bluuebunny/crossref_metadata_2025 huggingface dataset:
Human gliomas and epileptic foci express high levels of a mRNA related to rat testicular sulfated glycoprotein 2, a purported marker of cell death.
Authors: Danik M , Chabot J G , Mercier C , Benabid A L , Chauvin C , Quirion R , Suh M
Published: May 2006

Clone pTB16 has been isolated by differential screening of a human glioma cDNA library. Northern blot analysis has shown that pTB16 expression is several times (greater than 11-fold) higher in gliomas than in a primitive neuroectodermal tumor. This observation was supported by in situ hybridization and extended to nine other gliomas. Expression was virtually absent in adenocarcinoma cells metastasized to brain. Malignant gliomas showed stronger hybridization than benign gliomas, while blood capillaries did not show hybridization. pTB16 mRNA was also shown to be expressed in established glioma cell lines and at high levels in epileptic foci, indicating that expression of the gene may be limited to certain cell types and that its upregulation is not merely a consequence of cellular proliferation. Nucleotide sequence analysis identified pTB16 as the human counterpart for rat testicular sulfated glycoprotein 2 (SGP-2), whose function in the reproductive system remains unknown. Although SGP-2 transcripts, and hence pTB16, were recently shown to be increased in neurodegenerative diseases such as scrapie in hamsters and Alzheimer disease in humans, our observations with brain tumors and epilepsy are suggestive of a role for pTB16 in neuropathologies in general and support the hypothesis of its involvement in tissue remodeling and cell death.
A different perception of the linear, nonthreshold hypothesis for low-dose irradiation.
Authors: Bond V P , Benary V , Sondhaus C A
Published: May 2006

Two equally useful dosimetric quantities, both of which are called dose, are used in toxicology. With radiation measurement, only one--the energy per unit mass D--is called dose. The other--the total energy in the irradiated system--is here distinguished from D by assigning it the name collective energy, epsilon. The collective energy is a more complete statement of dose because it is the product of the energy concentration D and the mass irradiated m. Especially in radioepidemiology, in which epsilon is the total energy imparted to all persons irradiated, the quantity m must be specified because it is situation specific and thus highly variable. At present, radioepidemiological dose-response curves are given only in terms of the toxicological model--i.e., the fraction (probability) of radiation-attributable cancers occurring as a function of D. Because this relation does not involve the number of persons at each value of D, it fosters the illusion that any dose, no matter how small, can result in cancer. However, we show that if the dose-response relationship is expressed in terms of the absolute number of attributable cancers as a function of epsilon, cancer occurs, on average, only if the collective energy exceeds a relatively large minimum value, the magnitude of which will be estimated. Therefore, we conclude that the nonthreshold aspect of the linear hypothesis is misleading and quite probably invalid. For example, in or around a facility in which exposure of humans to relatively low values of D occurs, attributable cancers are most unlikely to appear unless the epsilon to the irradiated population exceeds this minimum value.
Tonic inhibition and rebound facilitation of a neuronal calcium channel by a GTP-binding protein.
Authors: Kasai H
Published: May 2006

A significant fraction of differentiated NG108-15 neuroblastoma/glioma cells have Ca2+ channel current different from that of undifferentiated cells. In the former cells, the Ca2+ channel sensitive to omega-conotoxin GVIA had slowed activation kinetics and was facilitated by depolarizing prepulses. These kinetic features are identical to those produced by inhibition of the channel by G proteins. Prolonged treatment with prostaglandin E1 and theophylline, agents that cause cellular differentiation, promoted incidence and extent of the tonic inhibition. Intracellular guanosine 5'-[beta-thio]diphosphate removed the tonic inhibition, suggesting sustained activation of a G protein, but pertussis toxin did not block it. A sulfhydryl alkylating agent, N-ethylmaleimide (0.1 mM), rapidly eliminated agonist-induced inhibition, whereas N-ethylmaleimide spared the tonic inhibition and the one induced by intracellular guanosine 5'-[gamma-thio]triphosphate. An agonist could further inhibit the Ca2+ channel that was already tonically inhibited. After washout of an inhibitory agonist, the tonic inhibition was temporarily removed. This "rebound facilitation" gradually faded within a few minutes. Pertussis toxin or N-ethylmaleimide prevented the rebound facilitation, whereas phorbol ester, forskolin, or arachidonic acid induced neither the rebound facilitation nor the tonic inhibition. Whatever its mechanism, the tonic inhibition of Ca2+ channels may serve as the basis for long-term and bidirectional regulation of activity of neuronal Ca2+ channels.
Solution conformations of the gamma-carboxyglutamic acid domain of bovine prothrombin fragment 1, residues 1-65.
Authors: Charifson P S , Darden T , Tulinsky A , Hughey J L , Hiskey R G , Pedersen L G
Published: May 2006

Molecular dynamics simulations have been performed (AMBER version 3.1) on solvated residues 1-65 of bovine prothrombin fragment 1 (BF1) by using the 2.8-A resolution crystallographic coordinates as the starting conformation for understanding calcium ion-induced conformational changes that precede experimentally observable phospholipid binding. Simulations were performed on the non-metal-bound crystal structure, the form resulting from addition of eight calcium ions to the 1-65 region of the crystal structure, the form resulting from removal of calcium ions after 107 ps and continuing the simulation, and an isolated hexapeptide loop (residues 18-23). In all cases, the 100-ps time scale seemed adequate to sample an ensemble of solution conformers within a particular region of conformation space. The non-metal-containing BF1 did not unfold appreciably during a 106-ps simulation starting from the crystallographic geometry. The calcium ion-containing structure (Ca-BF1) underwent an interesting conformational reorganization during its evolution from the crystal structure: during the time course of a 107-ps simulation, Ca-BF1 experienced a trans----cis isomerization of the gamma-carboxyglutamic acid-21 (Gla-21)-Pro-22 peptide bond. Removal of the calcium ions from this structure followed by 114 ps of additional molecular dynamics showed significant unfolding relative to the final 20-ps average structure of the 107-ps simulation; however, the Gla-21-Pro-22 peptide bond remained cis. A 265-ps simulation on the termini-protected hexapeptide loop (Cys-18 to Cys-23) containing two calcium ions also did not undergo a trans----cis isomerization. It is believed that the necessary activation energy for the transitional event observed in the Ca-BF1 simulation was largely supplied by global conformational events with a possible assist from relief of intermolecular crystal packing forces. The presence of a Gla preceding Pro-22, the inclusion of Pro-22 in a highly strained loop structure, and the formation of two long-lived salt bridges prior to isomerization may all contribute to this finding.
DNase protection analysis of the stable synaptic complexes involved in Mu transposition.
Authors: Mizuuchi M , Baker T A , Mizuuchi K
Published: May 2006

Several critical steps in phage Mu transposition involve specialized protein-DNA complexes. Cleavage of Mu donor DNA by MuA protein leads to the formation of the stable cleaved donor complex (CDC), in which the two Mu DNA ends are held together by MuA. In the subsequent strand-transfer reaction the CDC attacks a target DNA to generate the strand-transfer complex, in which the donor and the target DNAs are covalently joined. We have carried out DNase I protection experiments on these protein-DNA complexes and found that only three MuA binding sites (L1, R1, and R2 of the six total) at the two Mu ends are stably bound by MuA to maintain the paired Mu end structure. The protection extends beyond the ends of the Mu sequence for different lengths (7-20 nucleotides) depending on the strand and the type of complex. After formation of the CDC, the other MuA binding sites (L2, L3, and R3) and internal activation sequence become dispensable for the subsequent strand-transfer reaction.
Phosphate-starvation response in plant cells: de novo synthesis and degradation of acid phosphatases.
Authors: Duff S M , Plaxton W C , Lefebvre D D
Published: May 2006

Induction of phosphatase activity is an important component of the plant cell response to phosphate deficiency. Suspension cell cultures of Brassica nigra contain two major inducible acid phosphatase (APase) isozymes; vacuolar phosphoenolpyruvate (PEP) APase and cell wall nonspecific APase. Polyclonal antibodies raised against purified PEP-APase crossreacted specifically with both isozymes. Furthermore, anti-(PEP-APase) IgG detected proteins from a wide range of higher plants, suggesting that the major plant APase isozymes have diverged from a common ancestral form. Quantification on immunoblots indicated that in B. nigra suspension cells experiencing transition from Pi sufficiency to deficiency or vice versa, the amount of total antigenic APase protein correlated closely with total enzyme activity. This was also shown in intact plant roots. Therefore, the activity was governed by the synthesis and degradation of APases. Increases in the amounts of both major APase isozymes occurred simultaneously following Pi deprivation of B. nigra suspension cells, suggesting the involvement of a common regulatory mechanism.
Alternative splicing and genomic structure of the Wilms tumor gene WT1.
Authors: Haber D A , Sohn R L , Buckler A J , Pelletier J , Call K M , Housman D E
Published: May 2006

The chromosome 11p13 Wilms tumor susceptibility gene WT1 appears to play a crucial role in regulating the proliferation and differentiation of nephroblasts and gonadal tissue. The WT1 gene consists of 10 exons, encoding a complex pattern of mRNA species: four distinct transcripts are expressed, reflecting the presence or absence of two alternative splices. Splice I consists of a separate exon, encoding 17 amino acids, which is inserted between the proline-rich amino terminus and the zinc finger domains. Splice II arises from the use of an alternative 5' splice junction and results in the insertion of 3 amino acids between zinc fingers 3 and 4. RNase protection analysis demonstrates that the most prevalent splice variant in both human and mouse is that which contains both alternative splices, whereas the least common is the transcript missing both splices. The relative distribution of splice variants is highly conserved between normal fetal kidney tissue and Wilms tumors that have intact WT1 transcripts. The ratio of these different WT1 mRNA species is also maintained as a function of development in the mouse kidney and in various mouse tissues expressing WT1. The conservation in structure and relative levels of each of the four WT1 mRNA species suggests that each encoded polypeptide makes a significant contribution to normal gene function. The control of cellular proliferation and differentiation exerted by the WT1 gene products may involve interactions between four polypeptides with distinct targets and functions.
Amino acid sequence of Fel dI, the major allergen of the domestic cat: protein sequence analysis and cDNA cloning.
Authors: Morgenstern J P , Griffith I J , Brauer A W , Rogers B L , Bond J F , Chapman M D , Kuo M C
Published: May 2006

The complete primary structure of Fel dI (International Union of Immunological Societies nomenclature), the major allergen produced by the domestic cat, Felis domesticus, was determined by protein sequence analysis and cDNA cloning. Protein sequencing of Fel dI from an immunoaffinity-purified extract of house dust revealed that the allergen is composed of two polypeptide chains. Degenerate oligonucleotides derived from the protein sequence were used in polymerase chain reaction amplification of cat salivary gland cDNA to demonstrate that the two chains are encoded by different genes. Chain 1 of Fel dI shares amino acid homology with rabbit uteroglobin, while chain 2 is a glycoprotein with N-linked oligosaccharides.
A determinant of disease symptom severity is located in the 3'-terminal noncoding region of the RNA of a plant virus.
Authors: Rodríguez-Cerezo E , Klein P G , Shaw J G
Published: May 2006

Inoculation of Nicotiana tabacum plants with RNA transcribed in vitro from a variant (pXBS8) of a cloned full-length DNA copy of tobacco vein mottling virus (TVMV) RNA resulted in attenuation of the vein mottling and blotching symptoms typically produced by transcripts of cloned wild-type cDNA (pXBS7). Similar amounts of virus were detected by ELISA (using anti-TVMV coat protein serum) in systemically infected leaves of plants inoculated with pXBS7 or pXBS8 transcripts. pXBS8 was shown to contain a 58-nucleotide segment in the sequence corresponding to the 3'-terminal untranslated region of TVMV RNA that was not present in pXBS7. This segment resulted in the appearance in pXBS8 transcripts of four adjacent direct repeats of a 14-nucleotide sequence, AUAAUUAUAUAUAU, that is present in the 3'-untranslated region of TVMV RNA, with two additional nucleotides (AU) between the first and second repeats. Insertion of restriction fragments containing the segment into pXBS7 and inoculation of plants with transcripts of the chimeric construct (pXBS78) resulted in the attenuated-symptom phenotype and was not accompanied by a reduced accumulation of virus in the plant as determined by ELISA and Northern blot analysis. When the extra nucleotides were removed from the variant clone, symptoms induced by transcripts of the cDNA (pXBS87) resembled those induced by wild-type transcripts. The results indicate that a noncoding region of the genome can have a direct effect on the induction of disease symptoms by an RNA virus.
Chronic myeloproliferative disease induced by site-specific integration of Abelson murine leukemia virus-infected hemopoietic stem cells.
Authors: Han X D , Wong P M , Chung S W
Published: May 2006

We recently showed that hemopoietic stem cells expressing the v-abl oncogene can cause leukemia when injected into lethally irradiated recipient mice. Progenitor cells expressing v-abl did not significantly contribute to disease development, and the leukemia was monoclonal in origin. By serially transplanting v-abl-transduced hemopoietic stem cells into normal, nonirradiated syngeneic recipients, we showed that multiple stem-cell clones do exist in some recipients. These cells fluctuated as normal stem cells do and could home to normal bone marrow. Based on the time course of disease, the recipients developed either an acute or a chronic phase of disorder. All recipients with the acute disease had stem-cell clones with random Abelson murine leukemia virus integration sites. All recipients with the chronic disorder had a specific Abelson murine leukemia virus integration site. We believe this abl-specific integration site, termed ASI, is important in abl-mediated stem-cell leukemogenesis.
Uptake of N-(4'-pyridoxyl)amines and release of amines by renal cells: a model for transporter-enhanced delivery of bioactive compounds.
Authors: Zhang Z M , McCormick D B
Published: May 2006

The importing of vitamin B6 by renal proximal tubular cells from the rat is facilitated and Na(+)-dependent and reflects specificity for the meta-phenolate pyridinium structure with a 5-hydroxymethyl function. This transporter can, however, accept competitively each of the natural nonphosphorylated vitamers (pyridoxine, pyridoxamine, and pyridoxal) and other B6 analogues differing only in the groups at position 4. A series of N-(4'-pyridoxyl)amines was synthesized by sodium borohydride or boro[3H]hydride reduction of aldimines formed by condensing the amines with pyridoxal. The unlabeled B6-secondary amine compounds were found to competitively inhibit the uptake of [4'-3H]pyridoxine by the renal cells. Moreover, the 3H-labeled N-(4'-pyridoxyl)amines were shown to enter the cells by the process facilitated by the B6 transporter. Upon entry the labeled compounds were converted to N-(5'-phospho-4'-pyridoxyl)amines in a reaction catalyzed by pyridoxal kinase, an enzyme that tolerates considerable functional variation in position 4 of the B6 structure. The 5'-phosphates were subsequently converted within the cell to pyridoxal 5'-phosphate with liberation of the original amine in a reaction catalyzed by pyridoxamine (pyridoxine) 5'-phosphate oxidase, an enzyme with broad specificity for 4'-substituted amines on the 5'-phospho-B6 structure. This system illustrates how knowledge of transporter specificity can permit design of a compound with potential biologic activity. A drug or other intracellular effector may be piggybacked onto a transported solute (e.g., vitamin or other nutrient) that gains facilitated entry to a cell and is, thereafter, metabolized to release the active compound.
Heat shock proteins can regulate expression of the Tla region-encoded class Ib molecule Qa-1.
Authors: Imani F , Soloski M J
Published: May 2006

T cells recognize foreign antigens in association with the highly polymorphic class I and class II molecules encoded in the major histocompatibility complex (MHC). In addition to these highly polymorphic molecules, the murine MHC also encodes, in the Qa/Tla region, several less polymorphic structures referred to as class I-like or class Ib molecules. Although no specific function has been assigned to these molecules, their overall structural similarities to the classical class I molecules and their association with beta 2-microglobulin suggest a role in antigen recognition. Recent data have suggested that the class Ib molecule Qa-1 may be involved in antigen presentation to T cells expressing gamma delta receptors. In addition, several reports have demonstrated that gamma delta T cells can respond to mycobacterial heat shock proteins. We report that transfection of a mouse fibroblast line with gene T23b leads to the surface expression of a molecule that is structurally identical to lymphocyte Qa-1b. In the transfected cells the predominant Qa-1 species was present in an immature intracellular form. The expression of mature cell surface Qa-1 was dramatically and selectively increased following heat shock. Furthermore, the addition of a tryptic digest of Mycobacterium bovis 65-kDa heat shock protein stabilized the surface expression of Qa-1b. These observations suggest that the Qa-1 molecule may be involved in the presentation of heat shock protein-derived peptides to the immune system.
Protection against endotoxic shock by a tumor necrosis factor receptor immunoadhesin.
Authors: Ashkenazi A , Marsters S A , Capon D J , Chamow S M , Figari I S , Pennica D , Goeddel D V , Palladino M A , Smith D H
Published: May 2006

Tumor necrosis factors (TNF) alpha and beta are structurally related cytokines that mediate a wide range of immunological, inflammatory, and cytotoxic effects. During bacterial infection of the bloodstream (sepsis), TNF-alpha induction by bacterial endotoxin is thought to be a major factor contributing to the cardiovascular collapse and critical organ failure that can develop. Despite antibiotic therapy, these consequences of sepsis continue to have a high mortality rate in humans. Here we describe a potent TNF antagonist, a TNF receptor (TNFR) immunoadhesin, constructed by gene fusion of the extracellular portion of human type 1 TNFR with the constant domains of human IgG heavy chain (TNFR-IgG). When expressed in transfected human cells, TNFR-IgG is secreted as a disulfide-bonded homodimer. Purified TNFR-IgG binds to both TNF-alpha and TNF-beta and exhibits 6- to 8-fold higher affinity for TNF-alpha than cell surface or soluble TNF receptors. In vitro, TNFR-IgG blocks completely the cytolytic effect of TNF-alpha or TNF-beta on actinomycin D-treated cells and is markedly more efficient than soluble TNFR (24-fold) or monoclonal anti-TNF-alpha antibodies (4-fold) in inhibiting TNF-alpha. In vitro, TNFR-IgG prevents endotoxin-induced lethality in mice when given 0.5 hr prior to endotoxin and provides significant protection when given up to 1 hr after endotoxin challenge. These results confirm the importance of TNF-alpha in the pathogenesis of septic shock and suggest a clinical potential for TNFR-IgG as a preventive and therapeutic treatment in sepsis.
Introduction of disease-related mitochondrial DNA deletions into HeLa cells lacking mitochondrial DNA results in mitochondrial dysfunction.
Authors: Hayashi J , Ohta S , Kikuchi A , Takemitsu M , Goto Y , Nonaka I
Published: May 2006

Mutant mitochondrial DNA with large-scale deletions (delta-mtDNA) has been frequently observed in patients with chronic progressive external ophthalmoplegia (CPEO), a subgroup of the mitochondrial encephalomyopathies. To exclude involvement of the nuclear genome in expression of the mitochondrial dysfunction characteristic of CPEO, we introduced the mtDNA of a CPEO patient into clonal mtDNA-less HeLa cells and isolated cybrid clones. Quantitation of delta-mtDNA in the cybrids revealed that delta-mtDNA was selectively propagated with higher levels of delta-mtDNA correlating with slower cellular growth rate. In these cybrid clones, translational complementation of the missing tRNAs occurred only when delta-mtDNA was less than 60% of the total mtDNA, whereas accumulation of delta-mtDNA to greater than 60% resulted in progressive inhibition of overall mitochondrial translation as well as reduction of cytochrome c oxidase activity throughout the organelle population. Because these cybrids shared the same nuclear background as HeLa cells, these results suggest that large-scale deletion mutations of mtDNA alone are sufficient for the mitochondrial dysfunction characteristic of CPEO.
Expression of GATA-binding proteins during embryonic development in Xenopus laevis.
Authors: Zon L I , Mather C , Burgess S , Bolce M E , Harland R M , Orkin S H
Published: May 2006

Proteins that recognize the core sequence GATA are important regulators of hematopoietic-specific gene transcription. We have characterized cDNAs encoding the Xenopus laevis homologues of three related transcription factors, designated GATA-1, -2, and -3. Comparative sequence analysis reveals strong conservation of the zinc-finger DNA-binding domain among all vertebrate GATA-binding proteins. GATA-2 and GATA-3 polypeptides are homologous throughout their entire sequences, whereas GATA-1 sequence is conserved only in the region responsible for DNA binding. In Xenopus, RNAs encoding GATA-binding proteins are expressed in both larval and adult erythroid cells. GATA-1, -2, and -3 RNAs are first detectable in early gastrula (Nieuwkoop developmental stage 11). This is earlier than the appearance of the early larval alpha T1 globin RNA (stage 15), beta T1 globin RNA (stage 26), or blood island formation (stage 30). The expression of GATA-1, -2, and -3 in early development may signal an early commitment of mesoderm to form hematopoietic tissue.
Reactions of the subunits of the class II major histocompatibility complex molecule IAd.
Authors: Tampé R , Tyvoll D , McConnell H M
Published: May 2006

Major histocompatibility complex (MHC) class II molecules are heterodimers formed by noncovalent linkage of alpha and beta chains. It has been shown that the subunits of the MHC class II molecules IAd and IEk bind antigenic peptides as well as antigenic peptides labeled with fluorescent probes. Laser scanning fluorescence microscopy on SDS/polyacrylamide gels demonstrates that the subunit-peptide complexes of IAd are stable over a wide pH range. Below pH 5.3 the heterodimer of IAd dissociates into the free chains, which still bind antigenic peptides such as the 18-amino acid peptide obtained by a tyrosine addition to a chicken ovalbumin peptide, Ova-(323-339)Y. The stability of preformed subunit complexes with fluorescein-labeled Ova-(323-339)Y was investigated by using high-performance size exclusion chromatography and epifluorescence microscopy. Each subunit forms a long-lived complex, both in detergent solutions and in reconstituted lipid bilayers. At 37 degrees C and pH 7.0 the dissociation half-time of the beta-subunit-peptide complex was determined to be 28 hr and that of the alpha-subunit-peptide complex was 10 hr. In contrast to the dissociation of the peptide from the IAd heterodimer, the half-times for dissociation of the peptide from the separate chains are not decreased at pH 5.0.
Toward an animal model of cystic fibrosis: targeted interruption of exon 10 of the cystic fibrosis transmembrane regulator gene in embryonic stem cells.
Authors: Koller B H , Kim H S , Latour A M , Brigman K , Boucher R C , Scambler P , Wainwright B , Smithies O
Published: May 2006

A gene-targeting construct was made containing 7.8 kilobases of DNA spanning exon 10 of the mouse cystic fibrosis transmembrane regulator (CFTR) gene in which part of the exon has been replaced by two neomycin-resistance (Neo) genes driven by different promoters. (This replacement introduces a chain-termination codon at amino acid position 489 in the CFTR sequence). A herpes simplex thymidine kinase gene was on each end of the construct, which was electroporated into embryonic stem (ES) cells. Colonies resistant to G418, or to G418 plus ganciclovir, were selected and screened by Southern blotting or by PCR amplification. Five pools of G418-resistant cells gave PCR products diagnostic of targeting. Four independent clones of ES cells with a disrupted CFTR gene have been isolated from these pools. The frequency of targeting was 1/2500 G418-resistant colonies. This low frequency is not the consequence of marginal expression of the Neo genes in the targeted cells. The CFTR targeting events were clustered among our experiments in a manner suggesting that some unidentified factor(s), possibly passage number, influences the recovery of CFTR-targeted cells.
Reversible conversion of nitroxyl anion to nitric oxide by superoxide dismutase.
Authors: Murphy M E , Sies H
Published: May 2006

Superoxide dismutase (SOD) rapidly scavenges superoxide (O2-) and also prolongs the vasorelaxant effects of nitric oxide (NO), thought to be the endothelium-derived relaxing factor. This prolongation has been ascribed to prevention of the reaction between O2- with NO. We report that SOD supports a reversible reduction of NO to NO-. When cyanamide and catalase were used to generate NO- in the presence of SOD, NO was measured by the conversion of HbO2 to MetHb. When SOD[Cu(I)] was exposed to NO anaerobically, NO- was trapped by MetHb forming nitrosylmyoglobin. When NO was generated by 3-morpholinosydnonimine hydrochloride in the presence of SOD, NO- or a similar reductant was formed, which reduced catalase compound II and promoted the formation of the catalase [Fe(III)]-NO complex. It is, therefore, conceivable that SOD may protect NO and endothelium-derived relaxing factor by a mechanism in addition to O2- scavenging and that NO- may be a physiologically important form of endothelium-derived relaxing factor.
Infrared spectroscopy of exfoliated human cervical cells: evidence of extensive structural changes during carcinogenesis.
Authors: Wong P T , Wong R K , Caputo T A , Godwin T A , Rigas B
Published: May 2006

Infrared spectra were obtained from exfoliated cervical cells from 156 females, of whom 136 were normal, 12 had cervical cancer, and 8 had dysplasia. The spectra of the normal women, essentially identical, differed from those obtained from patients with either cancer or dysplasia. In malignant samples we noted (i) significant changes in the intensity of the glycogen bands at 1025 cm-1 and 1047 cm-1, the bands at 1082 cm-1 and 1244 cm-1, the C--O stretching band at 1155 cm-1, and the band at 1303 cm-1, (ii) significant shifts of the peaks normally appearing at 1082 cm-1, 1155 cm-1, and 1244 cm-1, and (iii) an additional band at 970 cm-1. Further study of several of these bands, including the pressure dependence of their frequencies, revealed that in the malignant cervical tissue there were extensive changes in the degree of hydrogen bonding of phosphodiester groups of nucleic acids and C--OH groups of proteins, as well as changes in the degree of disorder of methylene chains of lipids. The IR spectra of samples with dysplasia demonstrated the same changes with cancer samples, except that the changes were of lesser magnitude and the phosphodiester peak normally appearing at 1082 cm-1 did not shift. These spectroscopic changes appear to progress in tandem with the morphological changes that lead normal cervical epithelium to cancer through the premalignant stage of dysplasia. The diagnostic potential of IR spectroscopy is discussed.
On the repulsion of energy eigenstates in the time domain.
Authors: Levine R D , Kinsey J L
Published: May 2006

The rate and extent of the exploration of the available phase space of a bound quantum mechanical system are shown to depend on the repulsion of energy eigenstates. Central to the argument is the Fourier transform relating the survival probability (in time) of an initially prepared nonstationary state and the (frequency) autocorrelation function of the excitation spectrum. Strong repulsion of states, as in the Wigner surmise, leads to a rapid dephasing of the initially coherently prepared state. The rate and extent of sampling of phase space depend not only on the spacing distribution but also on the intensity fluctuations. The rate of dephasing is equal to that inferred from the width of the spectral autocorrelation function.
Nitroxergic nerves mediate vagally induced relaxation in the isolated stomach of the guinea pig.
Authors: Desai K M , Zembowicz A , Sessa W C , Vane J R
Published: May 2006

Here we show that the relaxation induced by stimulation of the vagus nerve in the presence of cholinergic (muscarinic) and adrenergic blockade in the isolated stomach of the guinea pig is mediated by nitric oxide (NO). This is substantiated by inhibition of vagal relaxation by NG-monomethyl-L-arginine, an inhibitor of NO synthesis. The effect of NG-monomethyl-L-arginine was partially reversed by coincubation with L-arginine but not with D-arginine. NO activates soluble guanylate cyclase, and relaxation of the stomach induced by vagal stimulation was prevented by an inhibitor of soluble guanylate cyclase, methylene blue, further supporting our conclusions. The relaxant effect of vagal stimulation was also ablated by hexamethonium, an inhibitor of ganglionic nicotinic receptors, thereby showing that ganglionic transmission did not rely on NO, through its release from preganglionic neurons. However, hexamethonium did not inhibit the gastric relaxation brought about by increasing the intragastric pressure, which is also mediated by NO as previously described by us. The selective inhibition by hexamethonium of only the vagally mediated relaxation but not of the pressure-induced relaxation of the stomach indicates the existence of at least two separate neuronal pathways able to generate NO and bring about gastric accommodation of food or fluid.
Binary system for regulating transgene expression in mice: targeting int-2 gene expression with yeast GAL4/UAS control elements.
Authors: Ornitz D M , Moreadith R W , Leder P
Published: May 2006

We have developed a binary transgenic system that activates an otherwise silent transgene in the progeny of a simple genetic cross. The system consists of two types of transgenic mouse strains, targets and transactivators. A target strain bears a transgene controlled by yeast regulatory sequences (UAS) that respond only to the yeast transcriptional activator GAL4. A transactivator strain expresses an active GAL4 gene that can be driven by any selected promoter. The current paradigm uses the murine growth factor int-2 cDNA as the target gene and the GAL4 gene driven by the mouse mammary tumor virus long terminal repeat as the transactivator. Both target and transactivator strains are phenotypically normal. By contrast, the bigenic offspring of these two strains express high levels of the target int-2 gene in each organ expressing the GAL4 transactivator. They also display a characteristic dominant int-2 phenotype that consists of epithelial hyperplasia in mammary and salivary glands, as well as prostatic and epididymal hypertrophy, which results in male sterility.
Borrowed proteins in bacterial bioluminescence.
Authors: O'Kane D J , Woodward B , Lee J , Prasher D C
Published: May 2006

A library of Photobacterium phosphoreum DNA was screened in lambda 2001 for the lumazine protein gene, using two degenerate 17-mer oligonucleotide probes that were deduced from a partial protein primary sequence. The lumazine protein gene was localized to a 3.4-kilobase BamHI/EcoRI fragment in one clone. The fragment contained an open reading frame, encoding a 189-residue protein, that had a predicted amino acid sequence that concurred with the partial sequence determined for lumazine protein. Considerable sequence similarity was detected between lumazine protein, the yellow fluorescence protein from Vibrio fischeri, and the alpha subunit of riboflavin synthetase (EC 2.5.1.9). A highly conserved sequence in lumazine protein corresponds to the proposed lumazine binding sites in the alpha subunit of riboflavin synthetase. Several secondary structure programs predict the conformation of this site in lumazine protein to be a beta-sheet. A minimal model with three interactions between the ligand and this beta-sheet structure is proposed, which is consistent with the results of NMR and ligand binding studies.
Requirements for bypass of UV-induced lesions in single-stranded DNA of bacteriophage phi X174 in Salmonella typhimurium.
Authors: Slater S C , Maurer R
Published: May 2006

According to the current model for mutagenic bypass of UV-induced lesions, efficient bypass requires three proteins: activated RecA (RecA*) and either activated UmuD (UmuD') and UmuC or their plasmid-encoded analogues, MucA' and MucB. RecA* aids synthesis of UmuD' and UmuC (and MucA'/MucB) at two levels: by inactivation of the LexA transcriptional repressor of these genes and by cleavage of UmuD (and MucA) to produce the active fragments, UmuD' (MucA'). A third role for RecA is revealed when these two roles are otherwise satisfied in a suitably engineered strain. An often-suggested possible role for RecA in bypass is inhibition of editing by the epsilon subunit of DNA polymerase III. Here, by demonstrating that elimination of epsilon by deletion of its gene, dnaQ, does not relieve the requirement for the third function of RecA, we show that RecA must perform some function other than, or in addition to, inhibition of epsilon. We also show that elimination of epsilon does not relieve the requirement for either Muc protein. Moreover, we observed reactivation of irradiated phi X174 in unirradiated cells expressing MucA' and MucB. This finding makes it unlikely that the additional role of recA involves derepression of an unidentified gene or cleavage of an unidentified protein and makes it more likely that RecA participates directly in bypass.
The role of the superior colliculus in facilitating visual attention and form perception.
Authors: Sprague J M
Published: May 2006

When interhemispheric transfer in cats is studied from an intact hemisphere to a hemisphere with a suprasylvian cortical lesion, excellent transfer of grating discriminations, but no transfer of forms, is present. Stimuli with global, repetitive features covering a large visual field (gratings), which can be discriminated by preattentive vision, are transferred; perception of stimuli with local features (forms), which require serial exploration using focal vision, is defective in the hemisphere with cortical lesion and transfer is lacking. Influence of the midbrain in facilitating focal vision is shown by the restoration of form discriminations after section of the superior collicular commissure. It is hypothesized that the perceptual defect after lesion in the suprasylvian cortex is due to poor spatial attention and its restoration after midbrain lesion is due to improved function of those collicular cells that mediate orienting of attention.
Tissue acylation by the chlorofluorocarbon substitute 2,2-dichloro-1,1,1-trifluoroethane.
Authors: Harris J W , Pohl L R , Martin J L , Anders M W
Published: May 2006

Hydrochlorofluorocarbons (HCFCs) are being developed as substitutes for ozone-depleting chlorofluorocarbons (CFCs); because widespread human exposure to HCFCs may be expected, it is important to evaluate their toxicities thoroughly. Here we report studies on the bioactivation of the CFC substitute 2,2-dichloro-1,1,1-trifluoroethane (HCFC-123) to an electrophilic intermediate that reacts covalently with liver proteins. HCFC-123 and its analog halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) were studied in rats by 19F NMR spectroscopy, and we found that a trifluoroacetylated lysine adduct was formed with liver proteins. Also, the pattern of proteins immunoreactive with hapten-specific anti-trifluoroacetylprotein antibodies was identical in livers of HCFC-123- and halothane-exposed rats. Because halothane causes an idiosyncratic, and sometimes fatal, hepatitis that is associated with an immune response against several trifluoroacetylated liver proteins, the present findings raise the possibility that humans exposed to HCFC-123 or structurally related HCFCs may be at risk of developing an immunologically mediated hepatitis.
Very long charge runs in systemic lupus erythematosus-associated autoantigens.
Authors: Brendel V , Dohlman J , Blaisdell B E , Karlin S
Published: May 2006

Systemic lupus erythematosus and other chronic systemic autoimmune diseases are associated with circulating autoantibodies reactive with a limited set of mostly nuclear proteins. Using rigorous statistical methods we have identified segments of highly significant charge concentration in the majority of the characteristic nuclear and cytoplasmic autoantigens. Extremely long runs of charged residues, including some sequences of greater than 20 consecutive charged residues (purely acidic or mixed basic and acidic), occur in about a third of these proteins, whereas equivalent runs are found in less than 3% of other mammalian proteins. The other sequences have less extreme charge clusters, the type and location of which are often conserved between several otherwise nonsimilar antigens. We propose that supercharged surfaces render the targeted host proteins strongly immunogenic and that antinuclear antibody profiles might result from chronic exposure to intracellular contents, possibly in conjunction with crossreactive viral products. The limited number of potential systemic autoantigens may partly be due to the rarity of requisite charge properties.
Adhesion protein GMP140 inhibits superoxide anion release by human neutrophils.
Authors: Wong C S , Gamble J R , Skinner M P , Lucas C M , Berndt M C , Vadas M A
Published: May 2006

The respiratory burst of blood neutrophils has a critical role in the destruction of microorganisms and tissue damage in inflammation. Neutrophils adhere in a dose-dependent fashion to granule membrane protein 140 (GMP140), a member of the LEC-CAM (lectin/epidermal growth factor/complement-binding domain cell adhesion molecule) family of adhesion proteins when it is immobilized onto plastic surfaces. Adherence to GMP140 was associated with less superoxide anion generation than adherence to other surfaces, an effect that is especially remarkable after activation of neutrophils with tumor necrosis factor alpha, an agent that on other surfaces promotes adhesion and spreading. However, on GMP140 the cells fail to spread and instead remain rounded and refractile. Neutrophils adhering to GMP140 were also deficient in superoxide anion generation to formylmethionylleucylphenylalanine. Furthermore, fluid-phase GMP140 also inhibited the superoxide generation by neutrophils stimulated by tumor necrosis factor alpha. The effect of GMP140 was reversible by washing and was inhibited by anti-GMP140 Fab antibody. GMP140 appears to be a natural antiinflammatory molecule that may prevent the inappropriate activation of neutrophils in the circulation.
Identity of a second type of allatostatin from cockroach brains: an octadecapeptide amide with a tyrosine-rich address sequence.
Authors: Pratt G E , Farnsworth D E , Fok K F , Siegel N R , McCormack A L , Shabanowitz J , Hunt D F , Feyereisen R
Published: May 2006

An octadecapeptide that inhibits juvenile hormone synthesis has been isolated by HPLC from brain-retrocerebral complexes of the cockroach Diploptera punctata. The primary structure of this allatostatin has been elucidated by tandem mass spectrometry: Ala-Tyr-Ser-Tyr-Val-Ser-Glu-Tyr-Lys-Arg-Leu-Pro-Val-Tyr-Asn-Phe-Gly-Leu- NH2 (ASB2). The amidated three-residue C terminus of this type B allatostatin is identical to that of four known type A allatostatins, and the preceding three residues show close structural homology. ASB2 has over twice the activity of the type A tridecapeptide Ala-Pro-Ser-Gly-Ala-Gln-Arg-Leu-Tyr-Gly-Phe-Gly-Leu-NH2 (ASA1) in inhibiting juvenile hormone biosynthesis in corpora allata from females in early vitellogenesis (day 2), and its efficacy persists during pregnancy, but it is equally effective as ASA1 on glands from day-10 females (IC50 = 0.31 nM). The octadecapeptide is characterized by a potential dibasic cleavage site, Lys9-Arg10, the integrity of which is needed for high potency. The ASB2-(11-18)-octapeptide amide gives a full response at high concentrations at day 10 (IC50 = 48 nM), but the C-truncated (1-9)-, (1-11)-, and (1-17)-amide fragments of ASB2 are inactive. Thus, the endocrine message is located at the C terminus. N alpha-acetylation of the N-truncated (9-18), (10-18), and (11-18) fragments of ASB2 increases activity relative to the nonacetylated peptides. The site of action of type A and type B allatostatins is located before mevalonate kinase in the biosynthetic pathway for juvenile hormone.
Generation of diverse high-affinity human monoclonal antibodies by repertoire cloning.
Authors: Persson M A , Caothien R H , Burton D R
Published: May 2006

Combinatorial libraries of antibody heavy and light chains derived from the peripheral blood lymphocytes of an individual immunized with tetanus toxoid have been expressed in Escherichia coli by using phage lambda vectors. Screening of the libraries allowed identification of a large number of human monoclonal Fab fragments specific for tetanus toxoid. Initial studies suggested considerable sequence diversity in these antibodies. The method should allow the generation of many human monoclonal antibodies of interest and the dissection of human humoral immune responses.
The ubiquitous glucose transporter GLUT-1 belongs to the glucose-regulated protein family of stress-inducible proteins.
Authors: Wertheimer E , Sasson S , Cerasi E , Ben-Neriah Y
Published: May 2006

In mammals, glucose transport is mediated by five structurally related glucose transporters that show a characteristic cell-specific expression. However, the rat brain/HepG2/erythrocyte-type glucose transporter GLUT-1 is expressed at low levels in most cells. The reason for this coexpression is not clear. GLUT-1 is negatively regulated by glucose. Another family of proteins, glucose-regulated proteins (GRPs), is also ubiquitously expressed and stimulated by glucose deprivation and other cellular stresses. We therefore hypothesized that GLUT-1 may be a glucose-regulated stress protein. This was tested by subjecting L8 myocytes and NIH 3T3 fibroblasts to glucose starvation or exposure to the calcium ionophore A23187, 2-mercaptoethanol, or tunicamycin, all known to increase GRP levels. The mRNA for GLUT-1 was augmented by 50-300% in a time-dependent manner, similarly to the changes in GRP-78 mRNA. Ex vivo incubation of rat soleus muscles induced a marked and concomitant rise in the mRNA levels of GLUT-1 and GRP-78. Finally, calcium ionophore A23187 and 2-mercaptoethanol induced a 2- to 3-fold increase in the levels of the GLUT-1 protein and hexose uptake. In all instances in which GRP-78 and GLUT-1 responded to stress, the transcription of the cell-specific muscle/adipocyte-type insulin-responsive glucose transporter (GLUT-4) did not change. Thus, despite the lack of structural similarity, GLUT-1 and GRP-78 expression is regulated similarly, whereas the regulation of GLUT-4, which is structurally related to GLUT-1, is different. We propose that GLUT-1 belongs to the GRP family of stress proteins and that its ubiquitous expression may serve a specific purpose during cellular stress.
Murine erythroleukemia cell line GM979 contains factors that can activate silent chromosomal human gamma-globin genes.
Authors: Zitnik G , Hines P , Stamatoyannopoulos G , Papayannopoulou T
Published: May 2006

We introduced a normal chromosome 11 into GM979 murine erythroleukemia cells by fusing them with Epstein-Barr virus-transformed lymphocytes from a normal individual. In contrast to previous data obtained with other murine erythroleukemia cells, we detected activation of human chromosomal gamma-globin genes in GM979 cells. GM979, unlike previously used murine erythroleukemia cell lines, expresses murine embryonic globin in addition to adult globin. While all the hybrids expressed gamma- and beta-globin, they displayed a wide range of gamma-globin expression in relation to that of beta-globin. No correlation, however, was found in quantitative expression between murine embryonic globin and human gamma-globin in these hybrids, suggesting that the two globins are regulated independently, at least in this cell line. These data indicate that gamma-globin genes from normal, nonerythroid chromosomes are not irreversibly silenced, and they can be activated by a positive trans factor(s) present in GM979 cells.
Drosophila P-element transposase is a transcriptional repressor in vitro.
Authors: Kaufman P D , Rio D C
Published: May 2006

Mobility of P transposable elements in Drosophila melanogaster depends on the 87-kDa transposase protein encoded by the P element. Transposase recognizes a 10-base-pair DNA sequence that overlaps an A + T-rich region essential for transcription from the P-element promoter. We report here that transposase represses transcription from the P-element promoter in vitro. This transcriptional repression is blocked by prior formation of an RNA polymerase II transcription complex on the template DNA. Binding of transposase on the P-element promoter is blocked by prior binding of either the Drosophila RNA polymerase II complex or the yeast transcription factor TFIID. These data suggest that transposase represses transcription by preventing assembly of an RNA polymerase II complex at the P-element promoter.