bluuebunny/crossref_metadata_2025 huggingface dataset:
The Src family tyrosine kinases are required for platelet-derived growth factor-mediated signal transduction in NIH 3T3 cells.
Authors: Twamley-Stein G M , Pepperkok R , Ansorge W , Courtneidge S A
Published: May 2006

Three members of the Src family of protein tyrosine kinases Src, Fyn, and Yes associate with the activated platelet-derived growth factor (PDGF) receptor in vivo. This interaction requires the Src homology 2 (SH2) domain of the Src family member and causes activation of the intrinsic activity of the Src family kinases. We microinjected cells with DNA encoding catalytically inactive forms of the Src and Fyn proteins and examined their effects on PDGF-mediated signaling in vivo. Kinase-inactive Src and Fyn inhibited PDGF-stimulated entry of cells into S phase, whereas kinase-active forms of the proteins had no inhibitory effects. An intact SH2 domain was required for inhibition. Furthermore, when kinase-inactive Fyn was comicroinjected with a plasmid expressing activated Ras, the cells could enter S phase, indicating that the expression of kinase-inactive Fyn did not damage cell viability. Injection of an antibody specific for Src, Fyn, and Yes also reduced signal transduction through the PDGF receptor but only when injected within 8 hr of PDGF stimulation. Together these results indicate that the ubiquitously expressed Src family members are required for PDGF-induced mitogenic signaling.
RAD9-dependent G1 arrest defines a second checkpoint for damaged DNA in the cell cycle of Saccharomyces cerevisiae.
Authors: Siede W , Friedberg A S , Friedberg E C
Published: May 2006

Exposure of the yeast Saccharomyces cerevisiae to ultraviolet (UV) light, the UV-mimetic chemical 4-nitroquinoline-1-oxide (4NQO), or gamma radiation after release from G1 arrest induced by alpha factor results in delayed resumption of the cell cycle. As is the case with G2 arrest following ionizing radiation damage [Weinert, T. A. & Hartwell, L. H. (1988) Science 241, 317-322], the normal execution of DNA damage-induced G1 arrest depends on a functional yeast RAD9 gene. We suggest that the RAD9 gene product may interact with cellular components common to the G1/S and G2/M transition points in the cell cycle of this yeast. These observations define a checkpoint in the eukaryotic cell cycle that may facilitate the repair of lesions that are otherwise processed to lethal and/or mutagenic damage during DNA replication. This checkpoint apparently operates after the mating pheromone-induced G1 arrest point but prior to replicative DNA synthesis, S phase-associated maximal induction of histone H2A mRNA, and bud emergence.
Bilitranslocase and sulfobromophthalein/bilirubin-binding protein are both involved in the hepatic uptake of organic anions.
Authors: Torres A M , Lunazzi G C , Stremmel W , Tiribelli C
Published: May 2006

The hepatic uptake of cholephilic organic anions is a carrier-mediated process. Three distinct proteins [bilitranslocase (BTL), sulfobromophthalein (BSP)/bilirubin-binding protein (BBBP), and organic anion-binding protein] have been isolated from the basolateral plasma-membrane domain of the hepatocyte. To investigate the relative role of the first two of them in accounting for the hepatic uptake of organic anions, we measured the initial rates of uptake of 35S-labeled BSP into rat liver plasma-membrane vesicles. Because transport by BTL is electrogenic but transport by BBBP is electroneutral, studies were done either with or without a positive-inside membrane potential produced by adding valinomycin in the presence of an inwardly directed K+ gradient (outside K+ > inside K+). Both electrogenic and electroneutral transport systems followed saturation kinetics. Electroneutral uptake showed an apparent Km of 20 +/- 3 microM (mean +/- SD) and a Vmax of 1.0 +/- 0.13 nmol.(mg of prot)-1.15 sec-1, whereas the electrogenic portion of BSP uptake exhibited a Km of 5.2 +/- 0.8 microM and a Vmax of 1.1 +/- 0.1 nmol.(mg of prot)-1.15 sec-1. In this case, an overshoot was observed 15 sec after valinomycin addition. Electroneutral BSP uptake was inhibited by incubation with anti-BBBP antibody, whereas anti-BTL antibody did not show any inhibitory effect. Conversely, the electrogenic uptake was inhibited by anti-BTL antibody at a BSP concentration of 5 microM; no inhibition was seen either at 20 microM BSP or upon addition of anti-BBBP antibody. From these data we conclude that the hepatic uptake of organic ions occurs via two immunologically distinct carrier proteins (BTL and BBBP) operating in parallel. BTL is a higher affinity electrogenic transporting system of organic ions, whereas BBBP is a lower affinity electroneutral transporter.
A transcription inhibitor specific for unwound DNA in RNA polymerase-promoter open complexes.
Authors: Mazumder A , Perrin D M , Watson K J , Sigman D S
Published: May 2006

The kinetically component open complexes formed at prokaryotic and eukaryotic transcription start sites are efficiently nicked by the chemical nuclease activity of the 2:1 1,10-phenanthroline-copper(I) complex [(OP)2Cu+] and hydrogen peroxide. This reaction specificity has been attributed to the creation of a binding site(s) for redox-active tetrahedral (OP)2Cu+ when RNA polymerase form productive complexes with promoters. This proposal has been confirmed for the Escherichia coli lac UV-5 promoter by the demonstration that the 2:1 2,9-dimethyl-1,10-phenanthroline-copper(I) complex [(Me2OP)2Cu+], a redox-inactive isostere of (OP)2-Cu+, protects the transcription start site from scission by the chemical nuclease activity. (Me2OP)2Cu+ is also an effective inhibitor of transcription. The inhibition of transcription and the protection from scission of the open complex by (OP)2Cu+ exhibit the same dependence on the concentration of (Me2OP)2Cu+. This redox- and exchange-stable species is a previously undescribed transcription inhibitor that binds to a site generated by the interaction of RNA polymerase with the promoter. Unlike the intercalating agent proflavine, which is also an effective transcription inhibitor, it does not displace the enzyme from the promoter. The ability of (Me2OP)2Cu+ to inhibit transcription may be partially responsible for its potent cytotoxicity.
The isolation of molecular genetic markers for the identification of sex.
Authors: Griffiths R , Tiwari B
Published: May 2006

It is difficult to identify sex in many species of eukaryotic organism. This can considerably impede research into their biology. Fortunately, one sex often possesses a unique chromosome termed Y or W. When DNA markers are available for these chromosomes, then sex identification becomes straightforward. We describe a technique that facilitates the isolation of such markers. The procedure makes use of low-stringency PCR to screen randomly selected primers for their ability to amplify sex-specific loci.
FimC is a periplasmic PapD-like chaperone that directs assembly of type 1 pili in bacteria.
Authors: Jones C H , Pinkner J S , Nicholes A V , Slonim L N , Abraham S N , Hultgren S J
Published: May 2006

Biogenesis of the type 1 pilus fiber in Escherichia coli requires the product of the fimC locus. We have demonstrated that FimC is a member of the periplasmic chaperone family. The deduced primary sequence of FimC shows a high degree of homology to PapD and fits well with the derived consensus sequence for periplasmic chaperones, predicting that it has an immunoglobulin-like topology. The chaperone activity of FimC was demonstrated by purifying a complex that FimC forms with the FimH adhesion. A fimC1 null allele could be complemented by the prototype member of the chaperone superfamily, PapD, resulting in the production of adhesive type 1 pili. The general mechanism of action of members of the chaperone superfamily was demonstrated by showing that the ability of PapD to assemble both P and type 1 pili was dependent on an invariant arginine residue (Arg-8), which forms part of a conserved subunit binding site in the cleft of PapD. We suggest that the conserved cleft is a subunit binding feature of all members of this protein family. These studies point out the general strategies used by Gram-negative bacteria to assemble adhesins into pilus fibers, allowing them to promote attachment to eukaryotic receptors.
Asparagine 229 in thymidylate synthase contributes to, but is not essential for, catalysis.
Authors: Liu L , Santi D V
Published: May 2006

The conserved Asn-229 (N229) of thymidylate synthase (TS, EC 2.1.1.45) provides the only side chain that directly hydrogen bonds with the pyrimidine ring of the substrate dUMP. The carboxamide moiety forms a cyclic hydrogen bond network with the NH-3 and O-4 of the base and is a prime candidate for assisting proton-transfer reactions that occur at O-4 of the pyrimidine ring of dUMP. A complete replacement set of mutants at position 229 of Lactobacillus casei TS (N229 mutants) has been prepared, purified, and characterized. Fifteen of the 19 TS mutants were catalytically active. Steady-state kinetic parameters of N229 mutants varied 17- and 115-fold in the Km values for 5,10-methylene-5,6,7,8-tetrahydrofolate and dUMP, respectively, 1000-fold in kcat values, and 10,000-fold in kcat/Km values. Wild-type TS possesses lower Km and higher kcat and kcat/Km values than any of the TS N229 mutants. We conclude that N229 contributes to, but is not essential for, binding and catalysis. When the wild-type enzyme was not considered, there were excellent correlations between log kcat and the hydrophobicity of the side chains at position 229, in which the more hydrophobic side chains showed higher values. Our results suggest a unique interaction between N229 and the substrates that seems important in appropriately positioning the uracil heterocycle for catalysis. We propose that in the absence of N229, the electrophilic catalyst that transfers protons to the O-4 and stabilizes enol intermediates is a highly conserved molecule of water.
Recombinant human insulin-like growth factor I exerts a trophic action and confers glutamate sensitivity on glutamate-resistant cerebellar granule cells.
Authors: Calissano P , Ciotti M T , Battistini L , Zona C , Angelini A , Merlo D , Mercanti D
Published: May 2006

Cerebellar granule cells grown in the presence of a serum complex differentiate but are resistant to the lethal action of excitatory amino acids. When these cells are grown also in the presence of insulin-like growth factor I (IGF-I) they become fully susceptible to the toxic, lethal action of glutamate. The glutamate-sensitizing action of IGF-I is dependent on concentration (half-maximal effect at 2-4 ng/ml) and time (half-maximal effect at 2-4 days in vitro) and is paralleled by the appearance of functionally active, glutamate-activated, Ca2+ channels and of voltage-gated Na+ and late K+ channels. IGF-I-induced glutamate sensitivity is rapidly reversible (t1/2 = 30-60 min) after removal of this somatomedin. The action of IGF-I is not mimicked by IGF-II, nerve growth factor, basic or acidic fibroblast growth factor, platelet-derived growth factor, or tumor necrosis factor alpha. We postulate that the constitutive phenotype of cerebellar granule cells is glutamate-resistant and becomes responsive to excitatory amino acids under the action of epigenetic cues among which IGF-I may be one of those operative in vivo.
Physiological compensation in antisense transformants: specific induction of an "ersatz" glucan endo-1,3-beta-glucosidase in plants infected with necrotizing viruses.
Authors: Beffa R S , Neuhaus J M , Meins F
Published: May 2006

Plant class I glucan endo-1,3-beta-glucosidases (beta-1,3-glucanase; 1,3-beta-D-glucan glucanohydrolase, EC 3.2.1.39) have been implicated in development and defense against pathogen attack. Nevertheless, beta-1,3-glucanase deficiencies generated by antisense transformation of Nicotiana sylvestris and tobacco have little biological effect. We report here that another beta-1,3-glucanase activity is induced in these deficient mutants after infection with necrotizing viruses. Induction of class I beta-1,3-glucanase was markedly inhibited in leaves of N. sylvestris and tobacco antisense transformants infected with tobacco necrosis virus and tobacco mosaic virus, respectively. A serologically distinct beta-1,3-glucanase activity was present in the infected antisense transformants but was absent in both healthy and infected control plants and in antisense transformants treated with the stress hormone ethylene. Immunoblot analyses, localization studies, and measurements of antibody specificity indicate that this compensatory beta-1,3-glucanase activity is an intracellular enzyme different from known tobacco beta-1,3-glucanases. Therefore, plants can compensate for a deficiency in enzyme activity by producing a functionally equivalent replacement--i.e., "ersatz"--protein or proteins. The fact that compensation for beta-1,3-glucanase activity occurs in response to infection argues strongly for an important role of these enzymes in pathogenesis.
Amylin modulates beta-cell glucose sensing via effects on stimulus-secretion coupling.
Authors: Wagoner P K , Chen C , Worley J F , Dukes I D , Oxford G S
Published: May 2006

The release of insulin from the pancreatic beta cell is dependent upon a complex interplay between stimulators and inhibitors. Recently, amylin, a peptide secreted by pancreatic beta cells, has been implicated in the development of type II (noninsulin dependent) diabetes through its modulation of the peripheral effects of insulin. However, the effect of amylin on insulin secretion from the beta cell has remained controversial. It is reported here that in single beta cells exhibiting normal glucose sensing, amylin causes membrane hyperpolarization, increases in net outward current, and reductions in insulin secretion. In contrast, in cells with abnormal glucose sensing (e.g., from db/db diabetic mice), amylin has no effect on electrical activity or secretion. Thus, amylin's effects on excitation-secretion coupling in the beta cell of the pancreas appear to be linked to the cell's capacity for normal glucose sensing.
On the span of supersymmetric Young tableaux.
Authors: Rota G C , Stein J A
Published: May 2006

De Concini et al. [De Concini, C., Eisenbud, D. & Procesi, C. (1980) Invent. Math. 56, 129-165] have established for classical Young bitableaux the fact that the span of all bitableaux of shape lambda over the rationals includes all bitableaux of all shapes mu > lambda. We extend their result to the more general setting of supersymmetric Young tableaux. Our proof, even in the classical case, has the advantage of providing an explicit combinatorial algorithm for the computation of the coefficients.
The boundaries of partially edited transcripts are not conserved in kinetoplastids: implications for the guide RNA model of editing.
Authors: Landweber L F , Fiks A G , Gilbert W
Published: May 2006

We have studied partially edited molecules for the cytochrome-c oxidase subunit III (COIII) transcript from two species of the insect trypanosome Herpetomonas. We found unexpected patterns of editing, in which editing does not proceed strictly 3' to 5', in 24 of 61 partially edited clones. A comparison of the partially edited molecules between the two kinetoplastid species revealed an 8- to 10-nt shift in precisely defined editing boundaries, sites at which editing pauses before binding of the next guide RNA after formation of a stable duplex between a guide RNA and mRNA. This suggests that the region of base pairing between individual guide RNAs and the COIII transcript is not strictly conserved in kinetoplastids, implying gradual evolution of the editing process.
Superfamily Gondwanatherioidea: a previously unrecognized radiation of multituberculate mammals in South America.
Authors: Krause D W , Bonaparte J F
Published: May 2006

Multituberculates were the longest-lived order of the Class Mammalia and, during the Mesozoic and early Cenozoic, were among the most diverse and abundant representatives of the class. However, until the recent discovery of two Cretaceous teeth, one from South America and one from Africa, they were known only from northern continents. Additional material of the South American form Ferugliotherium has confirmed its multituberculate affinities and indicates that it may be a derived member of the Suborder Plagiaulacoidea. New specimens provide evidence that two other South American forms, Gondwanatherium and Sudamerica, are also multituberculates and that they are closely related to Ferugliotherium. Gondwanatherium and Sudamerica, each possessing highly specialized hypsodont molars, were previously thought to be the earliest known representatives of the Edentata, to be involved in the origin of edentates, or to represent a previously unknown higher taxon of mammals. However, there are detailed similarities in gross dental morphology, enamel microstructure, and inferred direction of jaw movement among Ferugliotherium, Gondwanatherium, and Sudamerica. All three genera are here regarded as representatives of a highly derived, endemic radiation of South American multituberculates and are allocated to the superfamily Gondwanatherioidea. Multituberculates were therefore more common elements of Late Cretaceous and early Paleocene mammalian faunas of South America than previously recognized.
Multiple Ca2+ channel types coexist to regulate synaptosomal neurotransmitter release.
Authors: Turner T J , Adams M E , Dunlap K
Published: May 2006

The regulation of excitation-secretion coupling by Ca2+ channels is a fundamental property of the nerve terminal. Peptide toxins that block specific Ca2+ channel types have been used to identify which channels participate in neurotransmitter release. Subsecond measurements of [3H]-glutamate and [3H]dopamine release from rat striatal synaptosomes showed that P-type channels, which are sensitive to the Agelenopsis aperta venom peptide omega-Aga-IVA, trigger the release of both transmitters. Dopamine (but not glutamate) release was also controlled by N-type, omega-conotoxin-sensitive channels. With strong depolarizations, where neither toxin was very effective alone, a combination of omega-Aga-IVA and omega-conotoxin produced a synergistic inhibition of 60-80% of Ca(2+)-dependent dopamine release. The results suggest that multiple Ca2+ channel types coexist to regulate neurosecretion under normal physiological conditions in the majority of nerve terminals. P- and N-type channels coexist in dopaminergic terminals, while P-type and a omega-conotoxin- and omega-Aga-IVA-resistant channel coexist in glutamatergic terminals. Such an arrangement could lend a high degree of flexibility in the regulation of transmitter release under diverse conditions of stimulation and modulation.
A 4.5-megabase yeast artificial chromosome contig from human chromosome 13q14.3 ordering 9 polymorphic microsatellites (22 sequence-tagged sites) tightly linked to the Wilson disease locus.
Authors: White A , Tomfohrde J , Stewart E , Barnes R , Le Paslier D , Weissenbach J , Cavalli-Sforza L , Farrer L , Bowcock A
Published: May 2006

We have previously performed a genetic analysis of multiply affected families to map a locus responsible for Wilson disease (WND) to a 0.3-centimorgan (cM) region within chromosome 13q14.3, between D13S31 and D13S59. Here we describe the construction of a contig of approximately 4.5 Mb, which spans this region and extends from D13S25 to D13S59. This contig consists of 28 genomic yeast artificial chromosome (YAC) clones. Five critical crossover events have been defined in this interval in two unaffected (Centre d'Etudes du Polymorphisme Humain) and three WND families. The combination of sequence tagged site content mapping of YACs with both polymorphic and nonpolymorphic markers and recombination breakpoint mapping resulted in the following order of polymorphic markers: centromere-RB1-D13S25-AFM205vh2-D13S31-D13S22 7-D13S228-AFM238vc3-D13S133- AFM084xc5-D13S137-D13S169, D13S155-D13S59-telomere. The recombination/physical distance ratio varies from approximately 3000 kb per cM in the region between D13S31 and D13S25 to 6000 kb per cM in the region between D13S31 and D13S59. Three WND families exhibiting recombination between the disease locus and D13S31 or D13S59 were genotyped for additional markers in this region and further refined the location of the WND gene to between D13S155 and D13S133. Nine of the markers in this region of < 1 cM are polymorphic microsatellites (seven have observed heterozygosities of 70% or above) that will be extremely useful in prenatal and preclinical diagnosis of this disease. This physical map is an essential step in the isolation of the WND gene and is a framework for the identification of candidate genes.
Probing the role of loop 2 in Ras function with unnatural amino acids.
Authors: Chung H H , Benson D R , Cornish V W , Schultz P G
Published: May 2006

The YDPT sequence motif (residues 32-35) in loop 2 (residues 32-40) of Ha-Ras p21 protein is conserved in the Ras protein family. X-ray crystal structures have revealed significant conformational differences in this region between the GTP- and GDP-bound forms. Moreover, mutations in this region block neoplastic transformation and prevent interaction with GTPase-activating protein (GAP), suggesting that this region may contribute to the effector function of Ras. To better understand the structural features required for GAP interaction and GTPase activity, the expanded repertoire of unnatural amino acid mutagenesis has been used to investigate the roles of the key residues, Pro-34, Thr-35, and Ile-36. A Pro-34-->methanoproline mutant, in which residue 34 is locked in the trans conformation, was found to retain high levels of intrinsic and GAP-activated GTPase activity, making unlikely conformational isomerization at this position. Deletion of a single methyl group from Ile (Ile-36-->norvaline) abolished GAP activation of Ras, revealing a remarkable specificity in this protein-protein interaction. Finally, replacement of Thr-35 with diastereomeric allo-threonine led to inactivation of Ras, demonstrating the importance of the orientation of this critical residue in Ras function.
Relationship between NMR transverse relaxation, trabecular bone architecture, and strength.
Authors: Chung H , Wehrli F W , Williams J L , Kugelmass S D
Published: May 2006

Structure, biomechanical competence, and incremental NMR line broadening (R'2) of water in the intertrabecular spaces of cancellous bone were examined on 22 cylindrical specimens from the lumbar vertebral bodies of 16 human subjects 24-86 years old (mean, 60 years old). A strong association (r = 0.91; P < 0.0001) was found between Young's modulus of elasticity and R'2 for a wide range of values corresponding to cancellous bone of very different morphologic composition. NMR line broadening is caused by the inhomogeneity of the magnetic field induced as a consequence of the coexistence of two adjacent phases of different diamagnetic susceptibility--i.e., mineralized bone and water in the marrow spaces. Structural analyses performed by means of NMR microscopy and digital image processing indicated that the variation in R'2 is closely related to the trabecular microstructure. Mean trabecular plate density measured along the direction of the magnetic field was found to play a major role in predicting R'2 (r = 0.74; P < 0.0001). This behavior was confirmed when the plate density was varied in individual specimens, which was achieved by rotating the specimen, making use of the bone's structural anisotropy. It is concluded that the NMR transverse relaxation rate in human cancellous bone of the spine is significantly determined by trabecular structural parameters relevant to biomechanical strength. The results further underscore the important role played by the transverse trabeculae in contributing to cancellous bone strength. The work has implications on possible in vivo use of quantitative magnetic resonance for the assessment of fracture risk in osteoporotic patients.
Charged-coupled detector sky surveys.
Authors: Schneider D P
Published: May 2006

Sky surveys have played a fundamental role in advancing our understanding of the cosmos. The current pictures of stellar evolution and structure and kinematics of our Galaxy were made possible by the extensive photographic and spectrographic programs performed in the early part of the 20th century. The Palomar Sky Survey, completed in the 1950s, is still the principal source for many investigations. In the past few decades surveys have been undertaken at radio, millimeter, infrared, and x-ray wavelengths; each has provided insights into new astronomical phenomena (e.g., quasars, pulsars, and the 3 degrees cosmic background radiation). The advent of high quantum efficiency, linear solid-state devices, in particular charged-coupled detectors, has brought about a revolution in optical astronomy. With the recent development of large-format charged-coupled detectors and the rapidly increasing capabilities of data acquisition and processing systems, it is now feasible to employ the full capabilities of electronic detectors in projects that cover an appreciable fraction of the sky. This talk reviews the first "large scale" charged-coupled detector survey. This program, designed to detect very distant quasars, reveals the powers and limitations of charged-coupled detector surveys.
Inhibition of insulitis and prevention of diabetes in nonobese diabetic mice by blocking L-selectin and very late antigen 4 adhesion receptors.
Authors: Yang X D , Karin N , Tisch R , Steinman L , McDevitt H O
Published: May 2006

Leukocyte adhesion to the endothelial venules in the pancreatic islets is thought to be one of the initial steps in the development of insulin-dependent diabetes mellitus. It has been suggested that leukocyte adhesion to endothelium is a sequential multistep process involving various different homing receptors. We report here that blocking different homing receptors--namely, L-selectin and very late antigen 4 (VLA-4)--which function during different stages of the adhesion process, by specific monoclonal antibodies inhibits insulitis and prevents diabetes in mice. Moreover, leukocyte attachment to the inflamed vessels within pancreatic sections could be inhibited by anti-L-selectin and anti-VLA-4 antibodies. Interestingly, anti-L-selectin or anti-VLA-4 antibody did not appear to influence the autoimmune response to a panel of pancreatic beta-cell autoantigens. These data suggest that L-selectin and VLA-4 receptors are involved in mediating leukocyte homing to the islets and that intervention of these two adhesion pathways may provide a novel approach for treatment of autoimmune diseases such as insulin-dependent diabetes mellitus.
Automatic enumeration and characterization of heterogeneous clonal progression in cell transformation.
Authors: Yao A , Rubin H
Published: May 2006

Most human tumors are clonal in origin, although the cells may be diverse in their properties. Since the tumors evolve through progressive stages over decades of time, it is possible that the conditions that induced the tumor transform many cells, but that selective overgrowth of the fastest growing lead to a clonal population of identified tumor cells. We studied the progression of neoplastic transformation in clones from a population in which about 10% of the cells formed well-defined transformed foci. A few of the clones produced many large foci, but most of the clones produced no foci or only one focus. Maintenance of the nonproducing and low-producing clones under the growth constraint of confluence and low serum concentration, which promotes transformation, led to the production of large numbers of small foci by all of them. Visual inspection revealed considerable heterogeneity in size and density among the foci from each clone, and this was quantitated by computer scanning. Subclonal analysis of focus formation was done on one of the clones after it had undergone further growth constraint to promote transformation. As in the original cloning, some of the subclones produced many large foci, but most produced none. Another round of growth constraint was imposed on the nonproducing subclones, which then became producers of many small foci varying in size and density. The results indicate that most if not all cells in the population respond to growth constraint by undergoing transformation. Though there is wide variation in the degree of transformation, the results are consistent with the view that an entire field of cells exposed to carcinogenic conditions in an animal undergoes some progression toward neoplasia but that heterogeneity of the response followed by selective neoplastic growth may lead to a clonal origin of the clinically detected tumor.
Identification of a domain required for oncogenic activity and transcriptional suppression by v-erbA and thyroid-hormone receptor alpha.
Authors: Damm K , Evans R M
Published: May 2006

v-erbA, a mutated version of the chicken thyroid hormone (TH) receptor type alpha, can inhibit hormonal induction of target genes. In addition, v-erbA acts as a constitutive repressor of the basal promoter activity. In vivo, v-erbA can arrest the differentiation of erythroid precursor cells and suppresses transcription of erythrocyte-specific genes. We show that the v-erbA protein of the transformation-defective avian erythroblastosis virus mutant (AEVtd359) fails to suppress basal transcription level and exhibits impaired ability in antagonizing the TH and retinoic acid response. The inactivating mutation is a 1-nt change leading to a Pro-->Arg replacement in the "hinge region" of v-erbA protein. Introducing this mutation in the context of TH receptor alpha selectively inactivates the suppressor function, while hormone-binding and transcriptional-activation properties are unaffected. These data suggest that trans-repression rather than a dominant negative block of TH-receptor or retinoic acid-receptor activation may represent the primary molecular property underlying erbA oncogenesis.
Inhibition of vascular endothelial cell growth factor activity by an endogenously encoded soluble receptor.
Authors: Kendall R L , Thomas K A
Published: May 2006

Vascular endothelial cell growth factor, a mitogen selective for vascular endothelial cells in vitro that promotes angiogenesis in vivo, functions through distinct membrane-spanning tyrosine kinase receptors. The cDNA encoding a soluble truncated form of one such receptor, fms-like tyrosine kinase receptor, has been cloned from a human vascular endothelial cell library. The mRNA coding region distinctive to this cDNA has been confirmed to be present in vascular endothelial cells. Soluble fms-like tyrosine kinase receptor mRNA, generated by alternative splicing of the same pre-mRNA used to produce the full-length membrane-spanning receptor, encodes the six N-terminal immunoglobulin-like extracellular ligand-binding domains but does not encode the last such domain, transmembrane-spanning region, and intracellular tyrosine kinase domains. The recombinant soluble human receptor binds vascular endothelial cell growth factor with high affinity and inhibits its mitogenic activity for vascular endothelial cells; thus this soluble receptor could act as an efficient specific antagonist of vascular endothelial cell growth factor in vivo.
The Swi5 zinc-finger and Grf10 homeodomain proteins bind DNA cooperatively at the yeast HO promoter.
Authors: Brazas R M , Stillman D J
Published: May 2006

SWI5 encodes a zinc-finger protein required for expression of the yeast HO gene. Using Swi5 protein that was purified from a bacterial expression system, we previously isolated a yeast factor that stimulates binding of Swi5 to the HO promoter. N-terminal amino acid sequence analysis identified the Swi5 stimulatory factor as the product of the GRF10 gene, which encodes a yeast homeodomain protein. GRF10, also known as PHO2 and BAS2, is a transcriptional activator of the PHO5 acid phosphatase gene and the HIS4 histidine biosynthesis gene. Grf10 protein purified from a bacterial expression system binds DNA cooperatively with Swi5 in vitro. Analysis of disassociation rates indicates that the Grf10-Swi5-DNA complex has a longer half-life than protein-DNA complexes that contain only Swi5 or Grf10. Finally, we show that HO expression is reduced in yeast strains containing grf10 null mutations and that full expression of a heterologous promoter containing a SWI5-dependent HO upstream activation sequence element requires GRF10.
Strings of contiguous modified pentanucleotides with increased DNA-binding affinity can be used for DNA sequencing by primer walking.
Authors: Azhikina T , Veselovskaya S , Myasnikov V , Potapov V , Ermolayeva O , Sverdlov E
Published: May 2006

Modified oligonucleotides containing 5-methylcytidine and/or 2-aminoadenosine form tighter hybrids with DNA and are, therefore, more efficient primers for DNA sequencing as compared to their natural counterparts. Strings of contiguous modified pentanucleotides can be used for DNA sequencing by primer walking.
Evolution and expression of a beta-like globin gene of the Australian marsupial Sminthopsis crassicaudata.
Authors: Cooper S J , Hope R M
Published: May 2006

A beta-like globin gene was isolated from the Australian dasyurid marsupial Sminthopsis crassicaudata. Nucleotide-sequence analysis of promoter and coding regions of the gene revealed that it was orthologous to eutherian early-expressed (epsilon, gamma, eta) beta-like globin genes. Comparison of the conceptually translated sequence of the gene with a partial amino acid sequence of the adult beta-globin chain from S. crassicaudata provided evidence that the gene was not expressed in adult tissues. In addition, Northern analysis of RNA isolated from an embryo, pouch young, and adult bone marrow indicated that the gene was expressed predominantly in embryonic tissues and that there was a significant reduction in the expression of the gene within a day of birth. These results provide strong support for the hypothesis of Koop and Goodman [Koop, B. F. & Goodman, M. (1988) Proc. Natl. Acad. Sci. USA 85, 3893-3897] that an embryonic beta-like globin gene existed prior to the divergence of the eutherian and marsupial lineages and that this gene was already differentiated with respect to its promoter regions and developmental expression. The observation that epsilon-globin mRNA was present at least until day 4 postpartum suggests that the epsilon-globin chain may play some role in influencing the physiological properties of hemoglobin in S. crassicaudata neonates.
Equilibration during mantle melting: a fractal tree model.
Authors: Hart S R
Published: May 2006

Many basalts from oceanic islands, ridges, and arcs show strong trace element evidence for melting at great depths, where garnet is a stable phase in mantle peridotites. If partial melts ascend to the surface by porous (intergranular) flow processes, the high-pressure garnet signature will be obliterated by diffusive reequilibration at shallower depths in the mantle. Spiegelman and Kenyon [Spiegelman, M. & Kenyon, P. (1992) Earth Planet Sci. Lett. 109, 611-620] argued that partial melts must therefore be focused into a coarser transport network, for high-speed delivery to the surface. Numerous natural network systems, such as rivers and the human vascular and bronchial systems, have fractal structures that are optimal for minimizing energy expenditure during material transport. I show here that a fractal magma "tree" with these optimal properties provides a network in which magma rapidly loses diffusive chemical "contact" with its host matrix. In this fractal network, magma conduits combine by twos, with the radius and flow velocities scaling as (2)n/3, where n is the generation number. For reasonable values of volume diffusivities, viscosities, and aspect ratios, melts will experience only limited diffusive reequilibration once they have traveled some hundreds of meters from their source. Melts thus represent rather local mantle domains, and there is little problem in delivering melts with deep (<100 km) geochemical signatures to the surface.
Developmental regulation and phytochrome-mediated induction of mRNAs encoding a proline-rich protein, glycine-rich proteins, and hydroxyproline-rich glycoproteins in Phaseolus vulgaris L.
Authors: Sheng J , Jeong J , Mehdy M C
Published: May 2006

We have studied developmental and light regulation of mRNAs encoding a putative cell wall proline-rich protein (PvPRP1), cell wall glycine-rich proteins (GRPs), and cell wall hydroxyproline-rich glycoproteins (HRGPs) in bean (Phaseolus vulgaris). Light increases the levels of these mRNAs 2- to 150-fold in highly spatially regulated patterns during seedling development. These mRNA changes include differential regulation of transcripts derived from the GRP and HRGP multigene families. In 6-day-old light-grown seedlings, the PvPRP1 and GRP1.0 mRNAs were most abundant in the apical region of hypocotyls, epicotyls, and roots. In contrast, several HRGP transcripts were most abundant in the mature region of hypocotyls and roots in light-grown seedlings. When etiolated 6-day-old seedlings were illuminated with white light for 8 hr, maximal accumulation of PvPRP1 and GRP1.0 mRNAs occurred in the apical hook, whereas HRGP and GRP1.8 mRNAs accumulated in the mature region of hypocotyls. Etiolated seedlings subjected to a pulse of red light accumulated PvPRP1, GRP, and HRGP mRNAs in the hypocotyls. Far-red light inhibited red light induction of these mRNAs, indicating a phytochrome-mediated process. The possible roles of PRPs, GRPs, and HRGPs in cell differentiation and photomorphogenesis are discussed.
Mechanism of superconductivity in K3C60.
Authors: Chen G , Goddard W A
Published: May 2006

Using electronic states and phonon states from the first-principles calculations and including both conventional electron-phonon charge coupling and Jahn-Teller coupling, we predict Tc and other superconducting properties. The only adjustable parameter in the theory is the screening length, Rsc. Using Rsc = 0.8-1.0 A, we find excellent agreement with experiment for Tc (16-18 K), pressure dependence of Tc (Delta Tc = -6 to -10 K for 1 GPa), and 12 C to 13 C isotope shift (alphaC = 0.2); experimental values: 19 K, -7 K, and 0.3, respectively.
Three-dimensional profiles from residue-pair preferences: identification of sequences with beta/alpha-barrel fold.
Authors: Wilmanns M , Eisenberg D
Published: May 2006

The three-dimensional profile method expresses the three-dimensional structure of a protein as a table, the profile, which represents the local environment of each residue. The score of an amino acid sequence, aligned with the three-dimensional profile, reflects its compatibility with the profiled structure. In the original implementation, each local environment was characterized by its polarity, the area buried of its side chain, and its secondary structure. Here we describe a modified three-dimensional profile algorithm that characterizes the local environment in terms of the statistical preferences of the profiled residue for neighbors of specific residue types, main-chain conformations, or secondary structure. Combined profiles of the original and the three new types were tested on beta/alpha-barrel protein structures. The method identified the following enzymes of unknown three-dimensional structure as probable beta/alpha-barrels, all of which catalyze reactions in the biosynthesis of aromatic amino acids: anthranilate phosphoribosyltransferase (trpD), glutamine amidotransferase (trpG), and phosphoribosylformimino-5-aminoimidazole carboxamide ribotide isomerase (hisA).
Sequencing two DNA templates in five channels by digital compression.
Authors: Nelson M , Zhang Y , Steffens D L , Grabherr R , Van Etten J L
Published: May 2006

By applying algebraic coding methods to the Sanger dideoxynucleotide procedure, DNA sequences of two templates can be determined simultaneously in only five reactions and data channels. A 5:2 data compression is accomplished by instantaneous source coding of nucleotide sequence pairs into one set of 5-bit block codes. A general algebraic expression, 2n-1 > or = 4f, describes conditions under which f DNA templates can be sequenced using n channels. Such compression sequencing is accurate and efficient, as demonstrated by manual 35S autoradiographic detection and automated on-line analysis using fluorescent-labeled primers. Symmetric 5:2 compression is especially useful when comparing two closely related sequences.
Mouse hepatitis virus strain A59 and blocking antireceptor monoclonal antibody bind to the N-terminal domain of cellular receptor.
Authors: Dveksler G S , Pensiero M N , Dieffenbach C W , Cardellichio C B , Basile A A , Elia P E , Holmes K V
Published: May 2006

Mouse hepatitis virus (MHV) strain A59 uses as cellular receptors members of the carcinoembryonic antigen family in the immunoglobulin superfamily. Recombinant receptor proteins with deletions of whole or partial immunoglobulin domains were used to identify the regions of receptor glycoprotein recognized by virus and by antireceptor monoclonal antibody CC1, which blocks infection of murine cells. Monoclonal antibody CC1 and MHV-A59 virions bound only to recombinant proteins containing the entire first domain of MHV receptor. To determine which of the proteins could serve as functional virus receptors, receptor-negative hamster cells were transfected with recombinant deletion clones and then challenged with MHV-A59 virions. Receptor activity required the entire N-terminal domain with either the second or the fourth domain and the transmembrane and cytoplasmic domains. Recombinant proteins lacking the first domain or its C-terminal portion did not serve as viral receptors. Thus, like other virus receptors in the immunoglobulin superfamily, including CD4, poliovirus receptor, and intercellular adhesion molecule 1, the N-terminal domain of MHV receptor is recognized by the virus and the blocking monoclonal antibody.
Poxvirus-induced alteration of arachidonate metabolism.
Authors: Palumbo G J , Glasgow W C , Buller R M
Published: May 2006

Recent evidence suggests that orthopoxviruses have an obligate requirement for arachidonic acid metabolites during replication in vivo and in vitro. Our report indicates that a virus family (Poxviridae) possesses multiple genes that function to regulate arachidonate metabolism. Analyses of BS-C-1 cells infected with cowpox virus or vaccinia virus detected enhanced arachidonate product formation from both the cyclooxygenase (specifically prostaglandins E2 and F2 alpha) and lipoxygenase (specifically 15-hydroxyeicosatetraenoic acid and 12-hydroxyeicosatetraenoic acid) pathways. In contrast, human parainfluenza type 3 or herpes simplex virus type 1 infections did not increase arachidonate metabolism. Results were consistent with a virus early-gene product either directly mediating or inducing a host factor that mediated the up-regulation of arachidonate metabolism, although vaccinia growth factor was not responsible. In addition, the cowpox virus 38-kDa protein-encoding gene, which is associated with inhibition of an inflammatory response, correlated with inhibition of formation of a product biochemically characteristic of (14R,15S)-dihydroxyeicosatetraenoic acid. We propose that orthopoxvirus-induced up-regulation of arachidonic acid metabolism during infection renders the infected cells susceptible to generation of inflammatory mediators from both the cyclooxygenase and the lipoxygenase pathways, and poxviruses, therefore, possess at least one gene (38K) that can alter the lipoxygenase-metabolite spectrum.
Protein dynamics in minimyoglobin: is the central core of myoglobin the conformational domain?
Authors: Di Iorio E E , Yu W , Calonder C , Winterhalter K H , De Sanctis G , Falcioni G , Ascoli F , Giardina B , Brunori M
Published: May 2006

The kinetics of CO binding to the horse myoglobin fragment Mb-(32-139), the so-called "mini-Mb," were investigated by laser flash photolysis in 0.1 M phosphate buffer and in buffer with 75% (vol/vol) glycerol. The reaction displays complex time courses that can be approximated satisfactorily only with a sum of five exponentials. The features of the kinetic components and a comparison of the deoxy-minus-carbonyl difference spectra of mini-Mb and horse Mb obtained under equilibrium conditions, with the kinetic difference spectra resulting from the global analysis of the traces recorded between 400 and 450 nm, show that CO binding to mini-Mb is accompanied by large structural changes. In view of the fact that mini-Mb is an approximation of the Mb-(31-105) fragment encoded by the central exon of the Mb gene, this finding is particularly relevant. On the basis of our data and previous reports [De Sanctis, G., Falcioni, G., Giardina, B., Ascoli, F. & Brunori, M. (1988) J. Mol. Biol. 200, 725-733; De Sanctis, G., Falcioni, G., Grelloni, F., Desideri, A., Polizo, F., Giardina, B., Ascoli, F. & Brunori, M. (1992) J. Mol. Biol. 222, 637-643], we propose that the protein fragment encoded by the central exon of the Mb gene is the domain responsible for ligand-linked conformational transitions, while the two terminal fragments dampen the amplitude of the structural changes that accompany ligand binding, thus rendering the protein stable and kinetically more efficient in its physiological function.
Relationships between the responses of triglyceride-rich lipoproteins in blood plasma containing apolipoproteins B-48 and B-100 to a fat-containing meal in normolipidemic humans.
Authors: Schneeman B O , Kotite L , Todd K M , Havel R J
Published: May 2006

The concentration of triglyceride-rich lipoproteins containing apolipoprotein (apo) B-48 (chylomicrons) and apo B-100 (very low density lipoproteins) was measured in blood plasma of healthy young men after an ordinary meal containing one-third of daily energy and fat. Plasma obtained in the postabsorptive state and at intervals up to 12 hr after the meal was subjected to immunoaffinity chromatography against a monoclonal antibody to apo B-100 that does not bind apo B-48 and a minor fraction of apo B-100 rich in apo E. Measurements of the concentrations of components of the total and unbound triglyceride-rich lipoproteins separated from plasma by ultracentrifugation showed that about 80% of the increase in lipoprotein particle number was in very low density lipoproteins containing apo B-100 and only 20% was in chylomicrons containing apo B-48 that carry dietary fat from the intestine. The maximal increments and the average concentrations of apo B-48 and B-100 during the 12 hr were highly correlated (r2 = 0.80), suggesting that preferential clearance of chylomicron triglycerides by lipoprotein lipase leads to accumulation of hepatogenous very low density lipoproteins during the alimentary period. The composition of the bulk of very low density lipoproteins that were bound to the monoclonal antibody changed little and these particles contained about 90% of the cholesterol and most of the apo E that accumulated in triglyceride-rich lipoproteins. The predominant accumulation of very low density lipoprotein rather than chylomicron particles after ingestion of ordinary meals is relevant to the potential atherogenicity of postprandial lipoproteins.