structure of PKC epsilon, NIH 3T3 cells were stably transfected to overexpress different epitope-tagged fragments of PKC epsilon. The overexpressed proteins each contain the epsilon-tag peptide at the C terminus to allow ready detection with an antibody specific for the tag. The holo-PKC epsilon was found to localize with the Golgi network and other compartments, whereas the zinc-finger domain localized exclusively at the Golgi. Golgi-specific glycosaminoglycan sulfation was strongly inhibited in cells overexpressing either holo-PKC epsilon or its zinc-finger domain, while the secretion of sulfated glycosaminoglycans into the medium was impaired in cells expressing the PKC epsilon zinc-finger domain. Thus, these results suggest that PKC epsilon may be involved in specifically regulating Golgi-related processes. Further, the results indicate that PKC epsilon domains other than the kinase domain may also have biological activity and that the zinc-finger domain may function as a subcellular localization signal.
Regulation of human retroviral latency by the NF-kappa B/I kappa B family: inhibition of human immunodeficiency virus replication by I kappa B through a Rev-dependent mechanism.
Authors: Wu B Y , Woffendin C , Duckett C S , Ohno T , Nabel G J
Published: May 2006

The cellular transcription factor NF-kappa B stimulates human immunodeficiency virus type 1 (HIV-1) transcriptional initiation, but its role in the retroviral life cycle has not been fully defined. In this report, we show that I kappa B alpha acts as a cellular inhibitor of human retroviral replication through a discrete mechanism, independent of its effect on HIV transcription. I kappa B alpha inhibited HIV replication and gp160 expression by negatively regulating Rev function, most likely acting through a cellular factor involved in Rev transactivation. A similar effect was observed with human T leukemia virus I, in which I kappa B alpha inhibited Rex function. In contrast, no effect was observed on the replication of a DNA virus, adenovirus type 5. The NF-kappa B/I kappa B regulatory pathway therefore modulates human retroviral replication by regulating a program of cellular gene expression required for several steps in the viral life cycle, including not only viral transcription but also RNA export. This interaction between cellular and viral gene products suggests that NF-kappa B plays a broader role in the regulation of human retroviral replication, providing a previously unrecognized link between two important regulators of HIV gene expression and common NF-kappa B-dependent programs of gene expression used by human retroviruses.
Cleavage without anchor addition accompanies the processing of a nascent protein to its glycosylphosphatidylinositol-anchored form.
Authors: Maxwell S E , Ramalingam S , Gerber L D , Udenfriend S
Published: May 2006

Rough microsomal membranes from most mammalian cells, in the presence of a translation system, process nascent proteins with appropriate COOH-terminal signal peptides to their mature glycosylphosphatidylinositol (GPI)-linked forms. The present study, using preprominiplacental alkaline phosphatase as substrate, shows that as much as 10% of the mature product is cleaved correctly but is not linked to GPI. Some of the factors that influence the relative proportions of GPI linked to free mini-placental alkaline phosphatase are the amounts of GPI in the cells and the amino acid substituent at the omega site of the nascent protein. A mechanism for explaining cleavage both with and without GPI addition is presented, which supports a transamidase type of enzyme as the catalyst.
Inhibition of cell proliferation by the somatostatin analogue RC-160 is mediated by somatostatin receptor subtypes SSTR2 and SSTR5 through different mechanisms.
Authors: Buscail L , Estève J P , Saint-Laurent N , Bertrand V , Reisine T , O'Carroll A M , Bell G I , Schally A V , Vaysse N , Susini C
Published: May 2006

Effects of the stable somatostatin analogue RC-160 on cell proliferation, tyrosine phosphatase activity, and intracellular calcium concentration were investigated in CHO cells expressing the five somatostatin receptor subtypes SSTR1 to -5. Binding experiments were performed on crude membranes by using [125I-labeled Tyr11] somatostatin-14; RC-160 exhibited moderate-to-high affinities for SSTR2, -3, and -5 (IC50, 0.17, 0.1 and 21 nM, respectively) and low affinity for SSTR1 and -4 (IC50, 200 and 620 nM, respectively). Cell proliferation was induced in CHO cells by 10% (vol/vol) fetal calf serum, 1 microM insulin, or 0.1 microM cholecystokinin (CCK)-8; RC-160 inhibited serum-induced proliferation of CHO cells expressing SSTR2 and SSTR5 (EC50, 53 and 150 pM, respectively) but had no effect on growth of cells expressing SSTR1, -3, or -4. In SSTR2-expressing cells, orthovanadate suppressed the growth inhibitory effect of RC-160. This analogue inhibited insulin-induced proliferation and rapidly stimulated the activity of a tyrosine phosphatase in only this cellular clone. This latter effect was observed at doses of RC-160 (EC50, 4.6 pM) similar to those required to inhibit growth (EC50, 53 pM) and binding to the receptor (IC50, 170 pM), implicating tyrosine phosphatase as a transducer of the growth inhibition signal in SSTR2-expressing cells. In SSTR5-expressing cells, the phosphatase pathway was not involved in the inhibitory effect of RC-160 on cell growth, since this action was not influenced by tyrosine and serine/threonine phosphatase inhibitors. In addition, in SSTR5-expressing cells, RC-160 inhibited CCK-stimulated intracellular calcium mobilization at doses (EC50, 0.35 nM) similar to those necessary to inhibit somatostatin-14 binding (IC50, 21 nM) and CCK-induced cell proliferation (EC50, 1.1 nM). This suggests that the inositol phospholipid/calcium pathway could be involved in the antiproliferative effect of RC-160 mediated by SSTR5 in these cells. RC-160 had no effect on the basal or carbachol-stimulated calcium concentration in cells expressing SSTR1 to -4. Thus, we conclude that SSTR2 and SSTR5 bind RC-160 with high affinity and mediate the RC-160-induced inhibition of cell growth by distinct mechanisms.
Possible proton relay pathways in cytochrome c oxidase.
Authors: Fetter J R , Qian J , Shapleigh J , Thomas J W , García-Horsman A , Schmidt E , Hosler J , Babcock G T , Gennis R B , Ferguson-Miller S
Published: May 2006

As the final electron acceptor in the respiratory chain of eukaryotic and many prokaryotic organisms, cytochrome c oxidase (EC 1.9.3.1) catalyzes the reduction of oxygen to water and generates a proton gradient. To test for proton pathways through the oxidase, site-directed mutagenesis was applied to subunit I of the Rhodobacter sphaeroides enzyme. Mutants were characterized in three highly conserved regions of the peptide, comprising possible proton loading, unloading, and transfer sites: an interior loop between helices II and III (Asp132Asn/Ala), an exterior loop between helices IX and X (His411Ala, Asp412Asn, Thr413Asn, Tyr414Phe), and the predicted transmembrane helix VIII (Thr352Ala, Pro358Ala, Thr359Ala, Lys362Met). Most of the mutants had lower activity than wild type, but only mutants at residue 132 lost proton pumping while retaining electron transfer activity. Although electron transfer was substantially inhibited, no major structural alteration appears to have occurred in D132 mutants, since resonance Raman and visible absorbance spectra were normal. However, lower CO binding (70-85% of wild type) suggests some minor change to the binuclear center. In addition, the activity of the reconstituted Asp132 mutants was inhibited rather than stimulated by ionophores or uncoupler. The inhibition was not observed with the purified enzyme and a direct pH effect was ruled out, suggesting an altered response to the electrical or pH gradient. The results support an important role for the conserved II-III loop in the proton pumping process and are consistent with the possibility of involvement of residues in helix VIII and the IX-X loop.
Mutants of Escherichia coli heat-labile toxin lacking ADP-ribosyltransferase activity act as nontoxic, mucosal adjuvants.
Authors: Douce G , Turcotte C , Cropley I , Roberts M , Pizza M , Domenghini M , Rappuoli R , Dougan G
Published: May 2006

A nontoxic mutant (LTK7) of the Escherichia coli heat-labile enterotoxin (LT) lacking ADP-ribosylating activity but retaining holotoxin formation was constructed. By using site-directed mutagenesis, the arginine at position 7 of the A subunit was replaced with lysine. This molecule, which was nontoxic in several assays, was able to bind to eukaryotic cells and acted as a mucosal adjuvant for co-administered proteins; BALB/c mice immunized intranasally with LTK7 and ovalbumin developed high levels of serum and local antibodies to ovalbumin and toxin. In addition, mice immunized intranasally with fragment C of tetanus toxin and LTK7 were protected against lethal challenge with tetanus toxin. Thus nontoxic mutants of heat-labile toxin can act as effective intranasal mucosal adjuvants.
Integration of human papillomavirus type 16 DNA into the human genome leads to increased stability of E6 and E7 mRNAs: implications for cervical carcinogenesis.
Authors: Jeon S , Lambert P F
Published: May 2006

In many cervical cancers, human papillomavirus type 16 (HPV-16) DNA genomes are found to be integrated into the host chromosome. In this study, we demonstrate that integration of HPV-16 DNA leads to increased steady-state levels of mRNAs encoding the viral oncogenes E6 and E7. This increase is shown to result, at least in part, from an increased stability of E6 and E7 mRNAs that arise specifically from those integrated viral genomes disrupted in the 3' untranslated region of the viral early region. Further, we demonstrate that the A+U-rich element within this viral early 3' untranslated region confers instability on a heterologous mRNA. We conclude that integration of HPV-16 DNA, as occurs in cervical cancers, can result in the increased expression of the viral E6 and E7 oncogenes through altered mRNA stability.
Detecting conserved regulatory elements with the model genome of the Japanese puffer fish, Fugu rubripes.
Authors: Aparicio S , Morrison A , Gould A , Gilthorpe J , Chaudhuri C , Rigby P , Krumlauf R , Brenner S
Published: May 2006

Comparative vertebrate genome sequencing offers a powerful method for detecting conserved regulatory sequences. We propose that the compact genome of the teleost Fugu rubripes is well suited for this purpose. The evolutionary distance of teleosts from other vertebrates offers the maximum stringency for such evolutionary comparisons. To illustrate the comparative genome approach for F. rubripes, we use sequence comparisons between mouse and Fugu Hoxb-4 noncoding regions to identify conserved sequence blocks. We have used two approaches to test the function of these conserved blocks. In the first, homologous sequences were deleted from a mouse enhancer, resulting in a tissue-specific loss of activity when assayed in transgenic mice. In the second approach, Fugu DNA sequences showing homology to mouse sequences were tested for enhancer activity in transgenic mice. This strategy identified a neural element that mediates a subset of Hoxb-4 expression that is conserved between mammals and teleosts. The comparison of noncoding vertebrate sequences with those of Fugu, coupled to a transgenic bioassay, represents a general approach suitable for many genome projects.
Molecular cloning of a ligand for the EPH-related receptor protein-tyrosine kinase Htk.
Authors: Bennett B D , Zeigler F C , Gu Q , Fendly B , Goddard A D , Gillett N , Matthews W
Published: May 2006

Htk is a receptor protein-tyrosine kinase that is related to the EPH subfamily of tyrosine kinases. The receptor has a wide tissue distribution including expression in several myeloid hematopoietic cell lines. Using an Htk-Fc fusion protein, a protein ligand for this receptor was expression cloned from the murine kidney mesangial cell line SV40MES 13. The Htk ligand cDNA encodes a transmembrane protein of 336 amino acids. Binding competition experiments demonstrated a Kd of 535 pM for binding of Htk-Fc to the Htk ligand. Incubation of 3T3 cells expressing Htk with COS-7 cells expressing the ligand resulted in tyrosine phosphorylation of Htk. The ligand, like its receptor, is widely expressed and may function in a variety of tissues. However, we localized hematopoietic expression of Htk to the monocytic lineage, suggesting that the ligand may play a role in differentiation and/or proliferation of these cells.
Gonadotrophin-releasing hormone receptor agonist-mediated down-regulation of Gq alpha/G11 alpha (pertussis toxin-insensitive) G proteins in alpha T3-1 gonadotroph cells reflects increased G protein turnover but not alterations in mRNA levels.
Authors: Shah B H , MacEwan D J , Milligan G
Published: May 2006

Prolonged exposure of alpha T3-1 pituitary gonadotrophs to a gonadotrophin-releasing hormone receptor agonist results in marked down-regulation of the pertussis toxin-insensitive G proteins Gq alpha and G11 alpha. The turnover of Gq alpha/G11 alpha was substantially accelerated in the presence of agonist. By contrast, the rate of degradation of the G protein Gi2 alpha was unaffected by agonist treatment. Analysis of Gq alpha/G11 alpha mRNA levels by reverse transcription-PCR demonstrated no detectable differences between control and agonist-treated cells. These studies indicate that gonadotrophin-releasing hormone receptor agonist-mediated down-regulation of Gq alpha/G11 alpha is a reflection of enhanced proteolysis of the activated G proteins.
Amyloid peptides are toxic via a common oxidative mechanism.
Authors: Schubert D , Behl C , Lesley R , Brack A , Dargusch R , Sagara Y , Kimura H
Published: May 2006

beta-Amyloid protein (A beta) is a member of a small group of proteins that accumulate as amyloid deposits in various tissues. It has recently been demonstrated that the toxicity of A beta toward some neural cells is caused by oxidative damage. Since all of the amyloid diseases are characterized by protein deposited in the antiparallel beta-sheet conformation, it was asked whether there is a common toxic mechanism. It is shown here that the protein components of other human amyloidoses, including amylin, calcitonin, and atrial natriuretic peptide, are all toxic to clonal and primary cells. The toxicity is mediated via a free radical pathway indistinguishable from that of A beta. Experiments with synthetic peptides suggest that it is the amphiphilic nature of the peptides generated by their beta structure rather than their beta structure per se that causes toxicity. These results tend to rule out the alternative that amyloid toxicity is exclusively mediated via specific cell surface receptors.
A segment of the C-terminal half of the G-protein beta 1 subunit specifies its interaction with the gamma 1 subunit.
Authors: Katz A , Simon M I
Published: May 2006

The beta and gamma subunits of the heterotrimeric guanine nucleotide binding (G protein) act as a dimer and directly regulate various signal transduction pathways. By using cotransfection assays, we tested the ability of several beta gamma combinations to activate inositol phospholipid-specific phospholipase C (PI-PLC)-beta 2. Our findings indicate that only beta gamma combinations that form dimers will activate PI-PLC-beta 2. Since G beta 1 interacts with G gamma 1, while G beta 2 cannot, chimeras between G beta 1 and G beta 2 were used to identify the regions in beta 1 that determine its specific association with gamma 1. Our evidence demonstrates that a chimera between beta 2 and beta 1 that contains the C-terminal 173 amino acids of beta 1 can interact and activate PI-PLC-beta 2 with gamma 1. Chimeras that contain portions of the beta 1 C-terminal region display a weaker association with gamma 1. Furthermore, the contribution of each of these regions depends on the sequence context of each chimeric protein. However, the segment between residues 210 and 293 of beta 1 consistently plays a critical role in specifying association with gamma 1.
Previously identified protein of uncertain function is karyopherin alpha and together with karyopherin beta docks import substrate at nuclear pore complexes.
Authors: Moroianu J , Blobel G , Radu A
Published: May 2006

Previously, we had purified a cytosolic protein complex, termed karyopherin, that functions in docking import substrate at the nuclear envelope in digitonin-permeabilized cells and also had molecularly cloned and sequenced its 97-kDa beta subunit. We now report that the karyopherin alpha subunit is the previously identified protein NPI-1/SRP-1 of hitherto uncertain function. Using purified recombinant karyopherin alpha or beta subunit, we showed that neither karyopherin alpha nor karyopherin beta alone was sufficient for docking of import substrate at the nuclear envelope. Docking occurred only when both subunits were present. Moreover, docking of import substrate by the two recombinant karyopherin subunits was productive, as it led to nuclear internalization of the docked substrate in the presence of additional, previously characterized cytosolic factors. In a binding assay using immobilized karyopherin alpha and beta subunits and import substrate as a ligand, we found that only karyopherin alpha bound ligand. We suggest that karyopherin beta functions as an adaptor that binds both to karyopherin alpha and to any of a large number of docking sites that are represented by a repetitive peptide motif containing nucleoporins on both the cytoplasmic and nucleoplasmic side of the nuclear pore complex (NPC), bidirectionally ferrying a complex of karyopherin alpha-substrate across the NPC.
Random amplified polymorphic DNA markers tightly linked to a gene for resistance to white pine blister rust in sugar pine.
Authors: Devey M E , Delfino-Mix A , Kinloch B B , Neale D B
Published: May 2006

We have genetically mapped a gene for resistance to white pine blister rust (Cronartium ribicola Fisch.) in sugar pine (Pinus lambertiana Dougl.) by using an approach which relies on three factors: (i) the ability to assay for genetic markers in the haploid stage of the host's life cycle, using megagametophyte seed tissue; (ii) a simple and clearly defined pathosystem; and (iii) the use of random amplified polymorphic DNA (RAPD) markers that can be quickly and efficiently evaluated. Resistance to white pine blister rust in sugar pine is known to be controlled by a single dominant gene (R). Maternal segregation of R and dominant RAPD markers were scored simultaneously following collection of megagametophytes for DNA assays and seedling inoculation with C. ribicola. Bulked samples of haploid megagametophyte DNA from resistant and susceptible offspring of segregating full-sib and half-sib families were used to evaluate 800 random decanucleotide primers. Ten loci linked with the gene for resistance to white pine blister rust were identified and segregation data were obtained from five families. Six of the linked markers were within 5 centimorgans of the gene, and one marker was 0.9 centimorgan from R. These and other markers derived by this approach may provide starting points for map-based cloning of this important gene.
cDNA sequence and expression pattern of the putative pheromone carrier aphrodisin.
Authors: Mägert H J , Hadrys T , Cieslak A , Gröger A , Feller S , Forssmann W G
Published: May 2006

The cDNA sequence for aphrodisin, a lipocalin from hamster vaginal discharge which is involved in pheromonal activity, has been determined. Corresponding genomic clones were isolated and the promoter region was identified. Primer extension analysis revealed an adenosine residue as the main transcription initiation site, located 50 bp upstream of the translation start codon ATG, which is surrounded by a typical Kozak sequence. However, data from polymerase chain reaction analysis suggest the existence of at least one alternative transcription initiation site. The aphrodisin cDNA is 732 bp long and codes for the mature 151-aa aphrodisin and an additional N-terminal 16-aa secretory signal peptide. The 3' nontranslated region is 228 bp long. Among the known sequences, the aphrodisin cDNA shares the highest homology with the rat odorant-binding protein cDNA (45%), which verifies the protein data. Vaginal tissue and Bartholin's glands are the main aphrodisin gene-expressing tissues of the female hamster genital tract, as demonstrated by Northern blot analysis. Under less stringent hybridization conditions, RNA isolated from rat Bartholin's glands also showed a signal, indicating the occurrence of aphrodisin-related mRNA in this species.
HypB protein of Bradyrhizobium japonicum is a metal-binding GTPase capable of binding 18 divalent nickel ions per dimer.
Authors: Fu C , Olson J W , Maier R J
Published: May 2006

Bradyrhizobium japonicum hypB encodes a protein containing an extremely histidine-rich region (24 histidine residues within a 39-amino-acid stretch) and guanine nucleotide-binding domains. The product of the hypB gene was overexpressed in Escherichia coli and purified by Ni(2+)-charged metal chelate affinity chromatography (MCAC) in a single step. In SDS/PAGE, HypB migrated at 38 kDa--slightly larger than the calculated molecular mass (32.8 kDa). Purified HypB has GTPase activity with a kcat of 0.18 min-1 and a Km for GTP of 7 microM, and it has dGTPase activity as well. HypB exists as a dimer of molecular mass 78 kDa in native solution as determined by fast protein liquid chromatography on Superose 12. It binds 9.0 +/- 0.14 divalent nickel ions per monomer (18 Ni2+ per dimer) with a Kd of 2.3 microM; it also binds Zn2+, Cu2+, Co2+, Cd2+, and Mn2+. In-frame deletion of the histidine-rich region (deletion of 38 amino acids including 23 histidine residues) resulted in a truncated HypB that did not bind to the MCAC column, whereas in-frame deletion of 14 amino acids including 8 histidine residues within HypB resulted in a truncated HypB that still bound to the column. The results indicate that the histidine residues within the histidine-rich region of HypB are involved in metal binding.
DNA recombination is sufficient for retroviral transduction.
Authors: Schwartz J R , Duesberg S , Duesberg P H
Published: May 2006

Oncogenic retroviruses carry coding sequences that are transduced from cellular protooncogenes. Natural transduction involves two nonhomologous recombinations and is thus extremely rare. Since transduction has never been reproduced experimentally, its mechanism has been studied in terms of two hypotheses: (i) the DNA model, which postulates two DNA recombinations, and (ii) the RNA model, which postulates a 5' DNA recombination and a 3' RNA recombination occurring during reverse transcription of viral and protooncogene RNA. Here we use two viral DNA constructs to test the prediction of the DNA model that the 3' DNA recombination is achieved by conventional integration of a retroviral DNA 3' of the chromosomal protooncogene coding region. For the DNA model to be viable, such recombinant viruses must be infectious without the purportedly essential polypurine tract (ppt) that precedes the 3' long terminal repeat (LTR) of all retroviruses. Our constructs consist of a ras coding region from Harvey sarcoma virus which is naturally linked at the 5' end to a retroviral LTR and artificially linked at the 3' end either directly (construct NdN) or by a cellular sequence (construct SU) to the 5' LTR of a retrovirus. Both constructs lack the ppt, and the LTR of NdN even lacks 30 nucleotides at the 5' end. Both constructs proved to be infectious, producing viruses at titers of 10(5) focus-forming units per ml. Sequence analysis proved that both viruses were colinear with input DNAs and that NdN virus lacked a ppt and the 5' 30 nucleotides of the LTR. The results indicate that DNA recombination is sufficient for retroviral transduction and that neither the ppt nor the complete LTR is essential for retrovirus replication. DNA recombination explains the following observations by others that cannot be reconciled with the RNA model: (i) experimental transduction is independent of the packaging efficiency of viral RNA, and (ii) experimental transduction may invert sequences with respect to others, as expected for DNA recombination during transfection.
Crystal structure of an H-2Kb-ovalbumin peptide complex reveals the interplay of primary and secondary anchor positions in the major histocompatibility complex binding groove.
Authors: Fremont D H , Stura E A , Matsumura M , Peterson P A , Wilson I A
Published: May 2006

Sequence analysis of peptides naturally presented by major histocompatibility complex (MHC) class I molecules has revealed allele-specific motifs in which the peptide length and the residues observed at certain positions are restricted. Nevertheless, peptides containing the standard motif often fail to bind with high affinity or form physiologically stable complexes. Here we present the crystal structure of a well-characterized antigenic peptide from ovalbumin [OVA-8, ovalbumin-(257-264), SIINFEKL] in complex with the murine MHC class I H-2Kb molecule at 2.5-A resolution. Hydrophobic peptide residues Ile-P2 and Phe-P5 are packed closely together into binding pockets B and C, suggesting that the interplay of peptide anchor (P5) and secondary anchor (P2) residues can couple the preferred sequences at these positions. Comparison with the crystal structures of H-2Kb in complex with peptides VSV-8 (RGYVYQGL) and SEV-9 (FAPGNYPAL), where a Tyr residue is used as the C pocket anchor, reveals that the conserved water molecule that binds into the B pocket and mediates hydrogen bonding from the buried anchor hydroxyl group could not be likewise positioned if the P2 side chain were of significant size. Based on this structural evidence, H-2Kb has at least two submotifs: one with Tyr at P5 (or P6 for nonamer peptides) and a small residue at P2 (i.e., Ala or Gly) and another with Phe at P5 and a medium-sized hydrophobic residue at P2 (i.e., Ile). Deciphering of these secondary submotifs from both crystallographic and immunological studies of MHC peptide binding should increase the accuracy of T-cell epitope prediction.
Effects of yeast DNA topoisomerase III on telomere structure.
Authors: Kim R A , Caron P R , Wang J C
Published: May 2006

The yeast TOP3 gene, encoding DNA topoisomerase III, and EST1 gene, encoding a putative telomerase, are shown to be abutted head-to-head on chromosome XII, with the two initiation codons separated by 258 bp. This arrangement suggests that the two genes might share common upstream regulatory sequences and that their products might be functionally related. A comparison of isogenic pairs of yeast TOP3+ and delta top3 strains indicates that the G1-3T repetitive sequence tracks in delta top3 cells are significantly shortened, by about 150 bp. Cells lacking topoisomerase III also show a much higher sequence fluidity in the subtelomeric regions. In delta top3 cells, clusters of two or more copies of tandemly arranged Y' elements have a high tendency of disappearing due to the loss or dispersion of the elements; similarly, a URA3 marker embedded in a Y' element close to the chromosomal tip shows a much higher rate of being lost relative to that in TOP3+ cells. These results suggest that yeast DNA topoisomerase III might affect telomere stability, and plausible mechanisms are discussed.
Endothelin 1 mediates ex vivo coronary vasoconstriction caused by exogenous and endogenous cytokines.
Authors: Klemm P , Warner T D , Hohlfeld T , Corder R , Vane J R
Published: May 2006

Treatment of rats with cytokines has been associated with an increase in the circulating levels of endothelin 1 (ET-1). Here we show that administration of tumor necrosis factor alpha (TNF-alpha; 4 micrograms.kg-1) to anesthetized rats caused within 15 min a strong elevation in the circulating levels of ET-1. This was associated with a striking coronary vasoconstriction in hearts from these animals when they were removed and perfused in vitro by the Langendorff technique. This vasoconstriction was largely overcome by treatment with either the endothelin type A (ETA) receptor antagonist FR 139317 or antibody against ET-1. Furthermore, it was mimicked by in vivo exposure to exogenous ET-1. Endogenously produced TNF-alpha may also cause such a coronary vasoconstriction, for treatment with interleukin 2 (600 micrograms.kg-1) produced an increase in coronary perfusion pressure that correlated with the increases in circulating TNF-alpha. This coronary vasoconstriction was substantially reversed by treatment either with antibody against TNF-alpha or with FR 139317. We suggest, therefore, that cytokine-driven changes in the production of ET-1 are key events in the development of vascular pathologies.
Transferrin-antibody fusion proteins are effective in brain targeting.
Authors: Shin S U , Friden P , Moran M , Olson T , Kang Y S , Pardridge W M , Morrison S L
Published: May 2006

In the present study, the receptor binding potential of transferrin (Tf) was linked to an antibody binding specificity. Human Tf was fused to mouse-human chimeric IgG3 at three positions: at the end of heavy chain constant region 1 (CH1), after the hinge, and after CH3. The resulting Tf-antibody fusion proteins were able to bind antigen and the Tf receptor. The CH3-Tf fusion protein showed no complement-mediated cytolysis but possessed IgG receptor I (Fc gamma RI) binding activity. Most importantly, all of the fusion proteins demonstrated significant uptake into brain parenchyma, with 0.3% of the injected dose of the hinge-Tf fusion protein rapidly targeted to the brain. Recovery of iodinated CH3-Tf fusion protein from the brain parenchyma demonstrated that the fusion proteins can cross the blood-brain barrier intact. The binding specificity of these fusion proteins can be used for brain delivery of noncovalently bound ligands, such as drugs and peptides, or for targeting antigens present within the brain.
Effects of serotonergic agents on neuronal nicotinic acetylcholine receptors.
Authors: García-Colunga J , Miledi R
Published: May 2006

In Xenopus oocytes expressing neuronal nicotinic acetylcholine receptors (nAcChoRs), made up of alpha 2 and beta 4 subunits, acetylcholine (AcCho) elicited ionic membrane currents (AcCho currents) that were modulated by serotonergic agents. Both agonists and antagonists specific for various serotonin (5-hydroxytryptamine, 5HT) receptor subtypes interacted directly with alpha 2 beta 4 nAcChoRs: 5HT, (+/-)-8-hydroxy-2-(di-n-propylamino)tetralin, methysergide, spiperone, and ketanserin reversibly reduced the amplitude of AcCho currents and accelerated their decay. The AcCho-current time course decayed with two exponential functions. In the presence of 5HT, the fast time constant of current decay (tau f) was not greatly modified, but the slow time constant (tau s) was reduced. With AcCho and 5HT both at 100 microM, tau s was reduced from 140 s to 85 s. The order of potency for inhibition of AcCho current amplitudes was (+/-)-8-hydroxy-2-(di-n-propylamino)tetralin > methysergide > spiperone > ketanserin > 5HT. The inhibition was voltage-dependent but the magnitude of the voltage dependence for the different blockers did not correspond to their blocking potency: e.g., the block with spiperone was stronger than with 5HT, but it was less voltage-dependent. Our results suggest that serotonergic agents block neuronal nAcChoRs in a noncompetitive manner, similar to the block of muscle nAcChoR by curare and other substances. These results show that neuronal nAcChoR channels that have been activated by their specific neurotransmitter may be modulated by nonspecific neurotransmitters and their antagonists. These effects may help to better understand brain functions as well as the mode of action of the many serotonergic agents that are used in medical practice.
Temporally distinct accumulation of transcripts encoding enzymes of the prechorismate pathway in elicitor-treated, cultured tomato cells.
Authors: Görlach J , Raesecke H R , Rentsch D , Regenass M , Roy P , Zala M , Keel C , Boller T , Amrhein N , Schmid J
Published: May 2006

The accumulation of phenylalanine-derived phenolic compounds is a well-known element of a plant's defense in response to pathogen attack. Phenylalanine, as well as the other two aromatic amino acids, tyrosine and tryptophan, is synthesized by way of the shikimate pathway. The first seven steps of the shikimate pathway (the prechorismate pathway) are common for the biosynthesis of all three aromatic amino acids. We have studied transcript levels of six genes--i.e., two 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase genes, one shikimate kinase gene, one 5-enolpyruvylshikimate 3-phosphate synthase gene, and two chorismate synthase genes--corresponding to four steps of the prechorismate pathway, in cultured tomato cells exposed to fungal elicitors. The abundance of transcripts specific for some of these genes increased 10- to 20-fold within 6 h after elicitor treatment, as did the abundance of phenylalanine ammonialyase-specific transcripts and the synthesis of ethylene. Interestingly, transcript accumulation occurred more rapidly for shikimate kinase than for the enzymes preceding or following it in the prechorismate pathway. Neither the inhibition of ethylene biosynthesis by aminoethoxyvinylglycine nor inhibition of phenylalanine ammonia-lyase (EC 4.3.1.5) activity by 2-aminoindan-2-phosphonic acid affected the time course or extent of transcript accumulation. Thus, the increased demand for phenylalanine in the phenylpropanoid pathway required after elicitor treatment appears to be met by increased de novo synthesis of its biosynthetic enzymes.
Thrombopoietin, the Mp1 ligand, is essential for full megakaryocyte development.
Authors: Kaushansky K , Broudy V C , Lin N , Jorgensen M J , McCarty J , Fox N , Zucker-Franklin D , Lofton-Day C
Published: May 2006

The development of megakaryocytes (MKs) from their marrow precursors is one of the least understood aspects of hematopoiesis. Current models suggest that early-acting MK colony-stimulating factors, such as interleukin (IL) 3 or c-kit ligand, are required for expansion of hematopoietic progenitors into cells capable of responding to late-acting MK potentiators, including IL-6 and IL-11. Recently, the Mp1 ligand, or thrombopoietin (Tpo), has been shown to display both MK colony-stimulating factor and potentiator activities, at potencies far greater than that of other cytokines. In light of these findings, we tested the hypothesis that Tpo is absolutely necessary for MK development. In this report we demonstrate that neutralizing the biological activity of Tpo eliminates MK formation in response to c-kit ligand, IL-6, and IL-11, alone and in combination, but that these reagents only partially reduce MK formation in the presence of combinations of cytokines including IL-3. However, despite the capacity of IL-3 to support the proliferation and initial stages of MK differentiation, elimination of Tpo prevents the full maturation of IL-3-induced MK. These data indicate that two populations of MK progenitors can be identified: one that is responsive to IL-3 but can fully develop only in the presence of Tpo and a second that is dependent on Tpo for both proliferation and differentiation. Thus, our results strongly suggest that Tpo is the primary regulator of MK development and platelet production.
Habituation of an invertebrate escape reflex due to modulation by higher centers rather than local events.
Authors: Krasne F B , Teshiba T M
Published: May 2006

Learning is widely thought to result from altered potency of synapses within the neural pathways that mediate the learned behavior. Support for this belief, which pervades current physiological and computational thinking, comes especially from the analysis of cases of simple learning in invertebrates. Here, evidence is presented that in one such case, habituation of crayfish escape, the learning is more due to onset of tonic descending inhibition than to the intrinsic depression of circuit synapses to which it was previously attributed. Thus, the altered performance seems to depend at least as much on events in higher centers as on local plasticity.
Nitric oxide inhibits hypothalamic luteinizing hormone-releasing hormone release by releasing gamma-aminobutyric acid.
Authors: Seilicovich A , Duvilanski B H , Pisera D , Theas S , Gimeno M , Rettori V , McCann S M
Published: May 2006

Nitric oxide synthase (NOS)-containing neurons, termed NOergic neurons, occur in various regions of the hypothalamus, including the median eminence-arcuate region, which plays an important role in controlling the release of luteinzing hormone-releasing hormone (LHRH). We examined the effect of NO on release of gamma-aminobutyric acid (GABA) from medial basal hypothalamic (MBH) explants incubated in vitro. Sodium nitroprusside (NP) (300 microM), a spontaneous releaser of NO, doubled the release of GABA. This release was significantly reduced by incubation of the tissue with hemoglobin, a scavenger of NO, whereas hemoglobin alone had no effect on the basal release of GABA. Elevation of the potassium concentration (40 mM) in the medium increased GABA release 15-fold; this release was further augmented by NP. Hemoglobin blocked the increase in GABA release induced by NP but had no effect on potassium-induced release, suggesting that the latter is not related to NO. As in the case of hemoglobin, NG-monomethyl-L-arginine (NMMA), a competitive inhibitor of NOS, had no effect on basal release of GABA, which indicates again that NO is not significant to basal GABA release. However, NMMA markedly inhibited the release of GABA induced by high potassium, which indicates that NO plays a role in potassium-induced release of GABA. In conditions in which the release of GABA was substantially augmented, there was a reduction in GABA tissue stores as well, suggesting that synthesis of GABA in these conditions did not keep up with release of the amine. Although NO released GABA, there was no effect of the released GABA on NO production, for incubation of MBH explants with GABA had no effect on NO release as measured by [14C]citrulline production. To determine whether GABA had any effect on the release of LHRH from these MBH explants, GABA was incubated with the tissue and the effect on LHRH release was determined. GABA (10(-5) or 10(-6) M) induced a 70% decrease in the release of LHRH, indicating that in the male rat GABA inhibits the release of this hypothalamic peptide. This inhibition in LHRH release induced by GABA was blocked by NMMA (300 microM), which indicates that GABA converts the stimulatory effect of NO on LHRH release into an inhibitory one, presumably via GABA receptors, which activate chloride channels that hyperpolarize the cell. Previous results have indicated that norepinephrine stimulates release of NO from the NOergic neurons, which then stimulates the release of LHRH. The current results indicate that the NO released also induces release of GABA, which then inhibits further LHRH release. Thus, in vivo the norepinephrinergic-driven pulses of LHRH release may be terminated by GABA released from GABAergic neurons via NO.
Folding of a nascent polypeptide chain in vitro: cooperative formation of structure in a protein module.
Authors: De Prat Gay G , Ruiz-Sanz J , Neira J L , Itzhaki L S , Fersht A R
Published: May 2006

We have prepared a family of peptide fragments of the 64-residue chymotrypsin inhibitor 2, corresponding to its progressive elongation from the N terminus. The growing polypeptide chain has little tendency to form stable structure until it is largely synthesized, and what structures are formed are nonnative and lack, in particular, the native secondary structural elements of alpha-helix and beta-sheet. These elements then develop as sufficient tertiary interactions are made in the nearly full-length chain. The growth of structure in the small module is highly cooperative and does not result from the hierarchical accretion of substructures.