bluuebunny/crossref_metadata_2025 huggingface dataset:
At day 8-8.5 of mouse development the yolk sac, not the embryo proper, has lymphoid precursor potential in vivo and in vitro.
Authors: Palacios R , Imhof B A
Published: May 2006

We have studied both in vitro and in vivo the formation of lymphocyte progenitors before blood circulation (day 9 of gestation) has started in the mouse embryo, and we have determined the tissue where this occurs. The results demonstrate that the yolk sac of embryos at day 8 and day 8.5 of gestation contains precursor cells that can give rise, in vivo and in vitro, to mature T and B lymphocytes. No lymphoid precursors were found in the embryo proper at this stage of mouse development. The yolk sac cells with lymphocyte precursor potential are most likely multipotent stem cells rather than cell-lineage-determined T- and/or B-lymphocyte progenitors. The defined in vitro assays described here that support differentiation of yolk sac stem cells along the T- or B-lymphocyte pathways also may now facilitate the study of the molecular events leading to cell-lineage commitment of lymphocyte progenitors in the mouse embryo.
Human T-cell clonal anergy is induced by antigen presentation in the absence of B7 costimulation.
Authors: Gimmi C D , Freeman G J , Gribben J G , Gray G , Nadler L M
Published: May 2006

The maximal T-cell response to its antigen requires presentation of the antigen by a major histocompatibility complex class II molecule as well as the delivery of one or more costimulatory signals provided by the antigen-presenting cell (APC). Although a number of candidate molecules have been identified that are capable of delivering a costimulatory signal, increasing evidence suggests that one such critical pathway involves the interaction of the T-cell surface antigen CD28 with its ligand B7, expressed on APCs. In view of the number of potential costimulatory molecules that might be expressed on the cell surface of APCs, artificial APCs were constructed by stable transfection of NIH 3T3 cells with HLA-DR7, B7, or both. Here, we show that in a human antigen-specific model system, when tetanus toxoid peptide antigen is presented by cells cotransfected with HLA-DR7 and B7, optimal T-cell proliferation and interleukin 2 production result. In contrast, antigen presentation, in the absence of B7 costimulation, results in T-cell clonal anergy. These results demonstrate that it is possible to induce antigen-specific clonal tolerance in human T cells that have been previously sensitized to antigen. The artificial antigen-presenting system provides a useful model for the investigation of the biochemical events involved in the generation of tolerance and for the study of signals necessary to overcome tolerance.
A mutant alpha subunit of G12 potentiates the eicosanoid pathway and is highly oncogenic in NIH 3T3 cells.
Authors: Xu N , Bradley L , Ambdukar I , Gutkind J S
Published: May 2006

The discovery of GTPase-inhibiting mutations in genes for alpha subunits of Gs or G(i2) in certain human endocrine tumors has raised the possibility that heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) might contribute to neoplastic disease. Expression of GTPase-deficient alpha s or alpha i2 polypeptides in rodent fibroblasts increases or decreases cAMP, respectively, and induces certain alterations in cell growth but only a few of the phenotypic changes associated with cellular transformation. In contrast, an analogous mutation in the alpha subunit of Gq, which activates phosphatidylinositol (PI)-specific phospholipase C, is fully oncogenic. However, activated alpha q is cytotoxic and several orders of magnitude less potent as an oncogene than certain G protein-coupled receptors. Thus, G proteins other than those inducing PI hydrolysis might possess high transforming efficiency. In the present study, we explored the G12 family of G proteins for their oncogenic potential. Our results show that whereas overexpression of wild-type alpha 12 in NIH 3T3 cells is itself weakly transforming, an activated alpha 12 behaves as a remarkably potent oncogene. Transformation by alpha 12 correlates with alterations in the eicosanoid pathway but not with PI-specific phospholipase C or other G protein-linked second messengers.
Rare scleroderma autoantibodies to the U11 small nuclear ribonucleoprotein and to the trimethylguanosine cap of U small nuclear RNAs.
Authors: Gilliam A C , Steitz J A
Published: May 2006

We have identified a scleroderma serum (Ru) with a previously undescribed specificity to protein components of the U11 small nuclear ribonucleoprotein particle (snRNP), a low-abundance member of the Sm class of U RNPs. The U11 RNP can be specifically immunoprecipitated from sonicated HeLa cells with Ru serum. In nuclear extracts, a fraction of the U11 particle is found complexed to the U12 RNP, an even lower abundance Sm snRNP. In glycerol gradient fractions, Ru serum identifies a 65-kDa protein that cosediments with the U11-U12 complex and is shifted upon targeted degradation of the U12 RNA. The 65-kDa protein therefore appears to be a component of the U11-U12 snRNP complex, whereas another Ru-reactive (140 kDa) protein may be associated with the free U11 RNP. The Ru serum also contains autoantibodies directed against the trimethylguanosine cap of U RNAs. This rare specificity has been described previously in only three other scleroderma patients.
Properties and purification of an active biotinylated lactose permease from Escherichia coli.
Authors: Consler T G , Persson B L , Jung H , Zen K H , Jung K , Privé G G , Verner G E , Kaback H R
Published: May 2006

A simplified approach for purification of functional lactose permease from Escherichia coli is described that is based on the construction of chimeras between the permease and a 100-amino acid residue polypeptide containing the biotin acceptor domain from the oxaloacetate decarboxylase of Klebsiella pneumoniae [Cronan, J. E., Jr. (1990) J. Biol. Chem. 265, 10327-10333]. Chimeras were constructed with a factor Xa protease site and the biotin acceptor domain in the middle cytoplasmic loop (loop 6) or at the C terminus of the permease. Each construct catalyzes active lactose transport in cells and right-side-out membrane vesicles. Moreover, the constructs are biotinylated in vivo, and in both chimeras, the factor Xa protease site is accessible from the cytoplasmic surface of the membrane. Both biotinylated permeases bind selectively to immobilized monomeric avidin and are eluted with free biotin in a high state of purity, and the loop 6 chimera catalyzes active transport after reconstitution into proteoliposomes. The methodology described should be applicable to other membrane proteins.
The cAMP-response-element-binding protein interacts, but Fos protein does not interact, with the proenkephalin enhancer in rat striatum.
Authors: Konradi C , Kobierski L A , Nguyen T V , Heckers S , Hyman S E
Published: May 2006

The proenkephalin gene is a well-studied model of transcription factor-target gene interaction in the nervous system and has been proposed as a regulatory target of the protein product of the immediate-early gene c-fos. This regulatory mechanism has been proposed, in part, because the cAMP response element 2 (CRE-2) site, the key DNA regulatory element within the proenkephalin second-messenger-inducible enhancer, avidly binds AP-1 proteins, including Fos, in vitro. However, we observe a dissociation in the time course of activation of c-fos and proenkephalin mRNA in rat striatum after administration of the dopamine D2 receptor antagonist haloperidol. This result prompted us to investigate the composition of protein complexes in striatal nuclear extracts that bind to the CRE-2 site. Even though our striatal nuclear extracts had substantial basal and haloperidol-inducible AP-1-binding activities that contained Fos, we could not detect Fos in complexes bound to the CRE-2 element. Instead, as determined by antibody supershift analysis, we detect CRE-binding protein (CREB)-like proteins binding to CRE-2 in both basal and haloperidol-stimulated conditions. Finally, we show that haloperidol induces CREB protein phosphorylation in striatum.
Guanosine pentaphosphate phosphohydrolase of Escherichia coli is a long-chain exopolyphosphatase.
Authors: Keasling J D , Bertsch L , Kornberg A
Published: May 2006

An exopolyphosphatase [exopoly(P)ase; EC 3.6.1.11] activity has recently been purified to homogeneity from a mutant strain of Escherichia coli which lacks the principal exopoly(P)ase. The second exopoly(P)ase has now been identified as guanosine pentaphosphate phosphohydrolase (GPP; EC 3.6.1.40) by three lines of evidence: (i) the sequences of five tryptic digestion fragments of the purified protein are found in the translated gppA gene, (ii) the size of the protein (100 kDa) agrees with published values for GPP, and (iii) the ratio of exopoly(P)ase activity to GPP activity remains constant throughout a 300-fold purification in the last steps of the procedure. The enzyme liberates orthophosphate by processive hydrolysis of the phosphoanyhydride bonds of polyphosphate [poly(P)] chains (1000 residues) or by hydrolysis of the 5'-gamma-phosphate of guanosine 5'-triphosphate 3'-diphosphate (pppGpp) to guanosine 5'-diphosphate 3'-diphosphate (ppGpp or "magic spot"). The Km for long-chain poly(P) as a substrate (approximately 0.5 nM) is far lower than that for pppGpp (0.13 mM); the kcat for the poly(P)ase activity is 1.1 s-1 and that for pppGpp hydrolase is 0.023 s-1. These and other findings direct attention to possible functions of poly(P) in the response of E. coli to stresses and deprivations.
Stabilization and ribosome association of unspliced pre-mRNAs in a yeast upf1- mutant.
Authors: He F , Peltz S W , Donahue J L , Rosbash M , Jacobson A
Published: May 2006

Nonsense-mediated mRNA decay, the accelerated turnover of mRNAs transcribed from genes containing early nonsense mutations, is dependent on the product of the UPF1 gene in yeast. Mutations that inactivate UPF1 lead to the selective stabilization of mRNAs containing early nonsense mutations but have no effect on the half-lives of almost all other mRNAs. Since the transcripts of nonsense alleles are not typical cellular constituents, we sought to identify those RNAs that comprise normal substrates of the nonsense-mediated mRNA decay pathway. Many yeast pre-mRNAs contain early in-frame nonsense codons and we consider it possible that a role of this pathway is to accelerate the degradation of pre-mRNAs present in the cytoplasm. Consistent with this hypothesis, we find that, in a strain lacking UPF1 function, the CYH2, RP51B, and MER2 pre-mRNAs are stabilized 2- to 5-fold and are associated with ribosomes. We conclude that a major source of early nonsense codon-containing cytoplasmic transcripts in yeast is pre-mRNAs and that the UPF1 protein may be part of a cellular system that ensures that potentially deleterious nonsense fragments of polypeptides do not accumulate.
A base-pairing interaction between U2 and U6 small nuclear RNAs occurs in > 150S complexes in HeLa cell extracts: implications for the spliceosome assembly pathway.
Authors: Wassarman D A , Steitz J A
Published: May 2006

In mammalian cells, base pairing between the U2 and U6 small nuclear RNAs is required during pre-RNA splicing. We show by psoralen crosslinking of HeLa nuclear extract that U2.U6 base pairing occurs within abundant ribonucleoprotein complexes that sediment at > 150 S in glycerol gradients. All of the spliceosomal RNAs (U1, U2, U4, U5, and U6) cosediment with these large complexes, suggesting that they may be related to small nuclear RNA-containing structures called speckles/coiled bodies or snurposomes, which have been visualized in mammalian or amphibian nuclei, respectively. In contrast to nuclear extract, S100 extract, which is splicing-defective and lacks the > 150S complexes, does not contain base-paired U2.U6. However, U2.U6 base pairs form in S100 extract that has been made splicing-competent by supplementation with Ser/Arg-rich (SR) proteins, ATP, and an adenovirus splicing substrate. During splicing in supplemented S100 extract, U2.U6 base pairing precedes the appearance of splicing intermediates and occurs initially in an approximately 60S spliceosome complex but also in progressively larger (100-300 S) complexes. Possible functional relationships between the 60S spliceosome and the > 150S complexes are discussed.
The 31-kDa precursor of interleukin 1 alpha is myristoylated on specific lysines within the 16-kDa N-terminal propiece.
Authors: Stevenson F T , Bursten S L , Fanton C , Locksley R M , Lovett D H
Published: May 2006

The cytokine interleukin 1 alpha (IL-1 alpha) is a critical mediator of the immune and inflammatory responses. A unique determinant of its activity as compared with IL-1 beta may be its association with the plasma membrane. While the biologic activity of "membrane IL-1" has been extensively reported, the mechanism of membrane binding remains unclear. We report that the N terminus of the 31-kDa IL-1 alpha precursor is myristoylated on specific internal lysine residues. Immunoprecipitation of [3H]myristic acid-radiolabeled human monocyte lysates with IgG antibodies to the 31-kDa IL-1 alpha precursor recovered a protein with the physicochemical properties of the IL-1 alpha N-terminal propiece (16 kDa, pI 4.45). Glycyl N-myristoylation of this protein is precluded by the absence of a glycine residue at position 2, suggesting that the propiece is myristoylated on epsilon-amino groups of lysine. To determine which lysine(s) are acylated, a series of synthetic peptides containing all lysines found in the IL-1 alpha N-terminal propiece were used in an in vitro myristoylation assay containing peptide, myristoyl-CoA, and monocyte lysate as enzyme source. Analysis of the reaction products by reverse-phase HPLC and gas-phase sequencing demonstrated the specific myristoylation of Lys-82 and Lys-83, yielding predominantly monoacylated product. A conserved sequence in the IL-1 beta propiece was myristoylated with at least 8-fold less efficiency. Acylation of the IL-1 alpha precursor by a previously unrecognized lysyl epsilon-amino N-myristoyl-transferase activity may facilitate its specific membrane targeting.
Crystal structures of medium-chain acyl-CoA dehydrogenase from pig liver mitochondria with and without substrate.
Authors: Kim J J , Wang M , Paschke R
Published: May 2006

The three-dimensional structure of medium-chain acyl-CoA dehydrogenase from pig mitochondria in the native form and that of a complex of the enzyme and a substrate (product) have been solved and refined by x-ray crystallographic methods at 2.4-A resolution to R factors of 0.172 and 0.173, respectively. The overall polypeptide folding and the quaternary structure of the tetramer are essentially unchanged upon binding of the ligand, octanoyl (octenoyl)-CoA. The ligand binds to the enzyme at the rectus (re) face of the FAD in the crevice between the two alpha-helix domains and the beta-sheet domain of the enzyme. The fatty acyl chain of the thioester substrate is buried inside of the polypeptide and the 3'-AMP moiety is close to the surface of the tetrameric enzyme molecule. The alkyl chain displaces the tightly bound water molecules found in the native enzyme and the carbonyl oxygen of the thioester interacts with the ribityl 2'-hydroxyl group of the FAD and the main-chain carbonyl oxygen of Glu-376. The C alpha--C beta of the fatty acyl moiety lies between the flavin and the gamma-carboxylate of Glu-376, supporting the role of Glu-376 as the base that abstracts the alpha proton in the alpha--beta dehydrogenation reaction catalyzed by the enzyme. Trp-166 and Met-165 are located at the sinister (si) side of the flavin ring at the surface of the enzyme, suggesting that they might be involved in the interactions with electron transferring flavoprotein. Lys-304, the prevalent mutation site found in patients with medium-chain acyl-CoA dehydrogenase deficiency, is located approximately 20 A away from the active site of the enzyme.
Functional analysis of Arabidopsis thaliana rRNA gene and spacer promoters in vivo and by transient expression.
Authors: Doelling J H , Gaudino R J , Pikaard C S
Published: May 2006

In eukaryotes, RNA polymerase I transcription is controlled by DNA elements located within the spacers that separate the tandemly arranged rRNA genes. Unlike rRNA coding sequences, the intergenic spacers evolve rapidly and have little sequence similarity even among closely related species. Nonetheless, the arrangement of functional elements, such as spacer promoters and enhancers, is thought to be highly conserved. Here, we identify spacer promoters in the plant Arabidopsis thaliana, thereby demonstrating their existence in both the plant and animal kingdoms. We also use an Arabidopsis transient expression system to perform transcriptional analysis of the ribosomal gene promoter. Spacer promoters share sequence similarity with the gene promoter from -91 to +22 relative to the transcription start site, +1. Deletion analysis shows that sequences required for RNA polymerase I transcription reside within these boundaries. Spacer sequences upstream of the gene promoter have only a small positive effect on transcription in transfected protoplasts but can increase transcription from a Xenopus ribosomal gene promoter in injected frog oocytes. This trans-kingdom enhancer effect further suggests that the functional elements within eukaryotic ribosomal genes are highly conserved.
Amplification of a DEAD box protein gene in retinoblastoma cell lines.
Authors: Godbout R , Squire J
Published: May 2006

DEAD box proteins, characterized by the conserved motif Asp-Glu-Ala-Asp, are putative RNA helicases implicated in a number of cellular processes involving alteration of RNA secondary structure such as translation initiation, nuclear and mitochondrial splicing, and ribosome and spliceosome assembly. Based on their distribution patterns, some members of this family are believed to be involved in embryogenesis, spermatogenesis, and cellular growth and division. Here, we report that the mRNA encoding a DEAD box protein, designated HuDBP-RB, is present at elevated levels in two of six retinoblastoma (RB) cell lines tested and is preferentially expressed in fetal tissues of neuroectodermal origin. It is not possible to classify HuDBP-RB as a member of any of the DEAD box protein subgroups identified to date since the regions of amino acid similarity between HuDBP-RB and other DEAD box proteins are restricted to the conserved motifs found in all members of this family. The HuDBP-RB gene, which has been mapped to chromosome band 2p24, is amplified in the RB cell lines that overexpress HuDBP-RB RNA. Furthermore, the MYCN gene is also present in multiple copies in these two cell lines, suggesting coamplification of the two genes.
Transgenic mice containing a 248-kb yeast artificial chromosome carrying the human beta-globin locus display proper developmental control of human globin genes.
Authors: Peterson K R , Clegg C H , Huxley C , Josephson B M , Haugen H S , Furukawa T , Stamatoyannopoulos G
Published: May 2006

Transgenic mice were generated using a purified 248-kb yeast artificial chromosome (YAC) bearing an intact 82-kb human beta-globin locus and 148 kb of flanking sequence. Seventeen of 148 F0 pups were transgenic. RNase protection analysis of RNA isolated from the blood of 13 gamma- and beta-globin-positive founders showed that only the human beta-globin gene was expressed in the adult founders. Studies of F1 and F2 fetuses demonstrated that the genes of the beta-locus YAC displayed the proper developmental switches in beta-like globin gene expression. Expression of epsilon- and gamma-globin, but not beta-globin, was observed in the yolk sac, there was only minor gamma and mostly beta expression in the 14-day liver, and only beta mRNA in the blood of the adult animals. Structural data showed that the locus was intact. These results indicate that it is now possible to dissect regulatory mechanisms within the context of an entire locus in vivo by using the ability to perform mutagenesis efficiently in yeast via homologous recombination, followed by purification of the altered YAC and its introduction into mice.
Serine/threonine phosphorylation regulates binding of C hnRNP proteins to pre-mRNA.
Authors: Mayrand S H , Dwen P , Pederson T
Published: May 2006

The C hnRNP proteins bind to nascent pre-mRNA and are thought to participate in an early step of the pre-mRNA splicing pathway. We report here that C hnRNP proteins are phosphorylated by a casein kinase II activity in a HeLa cell nuclear extract and that dephosphorylation of C hnRNP proteins is inhibited by the specific protein-serine/threonine-phosphatase 1/2A inhibitor okadaic acid. We further find that dephosphorylation of C hnRNP proteins is required for their binding to adenovirus and human beta-globin pre-mRNAs. These results indicate that the participation of C hnRNP proteins in pre-spliceosome assembly is coupled to a dynamic cycle of their phosphorylation and dephosphorylation.
Polymer models for interphase chromosomes.
Authors: Hahnfeldt P , Hearst J E , Brenner D J , Sachs R K , Hlatky L R
Published: May 2006

The overall geometry of chromosomes in mammalian cells during interphase is analyzed. On scales larger than approximately 10(5) bp, a chromosome is modeled as a Gaussian polymer subjected to additional forces that confine it to a subvolume of the cell nucleus. An appropriate partial differential equation for the polymer Green's function leads to predictions for the average geometric length between two points on the chromosome. The model reproduces several of the experimental observations: (i) a square root dependence of average geometric distance between two marked chromosome locations on their genomic separation over genomic length scales from approximately 10(5) to approximately 10(6) bp; (ii) an approach of the geometric distance to a maximum value for still larger genomic separations of the two points; (iii) overall chromosome localization in subdomains of the cell nucleus, as suggested by fluorescent labeling of whole chromosomes and by radiobiological evidence. The model is also consistent with known properties of the 30-nm chromatin fiber. It makes a testable prediction: that for two markers a given number of base pairs apart on a given chromosome, the average geometric separation is larger if the configuration is near one end of the chromosome than if it is near the center of the chromosome.
Channel gating kinetics and synaptic efficacy: a hypothesis for expression of long-term potentiation.
Authors: Ambros-Ingerson J , Lynch G
Published: May 2006

A kinetic model of the glutamate DL-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor/channel complex was used to test whether changes in the rate constants describing channel behavior could account for various features of long-term potentiation (LTP). Starting values for the kinetic parameters were set to satisfy experimental data (e.g., affinity, mean open time, mean burst length, etc.) and physical constraints (i.e., microreversibility). The resultant model exhibited a variety of dynamic properties known to be associated with the receptor. Increasing the rate constants governing opening/closing of the channel produced an unexpected increase in the probability of the channel being open shortly after transmitter binding. This would account for the enhanced response size with LTP. Increases in rate constants produced two other aspects of LTP: (i) an alteration of the waveform of the synaptic response and (ii) an interaction with changes in desensitization kinetics. The results obtained with the model corresponded closely to those found in LTP experiments. Thus, an increase in opening/closing rates for the postsynaptic receptor channel provides a single explanation for diverse characteristics of LTP. Finally, the kinetic manipulation reduced the coefficient of variation of synaptic currents in a model involving 250 receptors. This calls into question the use of variance measures for distinguishing pre- vs. postsynaptic sites of potentiation.
Coordinated decreases in rRNA gene transcription factors and rRNA synthesis during muscle cell differentiation.
Authors: Larson D E , Xie W , Glibetic M , O'Mahony D , Sells B H , Rothblum L I
Published: May 2006

rRNA synthesis decreases significantly during the differentiation of rat L6 myoblasts to myotubes. Nuclear run-on assays demonstrated that the decrease was attributable to decreased rates of rRNA gene transcription. Immunoblot analysis indicated a marked reduction in amounts of the RNA polymerase I transcription factors UBF1 and UBF2 (upstream binding factors 1 and 2, respectively). The levels of these factors dropped in parallel with the down-shift in rRNA gene transcription. The amount of UBF does not fall due to a general decrease in cellular protein, as myosin heavy-chain protein accumulates markedly during this same time. RNA blots of total RNA isolated from myoblasts and differentiating myotubes showed a decrease in the mRNA for UBF, at the same time the mRNA for myogenin was accumulating. The down-shift in UBF mRNA levels preceded the decrease in the protein levels for UBF. There have been reports that the acute response of the rRNA gene transcription system to physiological signals in many systems involves an RNA polymerase I-associated factor. However, our results imply that the regulation of rRNA gene DNA transcription in response to physiological processes, such as differentiation, may involve multiple regulatory pathways.
Yeast and mammalian metallothioneins functionally substitute for yeast copper-zinc superoxide dismutase.
Authors: Tamai K T , Gralla E B , Ellerby L M , Valentine J S , Thiele D J
Published: May 2006

Copper-zinc superoxide dismutase catalyzes the disproportionation of superoxide anion to hydrogen peroxide and dioxygen and is thought to play an important role in protecting cells from oxygen toxicity. Saccharomyces cerevisiae strains lacking copper-zinc superoxide dismutase, which is encoded by the SOD1 gene, are sensitive to oxidative stress and exhibit a variety of growth defects including hypersensitivity to dioxygen and to superoxide-generating drugs such as paraquat. We have found that in addition to these known phenotypes, SOD1-deletion strains fail to grow on agar containing the respiratory carbon source lactate. We demonstrate here that expression of the yeast or monkey metallothionein proteins in the presence of copper suppresses the lactate growth defect and some other phenotypes associated with SOD1-deletion strains, indicating that copper metallothioneins substitute for copper-zinc superoxide dismutase in vivo to protect cells from oxygen toxicity. Consistent with these results, we show that yeast metallothionein mRNA levels are dramatically elevated under conditions of oxidative stress. Furthermore, in vitro assays demonstrate that yeast metallothionein, purified or from whole-cell extracts, exhibits copper-dependent antioxidant activity. Taken together, these data suggest that both yeast and mammalian metallothioneins may play a direct role in the cellular defense against oxidative stress by functioning as antioxidants.
Susceptibility to renal carcinoma in the Eker rat involves a tumor suppressor gene on chromosome 10.
Authors: Yeung R S , Buetow K H , Testa J R , Knudson A G
Published: May 2006

Germ-line mutations of tumor suppressor genes confer strong predisposition to tumor formation. In the rat, a form of dominantly inherited renal carcinoma (RC) results in multiple chromophobe cell tumors that resemble the human disease, and heterozygous carriers (RC/+) are highly susceptible to environmental agents (radiation and chemical carcinogens), making it a desirable model to study epithelial carcinogenesis. By linkage analysis, the locus of the inherited RC mutation was mapped to rat chromosomal band 10q12, near the protamine locus (logarithm of odds score = 17.96). Renal tumors also showed a loss of heterozygosity at this locus, lending support to the recessive nature of this putative tumor suppressor gene. Our result suggested that the human homolog of the RC gene may reside on human chromosome 16, not known to be altered commonly in human RC.
The E2A gene product contains two separable and functionally distinct transcription activation domains.
Authors: Aronheim A , Shiran R , Rosen A , Walker M D
Published: May 2006

The E2A gene encodes transcription factors of the helix-loop-helix (HLH) family which are implicated in cell-specific transcriptional control in several cell lineages, including pancreatic beta cells. In the present work, we show by deletion mapping of both the E2A protein itself and the Gal4-E2A fusion protein that the protein contains at least two distinct activation domains. One domain (located between amino acids 1 and 153) functions efficiently in a variety of mammalian cell lines. The second domain (located between amino acids 369 and 485) functions preferentially in pancreatic beta cell lines. The latter domain shows a pattern of heptad repeats of leucine residues characteristics of "leucine zipper" transcription factors; site-directed mutagenesis of leucines within this repeat led to substantial reductions in activity. The selective properties of this activation domain may contribute to cell-specific transcription directed by the E2A gene.
Radish, a Drosophila mutant deficient in consolidated memory.
Authors: Folkers E , Drain P , Quinn W G
Published: May 2006

We have characterized the behavior and genetics of the Drosophila mutant radish (rsh gene). Initial learning of radish flies in two olfactory discrimination tests is high, but subsequent memory decays rapidly at both early and late times after training. Anesthesia-resistant memory (consolidated memory) is undetectable in radish flies 3 hr after training. The mutant shows normal locomotor activity and normal sensitivity to the odor cues and electric-shock reinforcement used in the learning tests. The radish gene maps within a 180-kb interval in the 11D-E region of the X chromosome.
Glyceraldehyde-3-phosphate dehydrogenase on the surface of group A streptococci is also an ADP-ribosylating enzyme.
Authors: Pancholi V , Fischetti V A
Published: May 2006

We recently identified an enzymatically active glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12; GAPDH) as a major protein on the surface of group A streptococci (SDH), which exhibits multiple binding activity to various mammalian proteins. We now report that the SDH molecule also functions as an ADP-ribosylating enzyme, which, in the presence of NAD, is auto-ADP-ribosylated. In a crude cell wall extract of group A streptococci, SDH is the only protein that is ADP-ribosylated. SDH found in the streptococcal cytoplasmic fraction could not be ADP-ribosylated in the presence of NAD. Treatment of ADP-ribosylated SDH with the cytoplasmic fraction removed the ADP-ribose from SDH, suggesting the presence of an ADP-ribosyl hydrolase in the cytoplasmic compartment. The covalent linkage of ADP-ribose to SDH was stable to neutral hydroxylamine, sensitive to HgCl2, and inhibitable by free cysteine, indicating that the modification was at a cysteine residue of SDH. In addition to its auto-ADP-ribosylation activity, purified SDH or streptococcal cell wall extracts were able to transfer the ADP-ribose moiety of NAD specifically to free cysteine, resulting in a true thioglycosidic linkage. Treatment of purified SDH or the crude cell wall extract with sodium nitroprusside, which spontaneously generates nitric oxide, was found to stimulate the ADP-ribosylation of SDH in a time-dependent manner. ADP-ribosylation and nitric oxide treatment inhibited the GAPDH activity of SDH. Since ADP-ribosylation and nitric oxide are involved in signal transduction events, the ADP-ribosylating activity of SDH may enable communication between host and parasite during infection by group A streptococci.
Beta-galactosidase activity in single differentiating bacterial cells.
Authors: Russo-Marie F , Roederer M , Sager B , Herzenberg L A , Kaiser D
Published: May 2006

Myxococcus xanthus strains containing transcriptional fusions to lacZ were analyzed and fractionated by differences in their levels of beta-galactosidase expression. The fluorogenic substrate for beta-galactosidase, fluorescein di-beta-galactopyranoside, was introduced into M. xanthus cells during a rapid decrease in osmolarity of the medium followed by a return to isoosmolarity. Fluorescein, the product of hydrolysis, was retained within the cells and their viability was preserved. Fluorescence increased linearly with time and was proportional to beta-galactosidase activity. beta-Galactosidase expression in most fusion strains, though beginning at different phases of growth or development, was distributed unimodally amongst cells. However, fusion strain Tn5 lac omega 4473 was shown to be heterogeneous at 9 hr of development. It was possible to separate physically cells that expressed beta-galactosidase at a high level from other, still viable, cells with no expression. The approach described here could be adapted to study differentiation in plants and animals as well, where transcriptional fusions and fluorogenic substrates for enzyme probes of gene expression also can be used.
An androgen receptor mRNA isoform associated with hormone-induced cell proliferation.
Authors: Fischer L , Catz D , Kelley D
Published: May 2006

The larynx of male Xenopus laevis undergoes an androgen-driven developmental transformation that enables the adult to produce his complex mate attraction song. During the early postmetamorphic period, androgen directs proliferation and differentiation of laryngeal muscle and cartilage. To explore the cellular and molecular basis of androgen control, we have cloned an androgen receptor cDNA from juvenile larynx. Here we identify two androgen receptor mRNA isoforms, alpha and beta, differing within the A/B, or hypervariable, domain. Northern blot analyses reveal that the beta isoform is transiently expressed during early juvenile stages, whereas the alpha transcript is expressed throughout postmetamorphic life. Using in situ hybridization and [3H]thymidine autoradiography, we examined the expression of androgen receptor mRNA isoforms during androgen-evoked cell proliferation and differentiation. The alpha and beta transcripts are coexpressed in proliferating tissues of the juvenile larynx; in postmitotic differentiated tissues, only the alpha transcript is expressed. Because androgen receptor beta mRNA is specifically expressed during hormone-evoked cell proliferation, we propose that this developmentally regulated mRNA isoform is required for the masculine program of cell addition within the developing vocal organ.
Substrate-assisted catalysis in the cleavage of DNA by the EcoRI and EcoRV restriction enzymes.
Authors: Jeltsch A , Alves J , Wolfes H , Maass G , Pingoud A
Published: May 2006

The crystal structure analyses of the EcoRI-DNA and EcoRV-DNA complexes do not provide clear suggestions as to which amino acid residues are responsible for the activation of water to carry out the DNA cleavage. Based on molecular modeling, we have proposed recently that the attacking water molecule is activated by the negatively charged pro-Rp phosphoryl oxygen of the phosphate group 3' to the scissile phosphodiester bond. We now present experimental evidence to support this proposal. (i) Oligodeoxynucleotide substrates lacking this phosphate group in one strand are cleaved only in the other strand. (ii) Oligodeoxynucleotide substrates carrying an H-phosphonate substitution at this position in both strands and, therefore, lacking a negatively charged oxygen at this position are cleaved at least four orders of magnitude more slowly than the unmodified substrate. These results are supported by other modification studies: oligodeoxynucleotide substrates with a phosphorothioate substitution at this position in both strands are cleaved only if the negatively charged sulfur is in the RP configuration as shown for EcoRI [Koziolkiewicz, M. & Stec, W.J. (1992) Biochemistry 31, 9460-9466] and EcoRV (B. A. Connolly, personal communication). As the phosphate residue 3' to the scissile phosphodiester bond is not needed for strong DNA binding by both enzymes, these findings strongly suggest that this phosphate group plays an active role during catalysis. This proposal, furthermore, gives a straightforward explanation of why in the EcoRI-DNA and EcoRV-DNA complexes the DNA is distorted differently, but in each case the 3' phosphate group closely approaches the phosphate group that is attacked. Finally, an alternative mechanism for DNA cleavage involving two metal ions is unlikely in the light of our finding that both EcoRI and EcoRV need only one Mg2+ per active site for cleavage.
Acute leukemias of different lineages have similar MLL gene fusions encoding related chimeric proteins resulting from chromosomal translocation.
Authors: Corral J , Forster A , Thompson S , Lampert F , Kaneko Y , Slater R , Kroes W G , van der Schoot C E , Ludwig W D , Karpas A
Published: May 2006

The MLL gene, on human chromosome 11q23, undergoes chromosomal translocation in acute leukemias, resulting in gene fusion with AF4 (chromosome 4) and ENL (chromosome 19). We report here translocation of MLL with nine different chromosomes and two paracentric chromosome 11 deletions in early B cell, B- or T-cell lineage, or nonlymphocytic acute leukemias. The mRNA translocation junction from 22 t(4;11) patients, including six adult leukemias, and nine t(11;19) tumors reveals a remarkable conservation of breakpoints within MLL, AF4, or ENL genes, irrespective of tumor phenotype. Typically, the breakpoints are upstream of the zinc-finger region of MLL, and deletion of this region can accompany translocation, supporting the der(11) chromosome as the important component in leukemogenesis. Partial sequence of a fusion between MLL and the AFX1 gene from chromosome X shows the latter to be rich in Ser/Pro codons, like the ENL mRNA. These data suggest that the heterogeneous 11q23 abnormalities might cause attachment of Ser/Pro-rich segments to the NH2 terminus of MLL, lacking the zinc-finger region, and that translocations occur in early hematopoietic cells, before commitment to distinct lineages.
In vitro analysis of Ah receptor domains involved in ligand-activated DNA recognition.
Authors: Dolwick K M , Swanson H I , Bradfield C A
Published: May 2006

The Ah receptor (AHR) is a basic helix-loop-helix protein that mediates the effects of 2,3,7,8-tetrachloro-dibenzo-p-dioxin. In this report, we describe a rabbit reticulocyte system that allows functional expression of both the AHR and its dimeric partner, the AHR nuclear translocator protein (ARNT). By using this in vitro system, we were able to reconstitute agonist binding to the AHR and agonist-induced AHR-ARNT recognition of a cognate DNA enhancer sequence. Expression of AHR deletion mutants revealed the location of N-terminal domains responsible for ligand and DNA recognition and C-terminal domains that play roles in agonist-induced DNA recognition.
Expression of mammalian glutathione S-transferase 5-5 in Salmonella typhimurium TA1535 leads to base-pair mutations upon exposure to dihalomethanes.
Authors: Thier R , Taylor J B , Pemble S E , Humphreys W G , Persmark M , Ketterer B , Guengerich F P
Published: May 2006

Dihalomethanes can produce liver tumors in mice but not in rats, and concern exists about the risk of these compounds to humans. Glutathione (GSH) conjugation of dihalomethanes has been considered to be a critical event in the bioactivation process, and risk assessment is based upon this premise; however, there is little experimental support for this view or information about the basis of genotoxicity. A plasmid vector containing rat GSH S-transferase 5-5 was transfected into the Salmonella typhimurium tester strain TA1535, which then produced active enzyme. The transfected bacteria produced base-pair revertants in the presence of ethylene dihalides or dihalomethanes, in the order CH2Br2 > CH2BrCl > CH2Cl2. However, revertants were not seen when cells were exposed to GSH, CH2Br2, and an amount of purified GSH S-transferase 5-5 (20-fold excess in amount of that expressed within the cells). HCHO, which is an end product of the reaction of GSH with dihalomethanes, also did not produce mutations. S-(1-Acetoxymethyl)GSH was prepared as an analog of the putative S-(1-halomethyl)GSH reactive intermediates. This analog did not produce revertants, consistent with the view that activation of dihalomethanes must occur within the bacteria to cause genetic damage, presenting a model to be considered in studies with mammalian cells. S-(1-Acetoxymethyl)GSH reacted with 2'-deoxyguanosine to yield a major adduct, identified as S-[1-(N2-deoxyguanosinyl)methyl]GSH. Demonstration of the activation of dihalomethanes by this mammalian GSH S-transferase theta class enzyme should be of use in evaluating the risk of these chemicals, particularly in light of reports of the polymorphic expression of a similar activity in humans.
Heat shock of Escherichia coli increases binding of dnaK (the hsp70 homolog) to polypeptides by promoting its phosphorylation.
Authors: Sherman M Y , Goldberg A L
Published: May 2006

The "molecular chaperone", dnaK, is induced in Escherichia coli upon heat shock and promotes ATP-dependent refolding or degradation of damaged proteins. When cells were grown at 25 degrees C and disrupted, a small fraction of the dnaK bound to affinity columns containing unfolded polypeptides (e.g., a fusion protein named CRAG or casein) and could be dissociated by ATP-Mg2+. After shifting cells to 42 degrees C for 30 min, up to 5-fold more dnaK bound to these columns than after growth at 25 degrees C. This enhanced binding capacity was reversed after shifting cells back to 25 degrees C. It resulted from a covalent modification, which decreases dnaK's electrophoretic mobility and isoelectric point. This modification appears to be phosphorylation; after treatment with phosphatases, the ATP-eluted dnaK resembled the predominant form in electrophoretic and binding properties. In addition, after incubating cells with [32P]orthophosphate at 42 degrees C, the 32P-labeled dnaK bound quantitatively to the CRAG column, unlike the nonlabeled protein. Thus, the phosphorylated dnaK is a special form of the chaperone with enhanced affinity for unfolded proteins. Its accumulation at high temperatures may account for dnaK's function as the "cellular thermometer."
Enzyme crystal structure in a neat organic solvent.
Authors: Fitzpatrick P A , Steinmetz A C , Ringe D , Klibanov A M
Published: May 2006

The crystal structure of the serine protease subtilisin Carlsberg in anhydrous acetonitrile was determined at 2.3 A resolution. It was found to be essentially identical to the three-dimensional structure of the enzyme in water; the differences observed were smaller than those between two independently determined structures in aqueous solution. The hydrogen bond system of the catalytic triad is intact in acetonitrile. The majority (99 of 119) of enzyme-bound, structural water molecules have such a great affinity to subtilisin that they are not displaced even in anhydrous acetonitrile. Of the 12 enzyme-bound acetonitrile molecules, 4 displace water molecules and 8 bind where no water had been observed before. One-third of all subtilisin-bound acetonitrile molecules reside in the active center, occupying the same region (P1, P2, and P3 binding sites) as the specific protein inhibitor eglin c.
Chronic lymphocytic leukemia cells with allelic deletions at 13q14 commonly have one intact RB1 gene: evidence for a role of an adjacent locus.
Authors: Liu Y , Szekely L , Grandér D , Söderhäll S , Juliusson G , Gahrton G , Linder S , Einhorn S
Published: May 2006

We have previously shown that 30% of patients with B-cell chronic lymphocytic leukemia (B-CLL) have hemizygous deletions of the retinoblastoma (RB1) gene at 13q14. RB1 gene deletions may thus participate in malignant transformation of B-CLL, but it is also possible that a neighboring gene on 13q is the relevant one. To answer this question the remaining RB1 allele of eight clones with hemizygous deletions was studied by reverse transcription-polymerase chain reaction (RT-PCR), single-strand conformation polymorphism (SSCP) analysis, and immunofluorescense techniques. Cells from 10 patients without RB1 gene deletions were also studied by these methods. Lack of RB1 mRNA and RB protein expression was seen in leukemia cells from one of the patients. All other cases were found to be normal with regard to immunofluorescense, RT-PCR, and SSCP analysis, indicating at least one functional RB1 allele and supporting the importance of another gene in the 13q14 deletions. We then performed extended Southern blot analyses of the 13q region, using probes for 10 different loci. In 14 of 31 CLL clones (45%), deletions of a region telomeric to the RB1 gene (D13S25) were observed. In 4 of the cases the deletions were homozygous. Hemizygous deletions of the RB1 gene were observed in 11 of these patients and in none of the patients without D13S25 deletions. These data thus indicate that a gene(s) telomeric to RB1 is involved in the malignant transformation of CLL clones and that deletions of this region are a common event in this disease.
Differential usage of multiple brain-derived neurotrophic factor promoters in the rat brain following neuronal activation.
Authors: Metsis M , Timmusk T , Arenas E , Persson H
Published: May 2006

The rat brain-derived neurotropic factor (BDNF) gene consists of four 5' exons linked to separate promoters and one 3' exon encoding the prepro-BDNF protein. To gain insights into the regulation of BDNF mRNA expression, probes specific for the different 5' exons were used to study the expression of BDNF mRNA in the brain. Following a systemic injection of the glutamate analog kainic acid, exon I, II, and III mRNAs increased transiently in hippocampus and cerebral cortex. A modest increase was seen for exon IV, where a new transcription initiation site was induced by this treatment. Pretreatments with the N-methyl-D-aspartate (NMDA) receptor antagonist MK801 or the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist 2,3-dihydroxy-6-nitrosulfanoylbenzo(f)quinoxaline revealed two region-specific patterns of glutamate receptor-mediated regulation. The first pattern found in neocortex, piriform cortex, and amygdala involves regulation of BDNF exon I, II, and III mRNAs through NMDA and AMPA/kainate receptors. The second pattern found in the hippocampus involves regulation of BDNF exon I, II, and III mRNAs by high-affinity kainate or metabotropic receptors. Treatment with the gamma-aminobutyric acid subtype A (GABAA) receptor antagonist bicuculline increased exon I and III mRNAs in the denate gyrus, and the muscarinic receptor agonist pilocarpine increased exon I mRNA mainly in the neocortex. These data show that the four BDNF promoters allow multiple points of BDNF mRNA regulation and suggest that the activation of different subtypes of glutamate receptors differentially regulates the expression of BDNF exon-specific mRNAs in the brain.
Cell proliferation, DNA repair, and p53 function are not required for programmed death of prostatic glandular cells induced by androgen ablation.
Authors: Berges R R , Furuya Y , Remington L , English H F , Jacks T , Isaacs J T
Published: May 2006

Androgen ablation induces programmed death of androgen-dependent prostatic glandular cells, resulting in fragmentation of their genomic DNA and the cells themselves into apoptotic bodies. Twenty percent of prostatic glandular cells undergo programmed death per day between day 2 and 5 after castration. During this same period, < 1% of prostatic glandular cells enter the S phase of the cell cycle, documenting that > 95% of these die in G0. During the programmed death of these G0 glandular cells, a futile DNA repair process is induced secondary to the DNA fragmentation. This futile DNA repair is not required, however, since inhibition of this process by > 90% with an appropriately timed hydroxy-urea dosing regimen had no effect upon the extent of the programmed death of these cells after castration. Likewise, p53 gene expression is not required since the same degree of cell death occurred in prostates and seminal vesicles after castration of wild-type and p53-deficient mice.