bluuebunny/crossref_metadata_2025 huggingface dataset:
Channel formation by antiapoptotic protein Bcl-2
Authors: Schendel Sharon L. , Xie Zhihua , Montal Myrta Oblatt , Matsuyama Shigemi , Montal Mauricio , Reed John C.
Published: July 2002

Bcl-2 is the prototypical member of a large family of apoptosis-regulating proteins, consisting of blockers and promoters of cell death. The three-dimensional structure of a Bcl-2 homologue, Bcl-X
L
, suggests striking similarity to the pore-forming domains of diphtheria toxin and the bacterial colicins, prompting exploration of whether Bcl-2 is capable of forming pores in lipid membranes. Using chloride efflux from KCl-loaded unilamellar lipid vesicles as an assay, purified recombinant Bcl-2 protein exhibited pore-forming activity with properties similar to those of the bacterial toxins, diphtheria toxin, and colicins, i.e., dependence on low pH and acidic lipid membranes. In contrast, a mutant of Bcl-2 lacking the two core hydrophobic α-helices (helices 5 and 6), predicted to be required for membrane insertion and channel formation, produced only nonspecific effects. In planar lipid bilayers, where detection of single channels is possible, Bcl-2 formed discrete ion-conducting, cation-selective channels, whereas the Bcl-2 (Δh5, 6) mutant did not. The most frequent conductance observed (18 ± 2 pS in 0.5 M KCl at pH 7.4) is consistent with a four-helix bundle structure arising from Bcl-2 dimers. However, larger channel conductances (41 ± 2 pS and 90 ± 10 pS) also were detected with progressively lower occurrence, implying the step-wise formation of larger oligomers of Bcl-2 in membranes. These findings thus provide biophysical evidence that Bcl-2 forms channels in lipid membranes, suggesting a novel function for this antiapoptotic protein.
Conservation of the
Drosophila
lateral inhibition pathway in human lung cancer: A hairy-related protein (HES-1) directly represses achaete-scute homolog-1 expression
Authors: Chen Herbert , Thiagalingam Arunthathi , Chopra Hemi , Borges Michael W. , Feder John N. , Nelkin Barry D. , Baylin Stephen B. , Ball Douglas W.
Published: July 2002

The achaete-scute genes encode essential transcription factors in normal
Drosophila
and vertebrate nervous system development. Human achaete-scute homolog-1 (hASH1) is constitutively expressed in a human lung cancer with neuroendocrine (NE) features, small cell lung cancer (SCLC), and is essential for development of the normal pulmonary NE cells that most resemble this neoplasm. Mechanisms regulating achaete-scute homolog expression outside of
Drosophila
are presently unclear, either in the context of the developing nervous system or in normal or neoplastic cells with NE features. We now provide evidence that the protein hairy-enhancer-of-split-1 (HES-1) acts in a similar manner as its
Drosophila
homolog, hairy, to transcriptionally repress achaete-scute expression. HES-1 protein is detected at abundant levels in most non-NE human lung cancer cell lines which lack hASH1 but is virtually absent in hASH1-expressing lung cancer cells. Moreover, induction of HES-1 in a SCLC cell line down-regulates endogenous hASH1 gene expression. The repressive effect of HES-1 is directly mediated by binding of the protein to a class C site in the hASH1 promoter. Thus, a key part of the process that determines neural fate in
Drosophila
is conserved in human lung cancer cells. Furthermore, modulation of this pathway may underlie the constitutive hASH1 expression seen in NE tumors such as SCLC, the most virulent human lung cancer.
Cell-surface perturbations of the epidermal growth factor and vascular endothelial growth factor receptors by phosphorothioate oligodeoxynucleotides
Authors: Rockwell Patricia , O’Connor William J. , King Karen , Goldstein Neil I. , Zhang L. M. , Stein C. A.
Published: July 2002

Antisense oligodeoxynucleotides offer potential as therapeutic agents to inhibit gene expression. Recent evidence indicates that oligodeoxynucleotides designed to target specific nucleic acid sequences can interact nonspecifically with proteins. This report describes the interactive capabilities of phosphorothioate oligodeoxynucleotides of defined sequence and length with two essential protein tyrosine receptors, flk-1 and epidermal growth factor receptor (EGFR), and their effects on receptor signaling in a transfected and tumor cell line, respectively. Phosphorothioate oligodeoxynucleotides bound to the cell surface, as demonstrated by fluorescence-activated cell-sorter analyses (FACS), and perturbed receptor activation in the presence and absence of cognate ligands, EGF (EGFR) and vascular endothelial growth factor (flk-1), in phosphorylation assays. Certain phosphorothioate oligodeoxynucleotides interacted relatively selectively with flk-1 and partially blocked the binding of specific anti-receptor monoclonal antibodies to target sites. They stimulated EGFR phosphorylation in the absence of EGF but antagonized ligand-mediated activation of EGFR and flk-1.
In vivo
studies showed that a nonspecific phosphorothioate oligodeoxynucleotide suppressed the growth of glioblastoma in a mouse model of tumorigenesis. These results emphasize the capacity of phosphorothioate oligodeoxynucleotides to interact with cells in a sequence-selective nonantisense manner, while associating with cellular membrane proteins in ways that can inhibit cellular metabolic activities.
Crystal structure of the homo-tetrameric DNA binding domain of
Escherichia coli
single-stranded DNA-binding protein determined by multiwavelength x-ray diffraction on the selenomethionyl protein at 2.9-Å resolution
Authors: Raghunathan Srinivasan , Ricard Cynthia S. , Lohman Timothy M. , Waksman Gabriel
Published: July 2002

The crystal structure of the tetrameric DNA-binding domain of the single-stranded DNA binding protein from
Escherichia coli
was determined at a resolution of 2.9 Å using multiwavelength anomalous dispersion. Each monomer in the tetramer is topologically similar to an oligomer-binding fold. Two monomers each contribute three β-strands to a single six-stranded β-sheet to form a dimer. Two dimer–dimer interfaces are observed within the crystal. One of these stabilizes the tetramer in solution. The other interface promotes a superhelical structure within the crystal that may reflect tetramer–tetramer interactions involved in the positive cooperative binding of the single-stranded DNA-binding protein to single-stranded DNA.
A single dose of kainic acid elevates the levels of enkephalins and activator protein-1 transcription factors in the hippocampus for up to 1 year
Authors: Bing Guoying , Wilson Belinda , Hudson Pearlie , Jin Lei , Feng Zhehui , Zhang Wanqin , Bing Renjie , Hong Jau-Shyong
Published: July 2002

Neuronal plasticity plays a very important role in brain adaptations to environmental stimuli, disease, and aging processes. The kainic acid model of temporal lobe epilepsy was used to study the long-term anatomical and biochemical changes in the hippocampus after seizures. Using Northern blot analysis, immunocytochemistry, and Western blot analysis, we have found a long-term elevation of the proconvulsive opioid peptide, enkephalin, in the rat hippocampus. We have also demonstrated that an activator protein-1 transcription factor, the 35-kDa fos-related antigen, can be induced and elevated for at least 1 year after kainate treatment. This study demonstrated that a single systemic injection of kainate produces almost permanent increases in the enkephalin and an activator protein-1 transcription factor, the 35-kDa fos-related antigen, in the rat hippocampus, and it is likely that these two events are closely associated with the molecular mechanisms of induction of long-lasting enhanced seizure susceptibility in the kainate-induced seizure model. The long-term expression of the proenkephalin mRNA and its peptides in the kainate-treated rat hippocampus also suggests an important role in the recurrent seizures of temporal lobe epilepsy.
Quantitative detection of immune activation transcripts as a diagnostic tool in kidney transplantation
Authors: Strehlau Jürgen , Pavlakis Martha , Lipman Mark , Shapiro Michael , Vasconcellos Lauro , Harmon William , Strom Terry B.
Published: July 2002

Procedures to diagnose renal allograft rejection depend upon detection of graft dysfunction and the presence of a mononuclear leukocytic infiltrate; however, the presence of a modest cellular infiltrate is often not conclusive and can be detected in non-rejecting grafts. We have pursued a molecular approach utilizing reverse transcription (RT)-PCR to test the diagnostic accuracy of multiple immune activation gene analysis as means to diagnose renal allograft rejection. The magnitude of intragraft gene expression of 15 immune activation genes was quantified by competitive RT-PCR in 60 renal allograft core biopsies obtained for surveillance or to diagnose the etiology of graft dysfunction. Results were compared with a clinicopathological analysis based upon the histological diagnosis (Banff criteria) and the response to antirejection treatment. During acute renal allograft rejection intragraft expression of the interleukin (IL)-7 (P< 0.001), IL-10 (P< 0.0001), IL-15 (P< 0.0001), Fas ligand (P< 0.0001), perforin (P< 0.0001), and granzyme B (P< 0.0015), but not IL-2, interferon γ, or IL-4, genes is significantly heightened. Amplified RANTES and IL-8 gene transcripts are sensitive but nonspecific markers of rejection. A simultaneous RT-PCR evaluation of perforin, granzyme B, and Fas ligand identifies acute rejection, including cases with mild infiltration, with extraordinary sensitivity (100%) and specificity (100%). Effective antirejection therapy results in a rapid down-regulation of gene expression. The combined analysis of Fas ligand, perforin, and granzyme B gene expression by quantitative RT-PCR provides a reliable tool for diagnosis and follow-up of acute renal allograft rejection. Its accuracy and a potential rapid application within few hours suggest its use in the clinical management of renal transplant patients.
Murine hematopoietic stem cells committed to macrophage/dendritic cell formation: Stimulation by Flk2-ligand with enhancement by regulators using the gp130 receptor chain
Authors: Metcalf Donald
Published: July 2002

The stimulation by Flk2-ligand (FL) of blast colony formation by murine bone marrow cells was selectively potentiated by the addition of regulators sharing in common the gp130 signaling receptor–leukemia inhibitory factor (LIF), oncostatin M, interleukin 11, or interleukin 6. Recloning of blast colony cells indicated that the majority were progenitor cells committed exclusively to macrophage formation and responding selectively to proliferative stimulation by macrophage colony-stimulating factor. Reculture of blast colony cells initiated by FL plus LIF in cultures containing granulocyte/macrophage colony-stimulating factor plus tumor necrosis factor α indicated that at least some of the cells were capable of maturation to dendritic cells. The cells forming blast colonies in response to FL plus LIF were unrelated to those forming blast colonies in response to stimulation by stem cell factor and appear to be a distinct subset of mature hematopoietic stem cells.
Response-reinforcement learning is dependent on
N
-methyl-
d
-aspartate receptor activation in the nucleus accumbens core
Authors: Kelley Ann E. , Smith-Roe Stephanie L. , Holahan Matthew R.
Published: July 2002

The nucleus accumbens, a site within the ventral striatum, is best known for its prominent role in mediating the reinforcing effects of drugs of abuse such as cocaine, alcohol, and nicotine. Indeed, it is generally believed that this structure subserves motivated behaviors, such as feeding, drinking, sexual behavior, and exploratory locomotion, which are elicited by natural rewards or incentive stimuli. A basic rule of positive reinforcement is that motor responses will increase in magnitude and vigor if followed by a rewarding event. It is likely, therefore, that the nucleus accumbens may serve as a substrate for reinforcement learning. However, there is surprisingly little information concerning the neural mechanisms by which appetitive responses are learned. In the present study, we report that treatment of the nucleus accumbens core with the selective competitive
N
-methyl-
d
-aspartate (NMDA) antagonist 2-amino-5-phosphonopentanoic acid (AP-5; 5 nmol/0.5 μl bilaterally) impairs response-reinforcement learning in the acquisition of a simple lever-press task to obtain food. Once the rats learned the task, AP-5 had no effect, demonstrating the requirement of NMDA receptor-dependent plasticity in the early stages of learning. Infusion of AP-5 into the accumbens shell produced a much smaller impairment of learning. Additional experiments showed that AP-5 core-treated rats had normal feeding and locomotor responses and were capable of acquiring stimulus-reward associations. We hypothesize that stimulation of NMDA receptors within the accumbens core is a key process through which motor responses become established in response to reinforcing stimuli. Further, this mechanism, may also play a critical role in the motivational and addictive properties of drugs of abuse.
Identification of a family of low-affinity insulin-like growth factor binding proteins (IGFBPs): Characterization of connective tissue growth factor as a member of the IGFBP superfamily
Authors: Kim Ho-Seong , Nagalla Srinivasa R. , Oh Youngman , Wilson Elizabeth , Roberts Charles T. , Rosenfeld Ron G.
Published: July 2002

The insulin-like growth factor (IGF) binding proteins (IGFBPs) modulate the actions of the insulin-like growth factors in endocrine, paracrine, and autocrine settings. Additionally, some IGFBPs appear to exhibit biological effects that are IGF independent. The six high-affinity IGFBPs that have been characterized to date exhibit 40–60% amino acid sequence identity overall, with the most conserved sequences in their NH2and COOH termini. We have recently demonstrated that the product of the mac25/IGFBP-7 gene, which shows significant conservation in the NH2terminus, including an “IGFBP motif” (GCGCCXXC), exhibits low-affinity IGF binding. The closely related mammalian genes connective tissue growth factor (CTGF) gene,nov, andcyr61encode secreted proteins that also contain the conserved sequences and IGFBP motifs in their NH2termini. To ascertain if these genes, along with mac25/IGFBP-7, encode a family of low-affinity IGFBPs, we assessed the IGF binding characteristics of recombinant human CTGF (rhCTGF). The ability of baculovirus-synthesized rhCTGF to bind IGFs was demonstrated by Western ligand blotting, affinity cross-linking, and competitive affinity binding assays using125I-labeled IGF-I or IGF-II and unlabeled IGFs. CTGF, like mac25/IGFBP-7, specifically binds IGFs, although with relatively low affinity. On the basis of these data, we propose that CTGF represents another member of the IGFBP family (IGFBP-8) and that the CTGF gene, mac25/IGFBP-7,nov, andcyr61are members of a family of low-affinity IGFBP genes. These genes, along with those encoding the high-affinity IGFBPs 1–6, together constitute an IGFBP superfamily whose products function in IGF-dependent or IGF-independent modes to regulate normal and neoplastic cell growth.
Transient “deafness” accompanies auditory development during metamorphosis from tadpole to frog
Authors: Boatright-Horowitz Seth S. , Simmons Andrea Megela
Published: July 2002

During metamorphosis, ranid frogs shift from a purely aquatic to a partly terrestrial lifestyle. The central auditory system undergoes functional and neuroanatomical reorganization in parallel with the development of new sound conduction pathways adapted for the detection of airborne sounds. Neural responses to sounds can be recorded from the auditory midbrain of tadpoles shortly after hatching, with higher rates of synchronous neural activity and lower sharpness of tuning than observed in postmetamorphic animals. Shortly before the onset of metamorphic climax, there is a brief “deaf” period during which no auditory activity can be evoked from the midbrain, and a loss of connectivity is observed between medullary and midbrain auditory nuclei. During the final stages of metamorphic development, auditory function and neural connectivity are restored. The acoustic communication system of the adult frog emerges from these periods of anatomical and physiological plasticity during metamorphosis.
Targeted disruption of the mouse αA-crystallin gene induces cataract and cytoplasmic inclusion bodies containing the small heat shock protein αB-crystallin
Authors: Brady James P. , Garland Donita , Duglas-Tabor Yvonne , Robison W. Gerald , Groome Anne , Wawrousek Eric F.
Published: July 2002

αA-crystallin (αA) and αB-crystallin (αB) are among the predominant proteins of the vertebrate eye lens.
In vitro
, the α-crystallins, which are isolated together as a high molecular mass aggregate, exhibit a number of properties, the most interesting of which is their ability to function as molecular chaperones for other proteins. Here we begin to examine the
in vivo
functions of α-crystallin by generating mice with a targeted disruption of the αA gene. Mice that are homozygous for the disrupted allele produce no detectable αA in their lenses, based on protein gel electrophoresis and immunoblot analysis. Initially, the αA-deficient lenses appear structurally normal, but they are smaller than the lenses of wild-type littermates. αA
−/−
lenses develop an opacification that starts in the nucleus and progresses to a general opacification with age. Light and transmission electron microscopy reveal the presence of dense inclusion bodies in the central lens fiber cells. The inclusions react strongly with antibodies to αB but not significantly with antibodies to β- or γ-crystallins. In addition, immunoblot analyses demonstrate that a significant portion of the αB in αA
−/−
lenses shifts into the insoluble fraction. These studies suggest that αA is essential for maintaining lens transparency, possibly by ensuring that αB or proteins closely associated with this small heat shock protein remain soluble.
Prostate enlargement in mice due to fetal exposure to low doses of estradiol or diethylstilbestrol and opposite effects at high doses
Authors: Saal Frederick S. vom , Timms Barry G. , Montano Monica M. , Palanza Paola , Thayer Kristina A. , Nagel Susan C. , Dhar Minati D. , Ganjam V. K. , Parmigiani Stefano , Welshons Wade V.
Published: July 2002

On the basis of results of studies using high doses of estrogens, exposure to estrogen during fetal life is known to inhibit prostate development. However, it is recognized in endocrinology that low concentrations of a hormone can stimulate a tissue, while high concentrations can have the opposite effect. We report here that a 50% increase in free-serum estradiol in male mouse fetuses (released by a maternal Silastic estradiol implant) induced a 40% increase in the number of developing prostatic glands during fetal life; subsequently, in adulthood, the number of prostatic androgen receptors per cell was permanently increased by 2-fold, and the prostate was enlarged by 30% (due to hyperplasia) relative to untreated males. However, as the free serum estradiol concentration in male fetuses was increased from 2- to 8-fold, adult prostate weight decreased relative to males exposed to the 50% increase in estradiol. As a model for fetal exposure to man-made estrogens, pregnant mice were fed diethylstilbestrol (DES) from gestation days 11 to 17. Relative to controls, DES doses of 0.02, 0.2, and 2.0 ng per g of body weight per day increased adult prostate weight, whereas a 200-ng-per-g dose decreased adult prostate weight in male offspring. Our findings suggest that a small increase in estrogen may modulate the action of androgen in regulating prostate differentiation, resulting in a permanent increase in prostatic androgen receptors and prostate size. For both estradiol and DES, prostate weight first increased then decreased with dose, resulting in an inverted-U dose-response relationship.
Fig1, an interleukin 4-induced mouse B cell gene isolated by cDNA representational difference analysis
Authors: Chu Charles C. , Paul William E.
Published: July 2002

Interleukin 4 (IL-4) is a cytokine that regulates growth and differentiation of lymphoid and nonlymphoid cells. To study the molecular basis of IL-4 function, we used a cDNA subtraction approach based on the representational difference analysis method. This subtractive amplification technique allowed us to use small amounts of RNA from lipopolysaccharide ± IL-4-stimulated normal B cells to obtain IL-4-induced genes from these cells. We report here on one such gene,Fig1(interleukin-four induced gene 1), the first characterized immediate–early IL-4 inducible gene from B cells.Fig1expression is strikingly limited to the lymphoid compartment. B cells, but not T cells or mast cells, expressFig1in response to IL-4 within 2 hr in a cycloheximide resistant manner. IL-2, IL-5, and Il-6 do not induceFig1in culture.Fig1maps betweenKlk1andLdh3on mouse chromosome 7, near two loci involved with murine lupus,Sle3andLbw5. TheFig1cDNA sequence encodes a predicted 70-kDa flavoprotein with best homology to the monoamine oxidases, particularly in domains responsible for FAD binding.
Neuritin: A gene induced by neural activity and neurotrophins that promotes neuritogenesis
Authors: Naeve Gregory S. , Ramakrishnan Meena , Kramer Rainer , Hevroni Dana , Citri Yoav , Theill Lars E.
Published: July 2002

Neural activity and neurotrophins induce synaptic remodeling in part by altering gene expression. A cDNA encoding a glycosylphoshatidylinositol-anchored protein was identified by screening for hippocampal genes that are induced by neural activity. This molecule, named neuritin, is expressed in postmitotic-differentiating neurons of the developing nervous system and neuronal structures associated with plasticity in the adult. Neuritin message is induced by neuronal activity and by the activity-regulated neurotrophins BDNF and NT-3. Purified recombinant neuritin promotes neurite outgrowth and arborization in primary embryonic hippocampal and cortical cultures. These data implicate neuritin as a downstream effector of activity-induced neurite outgrowth.
γ-Tocopherol traps mutagenic electrophiles such as NO
x
and complements α-tocopherol: Physiological implications
Authors: Christen Stephan , Woodall Alan A. , Shigenaga Mark K. , Southwell-Keely Peter T. , Duncan Mark W. , Ames Bruce N.
Published: July 2002

Peroxynitrite, a powerful mutagenic oxidant and nitrating species, is formed by the near diffusion-limited reaction of ·NO and O
2⨪
during activation of phagocytes. Chronic inflammation induced by phagocytes is a major contributor to cancer and other degenerative diseases. We examined how γ-tocopherol (γT), the principal form of vitamin E in the United States diet, and α-tocopherol (αT), the major form in supplements, protect against peroxynitrite-induced lipid oxidation. Lipid hydroperoxide formation in liposomes (but not isolated low-density lipoprotein) exposed to peroxynitrite or the ·NO and O
2⨪
generator SIN-1 (3-morpholinosydnonimine) was inhibited more effectively by γT than αT. More importantly, nitration of γT at the nucleophilic 5-position, which proceeded in both liposomes and human low density lipoprotein at yields of ≈50% and ≈75%, respectively, was not affected by the presence of αT. These results suggest that despite αT’s action as an antioxidant γT is required to effectively remove the peroxynitrite-derived nitrating species. We postulate that γT acts
in vivo
as a trap for membrane-soluble electrophilic nitrogen oxides and other electrophilic mutagens, forming stable carbon-centered adducts through the nucleophilic 5-position, which is blocked in αT. Because large doses of dietary αT displace γT in plasma and other tissues, the current wisdom of vitamin E supplementation with primarily αT should be reconsidered.
Discovery of three genes specifically expressed in human prostate by expressed sequence tag database analysis
Authors: Vasmatzis George , Essand Magnus , Brinkmann Ulrich , Lee Byungkook , Pastan Ira
Published: July 2002

A procedure is described to discover genes that are specifically expressed in human prostate. The procedure involves searching the expressed sequence tag (EST) database for genes that have many related EST sequences from human prostate cDNA libraries but none or few from nonprostate human libraries. The selected candidate EST clones were tested by RNA dot blots to examine tissue specificity and by Northern blots to examine the transcript size of the corresponding mRNA. The computer analysis identified 15 promising genes that were previously unidentified. When seven of these were examined in an RNA hybridization experiment, three were found to be prostate specific. The genes identified could be useful in the targeted therapy of prostate cancer. The procedure can easily be applied to discover genes specifically expressed in other organs or tumors.
Genetic, biochemical, and crystallographic characterization of Fhit–substrate complexes as the active signaling form of Fhit
Authors: Pace Helen C. , Garrison Preston N. , Robinson Angela K. , Barnes Larry D. , Draganescu Alexandra , Rösler Angelika , Blackburn G. Michael , Siprashvili Zurab , Croce Carlo M. , Huebner Kay , Brenner Charles
Published: July 2002

Alterations in the
FHIT
gene at 3p14.2 occur as early and frequent events in the development of several common human cancers. The ability of human Fhit-negative cells to form tumors in nude mice is suppressed by stable reexpression of Fhit protein. Fhit protein is a diadenosine
P
1
,
P
3
-triphosphate (ApppA) hydrolase whose fungal and animal homologs form a branch of the histidine triad (HIT) superfamily of nucleotide-binding proteins. Because the His-96 → Asn substitution of Fhit, which retards ApppA hydrolase activity by seven orders of magnitude, did not block tumor-suppressor activity
in vivo
, we determined whether this mutation affected ApppA binding or particular steps in the ApppA catalytic cycle. Evidence is presented that His-96 → Asn protein binds ApppA well and forms an enzyme-AMP intermediate extremely poorly, suggesting that Fhit–substrate complexes are the likely signaling form of the enzyme. The cocrystal structure of Fhit bound to Ado-p-CH
2
-p-p
s
-Ado (IB2), a nonhydrolyzable ApppA analog, was refined to 3.1 Å, and the structure of His-96 → Asn Fhit with IB2 was refined to 2.6 Å, revealing that two ApppA molecules bind per Fhit dimer; identifying two additional adenosine-binding sites on the dimer surface; and illustrating that His-98 is positioned to donate a hydrogen bond to the scissile bridging oxygen of ApppA substrates. The form of Fhit bound to two ApppA substrates would present to the cell a dramatically phosphorylated surface, prominently displaying six phosphate groups and two adenosine moieties in place of a deep cavity lined with histidines, arginines, and glutamines.
Measuring genome evolution
Authors: Huynen Martijn A. , Bork Peer
Published: July 2002

The determination of complete genome sequences provides us with an
opportunity to describe and analyze evolution at the comprehensive
level of genomes. Here we compare nine genomes with respect to their
protein coding genes at two levels: (
i
) we compare
genomes as “bags of genes” and measure the fraction of orthologs
shared between genomes and (
ii
) we quantify correlations
between genes with respect to their relative positions in genomes.
Distances between the genomes are related to their divergence times,
measured as the number of amino acid substitutions per site in a set of
34 orthologous genes that are shared among all the genomes compared. We
establish a hierarchy of rates at which genomes have changed during
evolution. Protein sequence identity is the most conserved, followed by
the complement of genes within the genome. Next is the degree of
conservation of the order of genes, whereas gene regulation appears to
evolve at the highest rate. Finally, we show that some genomes are more
highly organized than others: they show a higher degree of the
clustering of genes that have orthologs in other genomes.
Identification of rat susceptibility loci for adjuvant-oil-induced arthritis
Authors: Lorentzen Johnny C. , Glaser Anna , Jacobsson Lena , Galli Joakim , Fakhrai-rad Hossein , Klareskog Lars , Luthman Holger
Published: July 2002

One intradermal injection of incomplete Freund’s adjuvant-oil induces a T cell-mediated inflammatory joint disease in DA rats. Susceptibility genes for oil-induced arthritis (OIA) are located both within and outside the major histocompatibility complex (MHC,Oia1). We have searched for disease-linked non-MHC loci in an F2intercross between DA rats and MHC-identical but arthritis-resistant LEW.1AV1 rats. A genome-wide scan with microsatellite markers revealed two major chromosome regions that control disease incidence and severity:Oia2on chromosome 4 (P= 4 × 10−13) andOia3on chromosome 10 (P= 1 × 10−6). All animals homozygous for DA alleles at both loci developed severe arthritis, whereas all those homozygous for LEW.1AV1 alleles were resistant. These results have general implications for situations where nonspecific activation of the immune system (e.g., incomplete Freund’s adjuvant-oil) causes inflammation and disease, either alone or in conjunction with specific antigens. They may also provide clues to the etiology of inflammatory diseases in humans, including rheumatoid arthritis.
Characterization of mice deficient in aromatase (ArKO) because of targeted disruption of the
cyp19
gene
Authors: Fisher Carolyn R. , Graves Kathy H. , Parlow Albert F. , Simpson Evan R.
Published: July 2002

The formation of estrogens from C
19
steroids is catalyzed by aromatase cytochrome P450 (P450arom), the product of the
cyp19
gene. The actions of estrogen include dimorphic anatomical, functional, and behavioral effects on the development of both males and females, considerations that prompted us to examine the consequences of deficiency of aromatase activity in mice. Mice lacking a functional aromatase enzyme (ArKO) were generated by targeted disruption of the
cyp19
gene. Male and female ArKO mice were born with the expected Mendelian frequency from F
1
parents and grew to adulthood. Female ArKO mice at 9 weeks of age displayed underdeveloped external genitalia and uteri. Ovaries contained numerous follicles with abundant granulosa cells and evidence of antrum formation that appeared arrested before ovulation. No corpora lutea were present. Additionally the stroma were hyperplastic with structures that appeared to be atretic follicles. Development of the mammary glands approximated that of a prepubertal female. Examination of male ArKO mice of the same age revealed essentially normal internal anatomy but with enlargement of the male accessory sex glands because of increased content of secreted material. The testes appeared normal. Male ArKO mice are capable of breeding and produce litters of approximately average size. Whereas serum estradiol levels were at the limit of detection, testosterone levels were elevated, as were the levels of follicle-stimulating hormone and luteinizing hormone. The phenotype of these animals differs markedly from that of the previously reported ERKO mice, in which the estrogen receptor α is deleted by targeted disruption.
Human CUL1 forms an evolutionarily conserved ubiquitin ligase complex (SCF) with SKP1 and an F-box protein
Authors: Lyapina Svetlana A. , Correll Craig C. , Kipreos Edward T. , Deshaies Raymond J.
Published: July 2002

The SCF ubiquitin ligase complex of budding yeast triggers DNA replication by catalyzing ubiquitination of the S phase cyclin-dependent kinase inhibitor SIC1. SCF is composed of three proteins—ySKP1, CDC53 (Cullin), and the F-box protein CDC4—that are conserved from yeast to humans. As part of an effort to identify components and substrates of a putative human SCF complex, we isolated
hSKP1
in a two-hybrid screen with
hCUL1,
the closest human homologue of
CDC53
. Here, we show that hCUL1 associates with hSKP1
in vivo
and directly interacts with both hSKP1 and the human F-box protein SKP2
in vitro
, forming an SCF-like particle. Moreover,
hCUL1
complements the growth defect of yeast
cdc53
ts
mutants, associates with ubiquitination-promoting activity in human cell extracts, and can assemble into functional, chimeric ubiquitin ligase complexes with yeast SCF components. Taken together, these data suggest that hCUL1 functions as part of an SCF ubiquitin ligase complex in human cells. Further application of biochemical assays similar to those described here can now be used to identify regulators/components of hCUL1-based SCF complexes, to determine whether the hCUL2–hCUL5 proteins also are components of ubiquitin ligase complexes in human cells, and to screen for chemical compounds that modulate the activities of the hSKP1 and hCUL1 proteins.
Control of interferon-τ gene expression by Ets-2
Authors: Ezashi Toshihiko , Ealy Alan D. , Ostrowski Michael C. , Roberts R. Michael
Published: July 2002

Expression of the multiple interferon-τ (IFN-τ) genes is restricted to embryonic trophectoderm of ruminant ungulate species for a few days in early pregnancy. The promoter regions of these genes are highly conserved. A proximal (bp −91 to −69) sequence has been implicated in controlling trophoblast-specific expression. Here it was used as a target for yeast one-hybrid screening of a day 13 conceptus cDNA library. Two transcription factors of the Ets family, Ets-2 and GABPα, were identified, consistent with the observation that active ovine IFN-τ genes contain a single 10-bp Ets motif (core: GGAA) in the proximal segment, whereas three known inactive ovine genes contain a mutated core motif (
T
GAA). Cotransfection of a promoter- (−126 to +50) luciferase reporter construct from an active gene (
bovineIFN-τ
1;
boIFNT
1) and an Ets-2 expression plasmid in human JAr cells provided up to a 30-fold increase in reporter expression, whereas promoters from inactive genes were not transactivated. GABPα alone was ineffective and had only a ≈2-fold positive effect when coexpressed with its partner GABPβ. Other Ets-related transcription factors, which were not detected in the genetic screen, also provided a range of lesser transactivation effects. Coexpression of Ets-2 and activated Ras failed to transactivate the
IFNT
promoter greater than Ets-2 alone in JAr cells. The presence of Ets-2 in nuclei of embryonic trophectoderm was confirmed immunocytochemically. Together, these data suggest that Ets-2 plays a role in the transient expression of the nonvirally inducible
IFNT
genes.
The proton collecting function of the inner surface of cytochromecoxidase fromRhodobacter sphaeroides
Authors: Marantz Yael , Nachliel Esther , Aagaard Anna , Brzezinski Peter , Gutman Menachem
Published: July 2002

The experiments presented in this study address the problem of how the cytoplasmic surface (proton-input side) of cytochromecoxidase interacts with protons in the bulk. For this purpose, the cytoplasmic surface of the enzyme was labeled with a fluorescein (Flu) molecule covalently bound to Cys223 of subunit III. Using the Flu as a proton-sensitive marker on the surface and φOH as a soluble excited-state proton emitter, the dynamics of the acid-base equilibration between the surface and the bulk was measured in the time-resolved domain. The results were analyzed by using a rigorous kinetic analysis that is based on numeric integration of coupled nonliner differential rate equations in which the rate constants are used as adjustable parameters. The analysis of 11 independent measurements, carried out under various initial conditions, indicated that the protonation of the Flu proceeds through multiple pathways involving diffusion-controlled reactions and proton exchange among surface groups. The surface of the protein carries an efficient system made of carboxylate and histidine moieties that are sufficiently close to each other as to form a proton-collecting antenna. It is the passage of protons among these sites that endows cytochromecoxidase with the capacity to pick up protons from the buffered cytoplasmic matrix within a time frame compatible with the physiological turnover of the enzyme.
Nitrilase and Fhit homologs are encoded as fusion proteins in
Drosophila melanogaster
and
Caenorhabditis elegans
Authors: Pekarsky Yuri , Campiglio Manuela , Siprashvili Zurab , Druck Teresa , Sedkov Yurii , Tillib Sergei , Draganescu Alexandra , Wermuth Peter , Rothman Joel H. , Huebner Kay , Buchberg Arthur M. , Mazo Alexander , Brenner Charles , Croce Carlo M.
Published: July 2002

The tumor suppressor gene
FHIT
encompasses the common human chromosomal fragile site at 3p14.2 and numerous cancer cell biallelic deletions. To study
Fhit
function we cloned and characterized
FHIT
genes from
Drosophila melanogaster
and
Caenorhabditis elegans
. Both genes code for fusion proteins in which the Fhit domain is fused with a novel domain showing homology to bacterial and plant nitrilases; the
D. melanogaster
fusion protein exhibited diadenosine triphosphate (ApppA) hydrolase activity expected of an authentic Fhit homolog. In human and mouse, the nitrilase homologs and Fhit are encoded by two different genes:
FHIT
and
NIT1
, localized on chromosomes 3 and 1 in human, and 14 and 1 in mouse, respectively. We cloned and characterized human and murine
NIT1
genes and determined their exon-intron structure, patterns of expression, and alternative processing of their mRNAs. The tissue specificity of expression of murine
Fhit
and
Nit1
genes was nearly identical. Because fusion proteins with dual or triple enzymatic activities have been found to carry out specific steps in a given biochemical or biosynthetic pathway, we postulate that Fhit and Nit1 likewise collaborate in a biochemical or cellular pathway in mammalian cells.
The degree of extra-pair paternity increases with genetic variability
Authors: Petrie Marion , Doums Claudie , Møller Anders Pape
Published: July 2002

The amount of extra-pair paternity in socially monogamous bird species varies from 0% to 76% extra-pair offspring. The causes of this remarkable interspecific variation are largely unknown, although intraspecific analyses suggest that females seek extra-pair matings to improve the genetic quality of their offspring. If this is a general explanation for the occurrence of extra-pair matings, then proportionally more females should seek to modify the paternity of their clutch when there is more variation among males in their genetic quality. Here we test this prediction in birds and show that interspecific variation in the proportion of extra-pair offspring is positively related to the proportion of polymorphic loci as measured by protein electrophoresis, even when controlling for potentially confounding variables. Genetic variability was also assessed, for sister pairs of species and populations differing significantly in extra-pair paternity, by using random priming, which provides an estimate of genome-wide diversity. We found that genetic diversity was higher in the populations with a higher level of extra-pair paternity. These results suggest that the amount of genetic variability in a population may be an important factor influencing mating patterns.
Hypoxia stimulates insulin-like growth factor binding protein 1 (IGFBP-1) gene expression in HepG2 cells: A possible model for IGFBP-1 expression in fetal hypoxia
Authors: Tazuke Salli I. , Mazure Natalie M. , Sugawara Junichi , Carland Grace , Faessen Gerry H. , Suen Lii-Feng , Irwin Juan C. , Powell David R. , Giaccia Amato J. , Giudice Linda C.
Published: July 2002

IGFBP-1 is elevated in fetuses with long-term, chronic hypoxia and intrauterine growth restriction. We investigated the hypothesis that hypoxia regulates IGFBP-1 in the human fetus
in vivo
and
IGFBP-1
gene expression and protein
in vitro
. Umbilical artery IGFBP-1 levels (mean ± SEM) from term babies with respiratory acidosis (acute hypoxia), normal babies, and those with mixed respiratory/metabolic acidosis (more profound and prolonged hypoxia) were measured using an immunoradiometric assay. IGFBP-1 levels were similar in normal (
n
= 12) and acutely hypoxic (
n
= 6) babies (189.1 ± 71.8 vs. 175.8 ± 45.9 ng /ml, respectively,
P
= 0.789). However, with more profound and prolonged hypoxia (
n
= 19), IGFBP-1 levels were markedly elevated (470.6 ± 80.0 ng /ml,
P
= 0.044). To investigate IGFBP-1 regulation by hypoxia
in vitro,
HepG2 cells were incubated under hypoxia (pO
2
= 2%) and normoxia (pO
2
= 20%). IGFBP-1 protein and mRNA increased 8- and 12-fold, respectively, under hypoxic conditions. Hypoxia did not affect protein or mRNA levels of IGFBP-2 or -4. IGFBP-5 and -6 mRNAs, undetectable in control cells, were not induced by hypoxia, whereas minimally expressed IGFBP-3 mRNA increased twofold. Investigation into
IGFBP-1
gene structure revealed three potential consensus sequences for the hypoxia response element (HRE) in the first intron. To investigate functionality, a 372-bp fragment of IGFBP-1 intron 1, containing putative HREs, was placed 5′ to a heterologous hsp70 promoter in a plasmid using luciferase as a reporter gene. Under hypoxia, reporter gene activity increased up to 30-fold. Mutations in the middle HRE abolished reporter activity in response to hypoxia, suggesting that this HRE is functional in the IGFBP-1 hypoxia response. Cotransfection of HRE reporter genes with a constitutively expressing hypoxia-inducible factor 1 plasmid in HepG2 cells resulted in a fourfold induction of reporter activity, suggesting a role for hypoxia-inducible factor 1 in hypoxia induction of
IGFBP-1
gene expression. These data support the hypothesis that hypoxia regulation of IGFBP-1 may be a mechanism operating in the human fetus to restrict insulin-like growth factor-mediated growth
in utero
under conditions of chronic hypoxia and limited substrate availability.
Identification of an UP element consensus sequence for bacterial promoters
Authors: Estrem Shawn T. , Gaal Tamas , Ross Wilma , Gourse Richard L.
Published: July 2002

The UP element, a component of bacterial promoters located upstream of the −35 hexamer, increases transcription by interacting with the RNA polymerase α-subunit. By using a modification of the SELEX procedure for identification of protein-binding sites, we selected
in vitro
and subsequently screened
in vivo
for sequences that greatly increased promoter activity when situated upstream of the
Escherichia coli rrnB
P1 core promoter. A set of 31 of these upstream sequences increased transcription from 136- to 326-fold
in vivo
, considerably more than the natural
rrnB
P1 UP element, and was used to derive a consensus sequence: −59 nnAAA(A/T)(A/T)T(A/T)TTTTnnAAAAnnn −38. The most active selected sequence contained the derived consensus, displayed all of the properties of an UP element, and the interaction of this sequence with the α C-terminal domain was similar to that of previously characterized UP elements. The identification of the UP element consensus should facilitate a detailed understanding of the α–DNA interaction. Based on the evolutionary conservation of the residues in α responsible for interaction with UP elements, we suggest that the UP element consensus sequence should be applicable throughout eubacteria, should generally facilitate promoter prediction, and may be of use for biotechnological applications.
A general procedure for locating and analyzing protein-binding sequence motifs in nucleic acids
Authors: O’Neill Michael C.
Published: July 2002

In the last decade, two tools, one drawn from information theory and the other from artificial neural networks, have proven particularly useful in many different areas of sequence analysis. The work presented herein indicates that these two approaches can be joined in a general fashion to produce a very powerful search engine that is capable of locating members of a given nucleic acid sequence family in either local or global sequence searches. This program can, in turn, be queried for its definition of the motif under investigation, ranking each base in context for its contribution to membership in the motif family. In principle, the method used can be applied to any binding motif, including both DNA and RNA sequence families, given sufficient family size.
Chromosomal transposition of a Tc1/mariner-like element in mouse embryonic stem cells
Authors: Luo Guangbin , Ivics Zoltán , Izsvák Zsuzsanna , Bradley Allan
Published: July 2002

Mouse has become an increasingly important organism for modeling human diseases and for determining gene function in a mammalian context. Unfortunately, transposon-tagged mutagenesis, one of the most valuable tools for functional genomics, still is not available in this organism. On the other hand, it has long been speculated that members of the
Tc1/mariner
-like elements may be less dependent on host factors and, hence, can be introduced into heterologous organisms. However, this prediction has not been realized in mice. We report here the chromosomal transposition of the
Sleeping Beauty
(
SB
) element in mouse embryonic stem cells, providing evidence that it can be used as an
in vivo
mutagen in mice.
Oligomerization domain-directed reassembly of active dihydrofolate reductase from rationally designed fragments
Authors: Pelletier Joelle N. , Campbell-Valois F.-X. , Michnick Stephen W.
Published: July 2002

Reassembly of enzymes from peptide fragments has been used as a strategy for understanding the evolution, folding, and role of individual subdomains in catalysis and regulation of activity. We demonstrate an oligomerization-assisted enzyme reassembly strategy whereby fragments are covalently linked to independently folding and interacting domains whose interactions serve to promote efficient refolding and complementation of fragments, forming active enzyme. We show that active murine dihydrofolate reductase (E.C. 1.5.1.3) can be reassembled from complementary N- and C-terminal fragments when fused to homodimerizing GCN4 leucine zipper-forming sequences as well as heterodimerizing protein partners. Reassembly is detected by an
in vivo
selection assay in
Escherichia coli
and
in vitro
. The effects of mutations that disrupt fragment affinity or enzyme activity were assessed. The steady–state kinetic parameters for the reassembled mutant (Phe-31 → Ser) were determined; they are not significantly different from the full-length mutant. The strategy described here provides a general approach for protein dissection and domain swapping studies, with the capacity both for rapid
in vivo
screening as well as
in vitro
characterization. Further, the strategy suggests a simple
in vivo
enzyme-based detection system for protein–protein interactions, which we illustrate with two examples: ras–GTPase and raf–ras-binding domain and FK506-binding protein-rapamycin complexed with the target of rapamycin TOR2.
CXCR4 and CD4 mediate a rapid CD95-independent cell death in CD4+T cells
Authors: Berndt Christina , Möpps Barbara , Angermüller Sabine , Gierschik Peter , Krammer Peter H.
Published: July 2002

AIDS is characterized by a progressive decrease of CD4+helper T lymphocytes. Destruction of these cells may involve programmed cell death, apoptosis. It has previously been reported that apoptosis can be induced even in noninfected cells by HIV-1 gp120 and anti-gp120 antibodies. HIV-1 gp120 binds to T cells via CD4 and the chemokine coreceptor CXCR4 (fusin/LESTR). Therefore, we investigated whether CD4 and CXCR4 mediate gp120-induced apoptosis. We used human peripheral blood lymphocytes, malignant T cells, and CD4/CXCR4 transfectants, and found cell death induced by both cell surface receptors, CD4 and CXCR4. The induced cell death was rapid, independent of known caspases, and lacking oligonucleosomal DNA fragmentation. In addition, the death signals were not propagated via p56lckand Giα. However, the cells showed chromatin condensation, morphological shrinkage, membrane inversion, and reduced mitochondrial transmembrane potential indicative of apoptosis. Significantly, apoptosis was exclusively observed in CD4+but not in CD8+T cells, and apoptosis triggered via CXCR4 was inhibited by stromal cell-derived factor-1, the natural CXCR4 ligand. Thus, this mechanism of apoptosis might contribute to T cell depletion in AIDS and might have major implications for therapeutic intervention.