bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic heterogeneity of hepatocellular carcinoma.
Authors: Unsal H , Yakicier C , Marçais C , Kew M , Volkmann M , Zentgraf H , Isselbacher K J , Ozturk M
Published: May 2006

We studied 80 hepatocellular carcinomas from three continents for p53 gene (TP53) mutations and hepatitis B virus (HBV) sequences. p53 mutations were frequent in tumors from Mozambique but not in tumors from South Africa, China, and Germany. Independent of geographic origin, most tumors were positive for HBV sequences. X gene coding sequences of HBV were detected in 78% of tumors, whereas viral sequences in the surface antigen- and core antigen-encoding regions were present in less than 45% of tumors. These observations indicate that hepatocellular carcinomas are genetically heterogeneous. Mozambican-type of hepatocellular carcinomas are characterized by a high incidence of p53 mutations related to aflatoxins. In other tumors, the rarity of p53 mutations combined with the frequent presence of viral X gene coding sequences suggests a possible interference of HBV with the wild-type p53 function.
One disulfide bond in front of the second heavy chain constant region is necessary and sufficient for effector functions of human IgG3 without a genetic hinge.
Authors: Michaelsen T E , Brekke O H , Aase A , Sandin R H , Bremnes B , Sandlie I
Published: May 2006

We have created four IgG3 mutants without a normal hinge region: (i) m0 without a genetic hinge; (ii) m0/C131S, where Cys-131 in m0 was mutated to Ser; (iii) m0/231C232 (formerly HM-1), where a Cys residue was inserted in m0 between Ala-231 and Pro-232; (iv) m0/C131S/231C232, which is a hybrid of m0/231C232 and m0/C131S. The wild-type IgG3 and all mutants bind 5-iodo-4-hydroxy-3-nitrophenacetyl groups. The wild type and mutants, m15 (with 15 aa in the hinge), m0/231C232, and m0/C131S/231C232, were all positive for complement-mediated lysis, antibody-dependent cellular cytotoxicity mediated by peripheral blood leukocytes, and phagocytosis by U937. m0/C131S/231C232 was only weakly positive and sometimes negative for respiratory burst activity mediated by peripheral blood neutrophils (polymorphonuclear leukocytes), whereas m15, m0/231C232, and wild-type IgG3 were strongly positive. The m0 and m0/C131S mutants were mainly negative for complement-mediated lysis, antibody-dependent cell-mediated cytotoxicity, and phagocytosis by U937 and polymorphonuclear leukocytes. The results indicate that a hinge spacer region is not necessary, but the correct alignment of the two second heavy chain constant regions in the IgG3 molecule by a minimum of one disulfide bond is necessary and sufficient for effector functions.
Characterization of the PP2A alpha gene mutation in okadaic acid-resistant variants of CHO-K1 cells.
Authors: Shima H , Tohda H , Aonuma S , Nakayasu M , DePaoli-Roach A A , Sugimura T , Nagao M
Published: May 2006

Okadaic acid (OA)-resistant variants of Chinese hamster ovary cells, clones CHO/OAR6-6 and CHO/OAR2-3, were isolated from a CHO-K1 culture. These variant cells were 17- to 26-fold more resistant to OA than the parental cells. The phosphorylase phosphatase activity of the variant cell extracts was 2- to 4-fold more resistant to OA than that of the parental cells in the presence of inhibitor 2, a specific inhibitor of type 1 protein serine/threonine phosphatase (PP1). Nucleotide sequencing of PP2A alpha (an isotype of PP2A catalytic subunit) cDNA demonstrated that both variants have a T-->G transversion at the first base of codon 269 (805 nt), which results in substitution of glycine for cysteine. We expressed in COS-1 cells a mutant PP2A alpha tagged with the influenza hemagglutinin epitope. The recombinant mutant PP2A alpha protein immunoprecipitated with an anti-influenza hemagglutinin antibody was more resistant than the wild type to OA, their IC50 values being 0.65 nM and 0.15 nM, and their IC80 values being 4.0 nM and 0.45 nM, respectively. The cysteine at residue 269 present only in highly OA-sensitive protein serine/threonine phosphatase catalytic subunit isozymes, PP2A alpha, PP2A beta, and PPX, is suggested to be involved in the binding of OA. CHO/OAR6-6 and CHO/OAR2-3 cells also overexpressed the P-glycoprotein, and the efflux of OA was more rapid. It is suggested that the PP2A alpha mutation in cooperation with a high level of P-glycoprotein makes the CHO-K1 variants highly resistant to OA.
Mechanism of high-affinity potassium uptake in roots of Arabidopsis thaliana.
Authors: Maathuis F J , Sanders D
Published: May 2006

Potassium is a major nutrient in higher plants, where it plays a role in turgor regulation, charge balance, leaf movement, and protein synthesis. Terrestrial plants are able to sustain growth at micromolar external K+ concentrations, at which K+ uptake across the plasma membrane of root cells must be energized despite the presence of a highly negative membrane potential. However, the mechanism of energization has long remained obscure. Therefore, whole-cell mode patch clamping has been applied to root protoplasts from Arabidopsis thaliana to characterize membrane currents resulting from the application of micromolar K+. Analysis of whole cell current/voltage relationships in the presence and absence of micromolar K+ enabled direct testing of K+ transport for possible energization by cytoplasmic ATP and the respective trans-membrane gradients of Na+, Ca2+, and H+. Subtracted current/voltage relations for K(+)-dependent membrane currents are independent of ATP and reverse at potentials that imply H(+)-coupled K+ transport with a ratio of 1 H+:K+. Furthermore, the reversal potential of the K+ current shifts negative as external H+ activity is decreased. K(+)-dependent currents saturate in the micromolar concentration range with an apparent Km of 30 microM, a value in close agreement with previously reported Km values for high-affinity K+ uptake. We conclude that our results are consistent with the view that high-affinity K+ uptake in higher plants is mediated by a H+:K+ symport mechanism, competent in driving K+ accumulation to equilibrium ratios in excess of 10(6)-fold.
Photoactive mitochondria: in vivo transfer of a light-driven proton pump into the inner mitochondrial membrane of Schizosaccharomyces pombe.
Authors: Hoffmann A , Hildebrandt V , Heberle J , Büldt G
Published: May 2006

The light-driven proton pump bacteriorhodopsin (bR) from Halobacterium salinarium has been genetically transferred into the inner mitochondrial membrane (IM) of the eukaryotic cell Schizosaccharomyces pombe, where the archaebacterial proton pump replaces or increases the proton gradient usually formed by the respiratory chain. For targeting and integration, as well as for the correct orientation of bR in the IM, the bacterioopsin gene (bop) was fused to signal sequences of IM proteins. Northern and Western blot analysis proved that all hybrid gene constructs containing the bop gene and a mitochondrial signal sequence were expressed and processed to mature bR. Fast transient absorption spectroscopy showed photocycle activity of bR integrated in the IM by formation of the M intermediate. Experiments with the pH-sensitive fluorescence dye 2',7'-bis(2-carboxyethyl)-5 (and -6)-carboxyfluorescein revealed bR-mediated proton pumping from the mitochondrial matrix into the intermembrane space. Glucose uptake measurements under anaerobic conditions showed that yeast cells containing photoactive mitochondria need less sugar under illumination. In summary, our experiments demonstrate the functional genetic transfer of a light energy converter to a naturally nonphotoactive eukaryotic organism.
A potential peptide vaccine against two different strains of influenza virus isolated at intervals of about 10 years.
Authors: Naruse H , Ogasawara K , Kaneda R , Hatakeyama S , Itoh T , Kida H , Miyazaki T , Good R A , Onoé K
Published: May 2006

We have developed a strategy for making synthetic peptide vaccines, in which a peptide, HA127-133, derived from the hemagglutinin (HA) of A/Aichi/2/68(H3N2) influenza virus (Aichi/68) is introduced into the Ab binding component consisting of 43-46 and 54-58 residues of a pigeon cytochrome c analogue peptide, 46F50V54A. Indeed, this hybrid peptide, 46F/HA127-133/54A, induced impressive T-cell responses and antibody production neutralizing infectivity of Aichi/68 in vitro. In a subsequent study we found that 46F/HA127-133/54A(18mer) peptide antigen, which had been prepared by substitution at the central five residues of 46F50V54A with HA127-133, generated T-cell responses and neutralizing antibody responses as well. On the basis of these prior findings, in the present study we analyzed immunopotency of 46F/HA127-133/54A(18mer) in vivo administered in several ways to I-Ab mice. We show herein that this peptide vaccine loaded in multilamellar liposomes without adjuvant protects the mice against infection with Aichi/68 within 2 weeks after final immunization. Further, this peptide vaccine was shown to be effective in preventing infection with a naturally occurring antigenic variant, A/Texas/1/77(H3N2), carrying the same sequence at 127-133 of the HA as Aichi/68 virus. Since this part of the HA is relatively conserved among H3 subtype influenza viruses, our peptide vaccine may become the basis for a new strategy to prepare effective vaccines that will overcome the ineffectiveness of classical vaccines attributable to antigenic drift of influenza viruses.
A cluster of mutations within a short triplet repeat in the C1 inhibitor gene.
Authors: Bissler J J , Cicardi M , Donaldson V H , Gatenby P A , Rosen F S , Sheffer A L , Davis A E
Published: May 2006

Mutations in the C1 inhibitor gene that result in low functional levels of C1 inhibitor protein cause hereditary angioneurotic edema. This disease is characterized by episodic edema leading to considerable morbidity and death. Among 60 unreported kindred with the disease, four patients were discovered to have mutations clustered within a 12-bp segment of exon 5 from nucleotide 8449 to nucleotide 8460. This short segment of DNA contains three direct repeats of the triplet CAA and is immediately preceded by a similar adenosine-rich sequence (CAAGAACAC). These triplet repeats make this region susceptible to mutation by a slipped mispairing mechanism. There are two other short triplet repeat elements in the coding region for this gene, but they have not become mutated in any kindred examined. This suggests that the apparent enhanced mutation rate in this region of exon 5 may be influenced by DNA structural characteristics.
Role of platelet activating factor in the intestinal epithelial secretory and Chinese hamster ovary cell cytoskeletal responses to cholera toxin.
Authors: Guerrant R L , Fang G D , Thielman N M , Fonteles M C
Published: May 2006

With the recent heightened concern about cholera around the world come new questions about the mechanism by which cholera toxin causes diarrhea. Peterson and Ochoa have suggested that prostaglandin synthesis is key to both the intestinal epithelial secretory and the CHO cell responses to cholera toxin [Peterson, J. W. and Ochoa, G. (1989) Science 245, 857-859]. Because platelet activating factor (PAF) can be a potent stimulus for prostaglandin synthesis, we examined its role in the intestinal and tissue culture effects of cholera toxin. We report that the specific PAF receptor antagonists BN 52021 and SR 27417 inhibit the effects of cholera toxin on intestinal secretion in rabbit ileal loops in vivo and on the cytoskeleton of Chinese hamster ovary cells in vitro. We also show that PAF itself can cause net fluid secretion in the rabbit model and that PAF potentiates the effects of cholera toxin on intestinal secretion. Finally, we demonstrate that cholera toxin stimulates significant PAF production (2.6-fold) in isolated T-84 intestinal epithelial cells. We conclude that cholera toxin stimulates PAF production and that PAF is involved in both the secretory and cytoskeletal responses to cholera toxin. These findings further support the involvement of additional mediators of cholera toxin effects other than mucosal cell cyclic AMP and help explain the effects of cholera toxin on prostaglandin synthesis.
Arabidopsis thaliana gamma-glutamylcysteine synthetase is structurally unrelated to mammalian, yeast, and Escherichia coli homologs.
Authors: May M J , Leaver C J
Published: May 2006

A mutant of Escherichia coli, JTG10, deficient in gamma-glutamylcysteine synthetase (gamma-ECS; EC 6.3.2.2) is unable to synthesize glutathione (GSH) and is sensitive to 8-hydroxyquinoline. This phenotype was exploited for the isolation of Arabidopsis thaliana gamma-ECS cDNAs by expression cloning, and clones were selected through functional complementation by growth on 8-hydroxyquinoline. High levels of gamma-ECS activity were detectable in extracts derived from cultures of JTG10 expressing the Arabidopsis gamma-ECS open reading frame, although these complemented mutants accumulated GSH to only 10% of the wild-type level. The derived amino acid sequence constitutes a polypeptide of 59.9 kDa and shows only 44-48% similarity with previously published sequences of rat kidney, human liver, yeast, and E. coli gamma-ECS. When the gamma-ECS cDNA was used as a probe, Southern blot analysis of Arabidopsis genomic DNA revealed that it is present as a low copy number gene. Furthermore, the Arabidopsis gamma-ECS cDNA probe failed to hybridize to maize and tobacco genomic DNA at low stringency, suggesting that heterogeneity in gamma-ECS structure exists between plant species. The activity of recombinant Arabidopsis gamma-ECS was inhibited by buthionine sulfoximine and GSH, indicating that, while differences in the primary and secondary structure of gamma-ECS from different sources exist, the enzymes may have similar active site structures.
A nuclear factor containing the leucine-rich repeats expressed in murine cerebellar neurons.
Authors: Matsuoka K , Taoka M , Satozawa N , Nakayama H , Ichimura T , Takahashi N , Yamakuni T , Song S Y , Isobe T
Published: May 2006

A nuclear protein, termed leucine-rich acidic nuclear protein (LANP), has been isolated from among rat cerebellar proteins whose expression was transiently increased during an early stage of postnatal development. The amino acid sequence, deduced from its cDNA, showed that LANP contains 247 amino acids consisting of two distinct structural domains: the N-terminal domain characterized by "leucine-rich repeat," which is found in many eukaryotic proteins and which potentially functions in mediating protein-protein interactions, and the C-terminal domain characterized by a cluster of acidic amino acids with a putative nuclear localization signal. Immunohistochemical study using an antibody against LANP revealed that the protein is localized mainly in nuclei of Purkinje cells. In the rat cerebellum on postnatal day 7, LANP mRNA was expressed moderately in the external granule and Purkinje cells and weakly in the internal granule cells. The expression in these cells, especially in Purkinje cells, increased in the second postnatal week and thereafter decreased to an adult level. The structural characteristics, localization, and the stage- and cell type-specific expression suggest a potential role of LANP in a signal transduction pathway that directs differentiation of cerebellar neurons.
Loss of all ndh genes as determined by sequencing the entire chloroplast genome of the black pine Pinus thunbergii.
Authors: Wakasugi T , Tsudzuki J , Ito S , Nakashima K , Tsudzuki T , Sugiura M
Published: May 2006

The complete nucleotide sequence (119,707 bp) of the black pine (Pinus thunbergii) chloroplast genome has been determined. It contains 4 rRNA genes and 32 tRNA genes. To our knowledge, the tRNAPro (GGG) gene has not been found in any other chloroplast genome analyzed. Sixty-one genes encoding proteins and 11 conserved open reading frames are also found. Extensive rearrangements are apparent in the chloroplast genome relative to those of other land plants. The most striking feature is the loss of all 11 functional genes (ndh genes) for subunits of a putative NADH dehydrogenase that are found in the chloroplast genomes of angiosperms and a bryophyte. Four ndh genes were completely lost and the other 7 genes remain as obvious pseudogenes. This unexpected finding raises the possibility that all ndh genes have been transferred to the nucleus or that an NADH dehydrogenase is not essential in black pine chloroplasts.
Effects of long-chain polyunsaturated fatty acids on the contraction of neonatal rat cardiac myocytes.
Authors: Kang J X , Leaf A
Published: May 2006

Because of the ability of certain long-chain polyunsaturated fatty acids (PUFAs) to prevent lethal cardiac arrhythmias, we have examined the effects of various long-chain fatty acids on the contraction of spontaneously beating, isolated, neonatal rat cardiac myocytes. The omega 3 PUFA from fish oils, eicosapentaenoic acid [EPA; C20:5 (n-3)] and docosahexaenoic acid [DHA; C22:6 (n-3)], at 2-10 microM profoundly reduced the contraction rate of the cells without a significant change in the amplitude of the contractions. The fatty acid-induced reduction in the beating rate could be readily reversed by cell perfusion with fatty acid-free bovine serum albumin. Addition of either oxygenase inhibitors or antioxidants did not alter the effect of the fatty acids. Arachidonic acid [AA; C20:4 (n-6)] produced two different effects on the beating rate, an increase or a decrease, or it produced no change. In the case of the increased or unchanged beating rate in the presence of AA, addition of AA oxygenase inhibitors subsequently reduced the contraction rate. The nonmetabolizable AA analog eicosatetraynoic acid (ETYA) always reduced the beating rate, as did EPA or DHA. Two other PUFAs, linoleic acid [C18:2 (n-6)] and linolenic acid [C18:3 (n-3)] also exhibited similar but less potent effects compared with EPA or ETYA. In contrast, neither the monounsaturated fatty acid oleic acid [C18:1 (n-9)] nor the saturated fatty acids stearic acid (C18:0), myristic acid (C14:0), and lauric acid (C12:0) affected the contraction rate. The inhibitory effect of these PUFAs on the contraction rate was similar to that produced by the class I antiarrhythmic drug lidocaine. The fatty acids that are able to reduce the beating rate, particularly EPA and DHA, could effectively prevent and terminate lethal tachyarrhythmias (contracture/fibrillation) induced by high extracellular calcium concentrations or ouabain. These results suggest that free PUFAs can suppress the automaticity of cardiac contraction and thereby exert their antiarrhythmic effects.
Indigeneity of organic matter in fossils: a test using stable isotope analysis of amino acid enantiomers in Quaternary mollusk shells.
Authors: Engel M H , Goodfriend G A , Qian Y , Macko S A
Published: May 2006

Comparison of the delta 13C values of D and L enantiomers of individual amino acids was used to evaluate the presence of amino acid contaminants in Quaternary land snails. Measurements of delta 13C values of amino acid D and L enantiomers determined by combined gas chromatography, combustion, isotope-ratio mass spectrometry are reported. Conventional combustion techniques, following separation of aspartic acid and glutamic acid enantiomers by liquid chromatography, were also used to determine delta 13C as well as delta 15N values. Thoroughly cleaned samples ranging in age from 7000 to > 100,000 yr B.P. are shown to have analytically identical delta 13C values for the D and L enantiomers of each amino acid, thus confirming that the amino acids are indigenous to the shells, even in Pleistocene samples. On the other hand, partially cleaned material shows divergence of isotopic values, thus indicating the presence of amino acid contaminants and emphasizing the importance of proper cleaning procedures. This approach provides a powerful method for assessing the indigeneity of amino acids in fossils.
Posttranscriptional silencing of reporter transgenes in tobacco correlates with DNA methylation.
Authors: Ingelbrecht I , Van Houdt H , Van Montagu M , Depicker A
Published: May 2006

Endogenous plant genes or transgenes can be silenced on introduction of homologous gene sequences. Here we document a reporter gene-silencing event in Nicotiana tabacum that has a distinctive combination of features--i.e., (i) silencing occurs by a posttranscriptional process, (ii) silencing correlates with DNA methylation, and (iii) this de novo methylation is not restricted to cytosines located in the symmetrical motifs CG and CXG.
Heme coordination of NO in NO synthase.
Authors: Wang J , Rousseau D L , Abu-Soud H M , Stuehr D J
Published: May 2006

A current question in nitric oxide (NO) biology is whether NO can act as a feedback inhibitor of NO synthase (NOS). We have approached this problem by examining the interaction of NO with neuronal NOS by optical absorption and resonance Raman scattering spectroscopies. Under an inert atmosphere NO coordinated to the heme iron in both the oxidized and reduced forms of NOS. The Soret and visible optical absorption transitions are detected at 436 and at 567 nm, respectively, in the Fe(2+)-NO heme complex and at 440 nm and at 549 and 580 nm, respectively, in the Fe(3+)-NO heme complex. In the resonance Raman spectrum of the ferrous complex the Fe-NO stretching mode is located at 549 cm-1 in the presence of L-arginine and at 536 cm-1 in the absence of L-arginine, whereas in the ferric enzyme the mode is located at 540 cm-1 (in the absence of L-arginine). The interaction between bound L-arginine and the NO indicates that L-arginine binds directly over the heme just as do the substrates in cytochrome P-450s. In the absence of L-arginine, NO readily oxidized the ferrous heme iron. The oxidation was prevented by the presence of bound L-arginine and enabled NOS to form a stable ferrous NO complex. Under oxygen-limited conditions, NO generated by neuronal NOS coordinated to its heme iron and formed a spectrally detectable ferrous-NO complex. Taken together, our results show that NO can bind to both ferric and ferrous NOS and may inhibit NO synthesis through its binding to the heme iron during catalysis.
Reevaluation of chloride's regulation of hemoglobin oxygen uptake: the neglected contribution of protein hydration in allosterism.
Authors: Colombo M F , Rau D C , Parsegian V A
Published: May 2006

We have measured hemoglobin oxygen uptake vs. the partial pressure of oxygen, with independently controlled activities of chloride and water. This control is effected by combining different concentrations of NaCl and sucrose in the bathing solution to achieve: (i) water activities were varied and Cl- activity was fixed, (ii) both water and Cl- activities were varied with a traditional NaCl titration, or (iii) Cl- activities were varied and water activity was fixed by adding compensating sucrose. Within this analysis, the Cl(-)-regulated loading of four oxygens can be described by the reaction Hb.Cl- + 4 O2 + 65 H2O in equilibrium with Hb.4O2.65H2O + Cl-. The dissociation of a neatly integral chloride, rather than the nonintegral 1.6 chlorides inferred earlier from simple salt titration, demonstrates the need to recognize the potentially large contribution from changes in water activity when titrating weakly binding solutes. The single-chloride result might simplify structural considerations of the action of Cl- in hemoglobin regulation.
D1A, D1B, and D1C dopamine receptors from Xenopus laevis.
Authors: Sugamori K S , Demchyshyn L L , Chung M , Niznik H B
Published: May 2006

Three distinct genes encoding members of the D1 dopamine receptor family were isolated from Xenopus laevis. Based on the deduced amino acid sequence, two of the receptors (Xen D1A and Xen D1B) appear to be homologues of mammalian D1/D1A and D5/D1B receptors. The third receptor, termed Xen D1C, displays equal overall amino acid and nucleotide sequence identity (approximately 55%) with mammalian D1A and D1B/D5 receptors. In agreement with their structural similarities, Xen D1A and D1B receptors, when expressed in COS-7 cells, displayed pharmacological profiles that paralleled those of their mammalian counterparts, with dopamine and 2-amino-6,7-dihydroxytetralin exhibiting 10-fold higher affinity for D1B than for D1A. The Xen D1C receptor displayed an overall rank order of potency and pharmacological profile clearly indicative of a D1-like receptor, with individual affinities for most agonists higher than those for either Xen or mammalian D1/D1A and D5/D1B receptors, whereas antagonist Ki values were intermediate to those for the D1/D1A and D5/D1B receptors. All three receptors stimulated adenylate cyclase activity in response to dopamine or SKF-82526. Xen D1A, D1B, and D1C receptor mRNAs were differentially distributed, with all three receptors expressed in brain and only D1B and D1C receptors expressed in kidney. The existence of a receptor which lacks appreciable overall sequence similarity to, but displays pharmacological homology with, mammalian D1-like receptors lends strong support to the contention that additional mammalian D1-like receptor gene products may exist to allow for the expression of the full spectrum of D1-like dopamine receptor-mediated events.
KorA protein of promiscuous plasmid RK2 controls a transcriptional switch between divergent operons for plasmid replication and conjugative transfer.
Authors: Jagura-Burdzy G , Thomas C M
Published: May 2006

The trfA and trb operons, encoding genes essential for replication and conjugative transfer of broad host range plasmid RK2, are transcribed divergently. Deletion analysis presented here indicates that trfAp and trbAp are arranged as face to face promoters. The presence of the korA gene, whose product is known to repress seven operons on RK2, including the trfA operon, is shown here to stimulate trbAp. The effect of korA on trbAp is mimicked by the trfAp-1 promoter down mutation, suggesting that a reduction in the activity of trfAp is required for derepression of trbAp activity. The trfAp-1 mutation reduces RNA polymerase binding and open complex formation at trfAp but does not stimulate melting at trbAp in vitro. Therefore, the inhibition of trbAp is most probably due to forward transcription initiated at trfAp. The simultaneous inhibition/stimulation by KorA is seen even in the presence of the other repressors KorB and TrbA, which act at this region, thus providing a dominant mode of coordinating plasmid replication and transfer. This may be one of the keys to understanding how the maintenance and spread of promiscuous plasmids are balanced in different environments.
Gene regulation by temperature-sensitive p53 mutants: identification of p53 response genes.
Authors: Buckbinder L , Talbott R , Seizinger B R , Kley N
Published: May 2006

The ability of the p53 protein to act as a sequence-specific transcriptional activator suggests that genes induced by p53 may encode critical mediators of p53 tumor suppression. Using a tetracycline-regulated p53 expression system and cDNA library subtraction procedure, we identified several p53-induced gene transcripts in human Saos-2 osteosarcoma cells that are novel on the basis of their size, regulation, and low abundance. Wild-type p53-dependent induction of these transcripts was observed in cells that are growth arrested by p53, as well as in cells that undergo apoptosis upon expression of an inducible wild-type p53 transgene. These results show that p53 activates the expression of numerous response genes and suggest that multiple effectors may play a role in mediating cellular functions of p53.
The BTB domain, found primarily in zinc finger proteins, defines an evolutionarily conserved family that includes several developmentally regulated genes in Drosophila.
Authors: Zollman S , Godt D , Privé G G , Couderc J L , Laski F A
Published: May 2006

The Drosophila bric à brac protein and the transcriptional regulators encoded by tramtrack and Broad-Complex contain a highly conserved domain of approximately 115 amino acids, which we have called the BTB domain. We have identified six additional Drosophila genes that encode this domain. Five of these genes are developmentally regulated, and one of them appears to be functionally related to bric à brac. The BTB domain defines a gene family with an estimated 40 members in Drosophila. This domain is found primarily at the N terminus of zinc finger proteins and is evolutionarily conserved from Drosophila to mammals.
Socioeconomic studies of high-level nuclear waste disposal.
Authors: White G F , Bronzini M S , Colglazier E W , Dohrenwend B , Erikson K , Hansen R , Kneese A V , Moore R , Page E B , Rappaport R A
Published: May 2006

The socioeconomic investigations of possible impacts of the proposed repository for high-level nuclear waste at Yucca Mountain, Nevada, have been unprecedented in several respects. They bear on the public decision that sooner or later will be made as to where and how to dispose permanently of the waste presently at military weapons installations and that continues to accumulate at nuclear power stations. No final decision has yet been made. There is no clear precedent from other countries. The organization of state and federal studies is unique. The state studies involve more disciplines than any previous efforts. They have been carried out in parallel to federal studies and have pioneered in defining some problems and appropriate research methods. A recent annotated bibliography provides interested scientists with a compact guide to the 178 published reports, as well as to relevant journal articles and related documents.
Specific interaction of the CD45 protein-tyrosine phosphatase with tyrosine-phosphorylated CD3 zeta chain.
Authors: Furukawa T , Itoh M , Krueger N X , Streuli M , Saito H
Published: May 2006

The CD45 transmembrane protein-tyrosine phosphatase (PTPase, EC 3.1.3.48) plays an essential role in T-cell activation by activating the Lck and/or Fyn protein-tyrosine kinases. However, numerous experiments have indicated that CD45 may have both stimulatory and inhibitory roles in T-cell activation. Thus, it is unlikely that the two kinases are the sole substrates of the CD45 PTPase. Furthermore, the complex regulation of the alternative splicing of the extracellular domain in various leukocyte lineages also suggests additional roles for the CD45 PTPase. To identify such functions, it is necessary to identify physiologically relevant substrates of the CD45 PTPase other than the two protein-tyrosine kinases. To this end, we searched for high-affinity substrates of the CD45 PTPase among the tyrosine-phosphorylated T-cell proteins by using purified glutathione S-transferase-CD45 fusion molecules. The enzymatically inactive CD45 C828S mutant protein, in which the cysteine residue at the catalytic center was changed to a serine residue, bound tightly to the phosphorylated CD3 zeta chain. This binding was specific to CD45 PTPase, as neither the leukocyte common antigen-related molecule (LAR) PTPase nor the CD45-LAR hybrid PTPases bound the phosphorylated CD3 zeta chain. Furthermore, phosphorylated CD3 zeta chain was preferentially dephosphorylated by the wild-type CD45 PTPase under conditions that did not significantly dephosphorylate other cellular proteins. Thus, the phosphorylated CD3 zeta chain is a specific and high-affinity substrate of the CD45 PTPase. These results suggest that CD45 is involved in the termination of the T-cell response via dephosphorylation of CD3 zeta chain.
The structure of the transition state for the association of two fragments of the barley chymotrypsin inhibitor 2 to generate native-like protein: implications for mechanisms of protein folding.
Authors: de Prat Gay G , Ruiz-Sanz J , Davis B , Fersht A R
Published: May 2006

Possible early events in protein folding may be studied by dissecting proteins into complementary fragments. Two fragments of chymotrypsin inhibitor 2 [CI2-(20-59) and CI2-(60-83)] associate to form a native-like structure in a second-order reaction that combines collision and rearrangement. The transition state of the reaction, analyzed by the protein engineering method on 17 mutants, is remarkably similar to that for the folding of the intact protein--a structure that resembles an expanded version of the folded structure with most interactions significantly weakened. The exception is that the N-terminal region of the single alpha-helix (the N-capping box) is completely formed in the transition state for association of the fragments, whereas it is reasonably well formed for the intact protein. Preliminary evidence on the structures of the individual fragments indicates that both are mainly nonnative, lacking native secondary structure and having regions of nonnative buried hydrophobic clusters. The association reaction does not result from the collision of a subpopulation of two fully native-like fragments but involves a considerable rearrangement of structure.
Syndecans, cell surface heparan sulfate proteoglycans, are induced by a proline-rich antimicrobial peptide from wounds.
Authors: Gallo R L , Ono M , Povsic T , Page C , Eriksson E , Klagsbrun M , Bernfield M
Published: May 2006

Cell surface heparan sulfate proteoglycans, such as the syndecans, are required for cellular responses to heparin-binding growth factors and extracellular matrix components. Expression of syndecan-1 and -4 is induced in mesenchymal cells during wound repair in the mouse, consistent with a role for syndecans in regulating cell proliferation and migration in response to these effectors. Here we show that wound fluid contains inductive activity that mimics the in vivo induction in time of appearance, specificity for mesenchymal cells, and selectivity for syndecan-1 and -4. We have purified and synthesized a 4.8-kDa proline-rich protein from wound fluid that reproduces this induction of syndecan-1 and -4 in cultured cells. This peptide, identical to the antibacterial peptide PR-39, is released into the wound by the cellular infiltrate and induces syndecan expression at the same peptide concentrations that lyse bacteria. These results indicate that wounds contain a multifunctional protein that induces mammalian cells to express cell surface heparan sulfate proteoglycans as part of the wound repair process and that kills bacteria as part of a nonimmune defense mechanism.
Mother's prior intrauterine position affects the sex ratio of her offspring in house mice.
Authors: Vandenbergh J G , Huggett C L
Published: May 2006

Sex ratio alterations related to environmental factors occur in several mammals, but no mechanism has been identified to explain the adjustment. Intrauterine position (IUP) may provide the context in which such alterations occur. Previous studies on house mice and gerbils reveal that the position of a fetus in the uterus in relation to the sex of its neighbors influences its later anatomy, physiology, and behavior. The anogenital distance (AGD) of females located between two males (2M) is longer than that of females not between two males (OM). We have found that the IUP, as determined by cesarean section and by an index of the AGD, correlates with the sex ratio of the litters produced by female mice. The sex ratio of the first litter born to 2M females was 58% males, for 1M females was 51% males and for OM females was 42% males. The effect on sex ratio continues into the second litter. The number of pups produced by mothers of different IUPs in her first two litters did not differ, suggesting that the sex ratio adjustment occurs prior to parturition. These results provide a basis for the natural variability observed in sex ratios of litter-bearing mammals and suggest that one or more intrauterine mechanisms may be responsible for environmentally related sex ratio alterations.
The high mobility group protein HMG I(Y) can stimulate or inhibit DNA binding of distinct transcription factor ATF-2 isoforms.
Authors: Du W , Maniatis T
Published: May 2006

The high mobility group protein HMG I(Y) stimulates the binding of a specific isoform of the activating transcription factor 2 (ATF-2(195)) to the interferon beta (IFN-beta) gene promoter. HMG I(Y) specifically interacts with the basic-leucine zipper region of ATF-2(195), and HMG I(Y) binds to two sites immediately flanking the ATF-2 binding site of the IFN-beta promoter. Here, we show that HMG I(Y) can stimulate the binding of ATF-2(195), at least in part, by promoting ATF-2 dimerization. In addition, we report the characterization of a naturally occurring isoform of ATF-2 (ATF-2(192)) that binds specifically to the IFN-beta promoter but is unable to interact with HMG I(Y). Remarkably, HMG I(Y) inhibits the binding of ATF-2(192) to the IFN-beta promoter. Thus, the ability of HMG I(Y) to specifically interact with ATF-2 correlates with its ability to stimulate ATF-2 binding to the IFN-beta promoter. Comparisons of the amino acid sequences of the basic-leucine zipper domains of ATF-2(195) and ATF-2(192) suggest that HMG I(Y) interacts with a short stretch of basic amino acids near the amino terminus of the basic-leucine zipper domain of ATF-2(195).
Regional induction of tumor necrosis factor alpha expression in the mouse brain after systemic lipopolysaccharide administration.
Authors: Breder C D , Hazuka C , Ghayur T , Klug C , Huginin M , Yasuda K , Teng M , Saper C B
Published: May 2006

Tumor necrosis factor alpha (TNF-alpha) is a cytokine that is responsible, in part, for several aspects of the acute-phase response to inflammation, including the generation of fever. TNF-alpha has direct effects on central nervous system neurons deep within the hypothalamus that are involved in producing the febrile response, but the blood-brain barrier prevents circulating TNF-alpha from having access to these sites. We therefore have hypothesized that TNF-alpha may be produced in the brain and used as a mediator in the cerebral components of the acute-phase response. We used in situ hybridization to determine the distribution of production of TNF-alpha mRNA in the mouse brain after systemic administration of lipopolysaccharide. During the initial phase of fever, hybridization was observed in perivascular cells and neurons in circumventricular organs, including the vascular organ of the lamina terminalis, median eminence, and area postrema, as well as along the ventral surface of the medulla; hybridization was also prominent over many cell in the meninges. During the late phase of the response, hybridization was observed over neurons in the pericircumventricular nuclei such as the anteroventral periventricular and arcuate nuclei of the hypothalamus and the nucleus of the solitary tract. TNF-alpha produced by a cascade of neurons within the brain may participate in the complex autonomic, neuroendocrine, metabolic, and behavioral responses to infection and inflammation.
A general method for facilitating heterodimeric pairing between two proteins: application to expression of alpha and beta T-cell receptor extracellular segments.
Authors: Chang H C , Bao Z , Yao Y , Tse A G , Goyarts E C , Madsen M , Kawasaki E , Brauer P P , Sacchettini J C , Nathenson S G
Published: May 2006

Generation of soluble T-cell receptor (TCR) molecules by a variety of genetic engineering methods has been hampered by inefficient pairing of alpha and beta subunits in the absence of their respective transmembrane regions and associated CD3 components. To overcome this obstacle, we have added 30-amino acid-long segments to the carboxyl termini of alpha and beta extracellular domains via a cleavable flexible linker. These peptide segments (BASE-p1 for alpha and ACID-p1 for beta) have been previously shown to selectively associate to form a stable heterodimeric coiled coil termed a leucine zipper. Homodimeric structures are not permitted due to electrostatic repulsion among amino acid side chains. Expression of a representative TCR-leucine zipper fusion protein in a baculovirus expression system results in production of alpha beta TCR heterodimer at 0.6-1.4 mg/liter. This yield is 5- to 10-fold greater than that of the TCR expressed in the absence of the synthetic leucine zipper sequence. The structure of the TCR component of the fusion heterodimer was judged to be native when probed with a panel of 17 mAbs specific for alpha and beta constant and variable domains. A mAb specific for the isolated BASE-p1/ACID-p1 coiled coil was also generated and shown to react with the TCR fusion protein. The above technology should be broadly useful in the efficient production and purification of TCRs as well as other heterodimeric proteins.
Nonviral and viral delivery of a human immunodeficiency virus protective gene into primary human T cells.
Authors: Woffendin C , Yang Z Y , Udaykumar  , Xu L , Yang N S , Sheehy M J , Nabel G J
Published: May 2006

Because AIDS has been refractory to traditional pharmacologic interventions, alternative approaches have been developed. Although the introduction of specific antiviral genes into T leukemia cells can provide relative resistance to human immunodeficiency virus (HIV) replication, the testing of such genes against primary viral isolates in human CD4+ lymphocytes has been limited, and safety questions remain regarding gene delivery into cells from HIV-infected patients. In this report, we evaluate the efficacy of a transdominant mutant protein, Rev M10, against cloned and primary HIV isolates in human peripheral blood lymphocytes and describe different methods of gene transfer into peripheral blood lymphocytes from HIV-infected individuals. We show that gold microparticles can mediate stable Rev M10 gene transfer into these cells. Introduction of Rev M10 by these techniques conferred resistance to HIV infection in vitro to cloned and clinical isolates. Nonviral delivery of HIV protective genes will facilitate the development of gene therapy for AIDS and the analysis of viral and cellular gene expression in human T lymphocytes.
Glutamate and N-methyl-D-aspartate affect release from crayfish axon terminals in a voltage-dependent manner.
Authors: Parnas H , Parnas I , Ravin R , Yudelevitch B
Published: May 2006

In the crayfish neuromuscular junction, the excitatory transmitter is glutamate. The present study shows that at concentrations as low as 5 x 10(-7) M, glutamate affects the depolarization-evoked release of neurotransmitter. Furthermore, the effect of glutamate on release is voltage-dependent and depends on the level of the depolarizing pulse. Nerve terminals were exposed to 5 x 10(-7) M tetrodotoxin and then depolarized to different levels by a macropatch electrode. Depending on the amplitude of the depolarizing pulse, glutamate (5 x 10(-7) to 1 x 10(-5) M) had a dual effect on release. At small depolarizing pulses, glutamate reduced release, whereas at large depolarizing pulses, it enhanced it. Glutamate at 10(-6) M had no significant effect on action-potential-induced release. At 10(-4) M glutamate, the action-potential-induced release was always inhibited. N-Methyl-D-aspartate was found to mimic one of the effects of glutamate: N-methyl-D-aspartate (10(-7) to 10(-5) M) reduced release at small depolarizing pulses but had no effect with larger depolarizations. 2-Amino-5-phosphonovaleric acid blocked the effect of N-methyl-D-aspartate.
Molecular dynamics simulation of the gramicidin channel in a phospholipid bilayer.
Authors: Woolf T B , Roux B
Published: May 2006

A molecular dynamics simulation of the gramicidin A channel in an explicit dimyristoyl phosphatidylcholine bilayer was generated to study the details of lipid-protein interactions at the microscopic level. Solid-state NMR properties of the channel averaged over the 500-psec trajectory are in excellent agreement with available experimental data. In contrast with the assumptions of macroscopic models, the membrane/solution interface region is found to be at least 12 A thick. The tryptophan side chains, located within the interface, are found to form hydrogen bonds with the ester carbonyl groups of the lipids and with water, suggesting their important contribution to the stability of membrane proteins. Individual lipid-protein interactions are seen to vary from near 0 to -50 kcal/mol. The most strongly interacting conformations are short-lived and have a nearly equal contribution from both van der Waals and electrostatic energies. This approach for performing molecular dynamics simulations of membrane proteins in explicit phospholipid bilayers should help in studying the structure, dynamics, and energetics of lipid-protein interactions.
Activation of rat brain phospholipase D by ADP-ribosylation factors 1,5, and 6: separation of ADP-ribosylation factor-dependent and oleate-dependent enzymes.
Authors: Massenburg D , Han J S , Liyanage M , Patton W A , Rhee S G , Moss J , Vaughan M
Published: May 2006

Two major forms of phospholipase D (PLD) activity, solubilized from rat brain membranes with Triton X-100, were separated by HPLC on a heparin-5PW column with buffer containing octyl glucoside. One form was completely dependent on sodium oleate for activity. The other, which was dramatically activated by the addition of ADP-ribosylation factor (ARF) 1 and guanine 5' [gamma-thio]triphosphate, required the presence of phosphatidylinositol 4,5-bisphosphate in the phosphatidylcholine substrate for demonstration of activity, as described by others. Oleate-dependent activity was unaffected by guanine 5' [gamma-thio]triphosphate, or phosphatidylinositol 4,5-bisphosphate. Both sodium oleate-and ARF-dependent activities catalyzed transphosphatidylation, thus identifying them as PLDs. ARF-dependent PLD was activated by recombinant ARF5 (class II) and ARF6 (class III), as well as ARF1 (class I). Myristoylated recombinant ARFs were more effective than their nonmyristoylated counterparts. ARFs were originally identified as activators of cholera toxin ADP-ribosyltransferase activity. The effects of recombinant ARF proteins from the three classes on cholera toxin activity (assayed under conditions identical to those used to assay PLD activity) did not, however, correlate with those on PLD, consistent with the notion that different aspects of ARF structure are involved in the two functions.
Lymphocyte proliferation in mice congenitally deficient in T-cell receptor alpha beta + cells.
Authors: Viney J L , Dianda L , Roberts S J , Wen L , Mallick C A , Hayday A C , Owen M J
Published: May 2006

In mice and humans, T cells are characterized on the basis of T-cell receptor (TcR) expression and divided into the major TcR alpha beta + and minor TcR gamma delta + populations. TcR alpha beta + cells are considered to be the primary regulators of the immune response, whereas the function of TcR gamma delta + cells is unclear. Mice congenitally deficient in TcR alpha beta-expressing cells provide an ideal model for analyzing the independent in vivo function of TcR gamma delta + cells in the absence of TcR alpha beta + cells. Here we report that lymphoid organs in TcR alpha mutant mice undergo substantial enlargement after being challenged by environmental antigens. This organ expansion can be attributed in part to increases in the relative proportions and absolute numbers of TcR gamma delta + cells, but an expansion of the recently described TcR beta + alpha - population also has a role. The expansion of the TcR gamma delta + population is polyclonal, as evidenced by the usage of multiple gamma and delta variable chain segments. Furthermore, a substantial proportion of the cells appears to be activated and these activated cells express surface activation markers. The results clearly demonstrate that TcR gamma delta + cells proliferate independently in response to a broad spectrum of challenges. Moreover, since the expansion of the lymphoid tissues and the TcR gamma delta + cell population is excessive relative to that seen in wild-type animals, one role of TcR alpha beta + cells is directly or indirectly to limit the responses of the other lymphoid components.