bluuebunny/crossref_metadata_2025 huggingface dataset:
Gene for an extracellular matrix receptor protein from Pneumocystis carinii.
Authors: Narasimhan S , Armstrong M Y , Rhee K , Edman J C , Richards F F , Spicer E
Published: May 2006

An initial and crucial step in the establishment of many microbial infections is the attachment of the pathogen to the host cells. Thus, adherence of Pneumocystis carinii (Pc) to type I pneumocytes is believed to be important in the induction of Pc pneumonia. Little is known about the nature of the attachment of Pc to type I cells, although extracellular matrix (ECM) proteins, such as fibronectin and laminin, have been implicated in the process. We report here the isolation of a Pc gene encoding a receptor protein that binds both fibronectin and laminin in vitro. A cDNA clone encoding the Pc ECM receptor was isolated from a Pc cDNA library and identified on the basis of sequence homology to the human colon carcinoma laminin receptor. Southern blot analysis of Pc genomic DNA confirmed that the cDNA was of Pc origin. Northern blot analysis of Pc total RNA showed a predominant mRNA of approximately 1400 nucleotides that hybridized to the ECM receptor gene. The ECM receptor predicted from the cDNA sequence is 295 amino acid residues long, with a molecular mass of 32.8 kDa. The C-terminal third of the polypeptide is highly negatively charged, whereas the N-terminal two-thirds contains hydrophobic segments that may play a role in membrane association. Sequence analysis and alignment of the N terminus with the laminin receptor cDNA sequence of human colon carcinoma support the conclusion that the Pc ECM receptor cDNA clone is a full-length clone. A Western blot of the overexpressed ECM receptor protein bound both laminin and fibronectin in vitro. Antibodies raised to the overexpressed receptor protein interacted with a 33-kDa protein in total Pc cell lysates. These findings raise the possibility that the Pc ECM receptor protein may mediate the organism's attachment to type I pneumocytes and, thus, may play a crucial role in Pc pathogenesis.
Extrachromosomal elements in tobacco plastids.
Authors: Staub J M , Maliga P
Published: May 2006

The plastid genome of higher plants is a circular double-stranded DNA molecule which is present in multiple identical copies. We report here an 868-bp plastid DNA minicircle, NICE1, that formed in tobacco (Nicotiana tabacum) plastids during transformation, as an unexpected product of homologous recombination. Such extrachromosomal elements are normally absent in plastids of higher plants. We have constructed shuttle plasmids containing NICE1 sequences which are maintained extrachromosomally when reintroduced into plastids by particle bombardment. Furthermore, recombination between homologous sequences in the shuttle plasmids and the main plastid genome occurs. Recombination products were characterized after recovery of the shuttle plasmids in Escherichia coli and of recombinant plastid genomes in the progeny of transformed plants. Our findings indicate that shuttle plasmids can be used to engineer plastid genes without concomitant integration of foreign DNA and to recover plastid mutations in E. coli.
A model of prenatal acquisition of speech parameters.
Authors: Seebach B S , Intrator N , Lieberman P , Cooper L N
Published: May 2006

An unsupervised neural network model inductively acquires the ability to distinguish categorically the stop consonants of English, in a manner consistent with prenatal and early postnatal auditory experience, and without reference to any specialized knowledge of linguistic structure or the properties of speech. This argues against the common assumption that linguistic knowledge, and speech perception in particular, cannot be learned and must therefore be innately specified.
The c-kit ligand suppresses apoptosis of human natural killer cells through the upregulation of bcl-2.
Authors: Carson W E , Haldar S , Baiocchi R A , Croce C M , Caligiuri M A
Published: May 2006

The bcl-2 protein plays a central role in the regulation of programmed cell death in a variety of tissues and is pivotal to the survival of lymphocytes in vivo. The growth factors responsible for survival of normal lymphocytes are unknown but are likely to maintain viability in part through the regulation of bcl-2 expression. A subset of human natural killer (NK) cells (CD3-CD56bright) are unique among lymphocytes in their constitutive expression of c-kit, a tyrosine kinase cell surface receptor that binds c-kit ligand (KL). Alone, KL does not promote proliferation or further differentiation of CD56bright NK cells. We now report that, in the absence of serum or additional growth factors, KL prevents apoptosis of cultured CD56bright NK cells, as assessed by DNA fragmentation studies, and maintains viability, as measured by biologic responses (i.e., proliferation and cytotoxicity) to the subsequent addition of other cytokines. Furthermore, we demonstrate that KL induces CD56bright NK cells to express the bcl-2 protein. In the presence of anti-c-kit antibody, the tyrosine kinase inhibitor genistein, or bcl-2 antisense oligonucleotide, the protective effect of KL on the survival of CD56bright NK cells is dramatically reduced. These data demonstrate that the binding of KL to its tyrosine kinase receptor results in the upregulation of bcl-2, thereby preventing apoptosis in this subset of normal human lymphocytes. As soluble KL is plentiful in normal human serum, this survival mechanism may be operative for CD56bright NK cells in vivo.
Analysis of the Streptomyces coelicolor sigE gene reveals the existence of a subfamily of eubacterial RNA polymerase sigma factors involved in the regulation of extracytoplasmic functions.
Authors: Lonetto M A , Brown K L , Rudd K E , Buttner M J
Published: May 2006

sigma E, an RNA polymerase sigma factor of apparent M(r) 28,000, was previously identified by its ability to direct transcription from the P2 promoter of the agarose gene (dagA) of Streptomyces coelicolor. A degenerate oligonucleotide probe, designed from the N-terminal sequence of purified sigma E, was used to isolate the sigma E gene (sigE). The predicted sequence of sigma E shows greatest similarity to sequences of seven other proteins: Myxococcus xanthus CarQ, Pseudomonas aeruginosa AlgU, Pseudomonas syringae HrpL, Escherichia coli sigma E, Alcaligenes eutrophus CnrH, E. coli FecI, and Bacillus subtilis SigX, a protein of unknown function. These eight proteins define a subfamily of eubacterial RNA polymerase factors sufficiently different from other sigma s that, in many cases, they are not identified by standard similarity searching methods. Available information suggests that all of them regulate extracytoplasmic functions and that they function as effector molecules responding to extracytoplasmic stimuli. A. eutrophus CnrH appears to be a plasmid-encoded factor.
Vaccinia protein kinase 2: a second essential serine/threonine protein kinase encoded by vaccinia virus.
Authors: Lin S , Broyles S S
Published: May 2006

The major protein kinase activity from vaccinia virus core particles was purified to near homogeneity. The protein kinase is a 50-kDa polypeptide that is shown here to phosphorylate primarily seryl residues in alpha-casein, a casein kinase I-specific peptide substrate, and itself through autophosphorylation. The sequence of four peptides derived from the protein kinase demonstrated that it is encoded by the vaccinia virus F10L gene. Expression of the F10L gene product in bacteria as a fusion with glutathione S-transferase confirmed that the vaccinia F10L gene encodes the protein kinase. We have termed this enzyme vaccinia protein kinase 2 (VPK2) to distinguish it from the protein kinase encoded by the vaccinia B1R gene. Targeted disruption of the VPK2 gene with a positive selectable marker demonstrated that all viruses with a disrupted gene also possessed a wild-type gene, suggesting that VPK2 is essential for viability. The discovery of a second essential protein kinase encoded by vaccinia virus, in addition to a protein phosphatase, underscores the importance of protein phosphorylation in poxvirus biogenesis.
Cell death and neuronal recruitment in the high vocal center of adult male canaries are temporally related to changes in song.
Authors: Kirn J , O'Loughlin B , Kasparian S , Nottebohm F
Published: May 2006

Adult male canaries modify their song every year. Most of these changes occur during late summer and early fall, after the end of the breeding season, and in late winter, immediately before the onset of the next breeding season. The high vocal center (HVC) is an important nucleus in the brain pathway that controls this learned behavior. New neurons continue to be added to the HVC of adult male canaries, where they replace older neurons that have died. The present report describes the monthly incidence of cell death and neuronal addition in the HVC of such birds. Different groups of 1- to 2-year-old male canaries were treated with [3H]thymidine, a marker of cell birth, during each month of the year and killed 27 days later. The ratio of 3H-labeled neurons to all neurons in the HVC showed seasonal peaks and troughs. This ratio was highest in October and March. Peaks in the ratio of pycnotic (dying) HVC cells to all neurons in HVC preceded the peaks in the ratio of 3H-labeled neurons. We suggest that seasonal peaks in cell loss and neuronal recruitment in HVC are related to endocrine changes and that all three play a role in the seasonality of song modification.
Design and synthesis of deoxynucleic guanidine: a polycation analogue of DNA.
Authors: Dempcy R O , Almarsson O , Bruice T C
Published: May 2006

The basic strategy is described for the connection of nucleosides by guanidinium (g) linkers to provide the positively charged deoxynucleic guanidine putative antigene agents. The synthetic procedures are provided for d(gT)n. Molecular modeling of double-stranded [d(gT)10.d(Ap)10] and the triple-helical hybrids [d(Tp)10.d(Ap)10.d(gT)10] and [d(gT)10.d(Ap)10.d(gT)10] suggest modes of interaction and anticipated structural features.
Independent in vitro assembly of a ribonucleoprotein particle containing the 3' domain of 16S rRNA.
Authors: Samaha R R , O'Brien B , O'Brien T W , Noller H F
Published: May 2006

Small (30S) subunits of Escherichia coli ribosomes are composed of 21 proteins and a 1542-nucleotide 16S rRNA, whose secondary structure is divided into three domains. An in vitro transcript of the 3' domain of 16S rRNA (residues 923-1542), assembles efficiently with 30S ribosomal proteins to form a compact ribonucleoprotein (RNP) particle. Isolated particles examined under the electron microscope have a globular appearance, similar in size and shape to the head of the 30S ribosomal subunit. Two-dimensional gel analysis of the particles indicates the presence of proteins S3, S7, S9, S10, S13, S14, and S19 and smaller amounts of S2, all of which have been localized to the head of the 30S subunit by immunoelectron microscopy and neutron diffraction and belong to the S7 assembly family. Interestingly, protein S4, which is believed to interact exclusively with the 5' domain, is also reproducibly found associated with the particles in significant amounts. Chemical probing of the RNA in the assembled particle reveals characteristic cleavage protection patterns, showing that the proteins assemble with the 3'-domain RNA similarly to the way in which they assemble with 16S rRNA, although some of the later steps of assembly appear to be incomplete. These results show that the 3' domain of 16S rRNA can indeed assemble independently of the rest of the 30S subunit into a particle that resembles its structure in the ribosome. In addition, the assembled particles are able to bind spectinomycin with an affinity comparable to that of 30S subunits.
Dephosphorylation of sperm midpiece antigens initiates aster formation in rabbit oocytes.
Authors: Pinto-Correia C , Poccia D L , Chang T , Robl J M
Published: May 2006

During fertilization in most mammals, the penetrating sperm organizes an aster of microtubules. We have investigated the mechanisms underlying this function of the sperm by a series of experiments based on microinjection of isolated sperm midpieces into unfertilized oocytes. These midpieces contain antigens recognized by the MPM-2 antibody. These antigens, which are absent from the rest of the tail fraction, correspond to three phosphorylated polypeptides of 77, 81, and 85 kDa. Dephosphorylation with alkaline phosphatase abolishes antigenicity on blots and in whole sperm. Reactivity to the antibody disappears between 1 and 3 hr after calcium stimulation of oocytes, following the decline in H1 kinase activity and coincident with aster formation. In unactivated oocytes, no aster forms and the antigen remains unchanged. MPM-2 treatment of midpieces prior to injection blocks their ability to form asters in oocytes activated by calcium stimulation. The epitope also disappears in 6-methyl-aminopurine-treated oocytes, implying that maintenance of the phosphorylated state requires kinase activity. A result that confirms this view is that sperm midpieces dephosphorylated by alkaline phosphatase can be rephosphorylated after injection into oocytes or by exposure in vitro to a Xenopus oocyte cytoplasmic fraction high in H1 kinase activity. We suggest that the microtubule nucleation activity of sperm midpieces after fertilization is triggered by the calcium-induced decrease in maturation promoting factor, which results in dephosphorylation of specific sperm centrosomal proteins.
Genomic structure and cloned cDNAs predict that four variants in the kinase domain of serine/threonine kinase receptors arise by alternative splicing and poly(A) addition.
Authors: Xu J , Matsuzaki K , McKeehan K , Wang F , Kan M , McKeehan W L
Published: May 2006

Heterodimers of types I and II serine/threonine kinase receptor monomers compose the active receptor complex for ligands of the transforming growth factor beta family. Here we show that the genomic organization of coding sequences for the intracellular domain of a widely expressed type I serine/threonine kinase receptor is similar to that of the activin type II receptor gene. The genomic structure and cDNA clones indicate that poly(A) addition to alternative exons at each of three carboxyl-terminal coding exon-intron junctions may be a common feature of both type I and II receptor genes. The predicted products are monomers truncated at kinase subdomains VII, IX, and X which vary in kinase activity and potential serine, threonine, and tyrosine phosphorylation sites. These results suggest that combinations of variants that affect the signal-transducing intracellular kinase domain of both type I and II receptor monomers within the transforming growth factor beta ligand family may add to the heterogeneity of biological effects of individual ligands in the family.
Reconstitution of SCID mice with human lymphoid and myeloid cells after transplantation with human fetal bone marrow without the requirement for exogenous human cytokines.
Authors: Kollmann T R , Kim A , Zhuang X , Hachamovitch M , Goldstein H
Published: May 2006

Investigation of human hematopoietic maturation has been hampered by the lack of in vivo models. Although engraftment of irradiated C.B-17 scid/scid (SCID) mice with human progenitor cells occurred after infusion with human pediatric bone marrow cells, significant engraftment of the mouse bone marrow with human cells was dependent upon continuous treatment with exogenous human cytokines. Furthermore, despite cytokine treatment, only minimal peripheral engraftment of these mice with human cells was observed. In the present study, after infusion of irradiated SCID mice with pre-cultured human fetal bone marrow cells (BM-SCID-hu mice), their bone marrow became significantly engrafted with human precursor cells and their peripheral lymphoid compartment became populated with human B cells and monocytes independently of the administration of extraneous human cytokines. Examination of the bone marrow of the BM-SCID-hu mice for human cytokine mRNA gene expression demonstrated human leukemia inhibitory factor mRNA and interleukin 7 mRNA in nine of nine BM-SCID-hu mice and macrophage-colony-stimulating factor mRNA in seven of eight BM-SCID-hu mice. This was an intriguing observation because these cytokines regulate different stages of human hematopoiesis. Since engraftment occurs in the absence of exogenous cytokine treatment, the BM-SCID-hu mouse model described should provide a useful in vivo system for studying factors important in the maturation of human myeloid and lymphoid cells in the bone marrow and the behavior of the mature human cells after dissemination into the peripheral lymphoid tissue.
The ctpA gene encodes the C-terminal processing protease for the D1 protein of the photosystem II reaction center complex.
Authors: Anbudurai P R , Mor T S , Ohad I , Shestakov S V , Pakrasi H B
Published: May 2006

The D1 protein of the photosystem II (PSII) complex in the thylakoid membrane of oxygenic photosynthetic organisms is synthesized as a precursor polypeptide (pD1) with a C-terminal extension. Posttranslational processing of the pD1 protein is essential to establish water oxidation activity of the PSII complex. We have recently identified a gene, ctpA, a mutation in which resulted in a loss of PSII activity in the cyanobacterium Synechocystis sp. PCC 6803. To study the function of the CtpA protein, we inactivated the ctpA gene by inserting a kanamycin-resistance gene into its coding sequence. The resultant mutant strain, T564, had no PSII-mediated water oxidation activity, but it had normal cytochrome b6f and photosystem I activities. Measurements of thermoluminescence profiles and rates of reduction of 2,6-dichlorophenolindophenol indicated that PSII complexes in the mutant cells had functional reaction centers that were unable to accept electrons from water. Immunoblot analysis showed that D1, D2, CP47, CP43, and the alpha subunit of cytochrome b559, five integral membrane proteins of PSII, were present in T564 cells. Interestingly, the D1 protein in the mutant cells was 2 kDa larger than that in wild-type cells, due to the presence of a C-terminal extension. We conclude that the CtpA protein is a processing enzyme that cleaves off the C-terminal extension of the D1 protein. Interestingly, the CtpA protein shows significant sequence similarity to the interphotoreceptor retinoid-binding proteins in the bovine, human, and insect eye systems.
Cold denaturation of a repressor-operator complex: the role of entropy in protein-DNA recognition.
Authors: Foguel D , Silva J L
Published: May 2006

The mechanisms by which regulatory proteins recognize specific DNA sequences are not fully understood. Here we examine the basis for the stability of a protein-DNA complex, using hydrostatic pressure and low temperature. Pressure converts the DNA-binding Arc repressor protein from a native state to a denatured, molten-globule state. Our data show that the folding and dimerization of Arc repressor in the temperature range 0-20 degrees C are favored by a large positive entropy value, so that the reaction proceeds in spite of an unfavorable positive enthalpy. On binding operator DNA, Arc repressor becomes extremely stable against denaturation. However, the Arc repressor-operator DNA complex is cold-denatured at subzero temperatures under pressure, demonstrating that the favorable entropy increases greatly when Arc repressor binds tightly to its operator sequence but not a nonspecific sequence. We show how an increase in entropy may operate to provide the protein with a mechanism to distinguish between a specific and a nonspecific DNA sequence. It is postulated that the formation of the Arc-operator DNA complex is followed by an increase in apolar interactions and release of solvent which would explain its entropy-driven character, whereas this solvent would not be displaced in nonspecific complexes.
N,N'-dicyclohexylcarbodiimide cross-linking suggests a central core of helices II in oligomers of URF13, the pore-forming T-toxin receptor of cms-T maize mitochondria.
Authors: Rhoads D M , Kaspi C I , Levings C S , Siedow J N
Published: May 2006

URF13 is a mitochondrially encoded, integral membrane protein found only in maize carrying the cms-T cytoplasm. URF13 is associated with cytoplasmic male sterility, Texas type, and causes susceptibility to the fungal pathogens Bipolaris maydis race T and Phyllosticta maydis. URF13 is predicted to contain three transmembrane alpha-helices and is a receptor for the pathotoxins (T-toxins) produced by B. maydis race T and P. maydis. Binding of T-toxin to URF13 leads to membrane permeability. Cross-linking of URF13 oligomers with N,N'-dicyclohexylcarbodiimide (DCCD) protects Escherichia coli cells expressing URF13 and cms-T mitochondria from the permeability caused by T-toxin or methomyl. Using mutated forms of URF13 expressed in E. coli cells, we determined the molecular mechanism of DCCD protection. We separately changed Lys-37 in helix II to isoleucine (K37I-URF13) and Lys-32 in the helix I/helix II loop region to alanine (K32A-URF13). DCCD treatment of K37I-URF13-expressing cells did not protect the cells from permeability caused by T-toxin or methomyl. DCCD cross-linking was greatly reduced in K37I-URF13 and in D39V-URF13-expressing cells, but it was unaffected in K32A-URF13-expressing cells. Binding of methomyl or T-toxin decreases DCCD cross-linking of URF13 oligomers expressed in either E. coli or cms-T mitochondria. We conclude that Asp-39 in helix II is cross-linked by DCCD to Lys-37 in helix II of an adjacent URF13 molecule and that this cross-linking protects against toxin-mediated permeabilization. Our results also indicate that helices II form a central core in URF13 oligomers.
Retroviral integration within the Fli-2 locus results in inactivation of the erythroid transcription factor NF-E2 in Friend erythroleukemias: evidence that NF-E2 is essential for globin expression.
Authors: Lu S J , Rowan S , Bani M R , Ben-David Y
Published: May 2006

Activation of either Fli-1 or Spi-1 members of the ets family of transcription factors as a result of retroviral insertion and mutational inactivation of the p53 tumor suppressor gene play essential roles in the multistage erythroleukemias induced in mice by various strains of Friend virus. We have previously identified another common site for provirus integration, designated Fli-2 (Friend leukemia integration 2), in some erythroleukemia clones induced either by Friend murine leukemia virus (F-MuLV) or by the polycythemia-inducing strain of Friend virus complex (FV-P). Here we show that genomic sequences adjacent to Fli-2 correspond to the coding region of the erythroid-specific DNA binding protein NF-E2 p45. In one erythroleukemia cell line the expression of NF-E2 p45 is undetectable due to proviral integration in one allele and loss of the other allele. The complete loss of NF-E2 p45 in this cell line is associated with a drastic reduction in expression of the alpha- and beta-globin genes that were partially restored by reintroduction of the NF-E2 p45 gene. Taken together, these results provide direct evidence that NF-E2 gene is essential for globin transcription and suggest that perturbation in expression of this transcription factor may contribute to erythroleukemia progression.
Dimeric structure of a human apolipoprotein B mRNA editing protein and cloning and chromosomal localization of its gene.
Authors: Lau P P , Zhu H J , Baldini A , Charnsangavej C , Chan L
Published: May 2006

Apolipoprotein B (apoB) mRNA editing consists of a posttranscriptional C-->U conversion involving the first base of the codon CAA encoding glutamine-2153 to UAA, a stop codon, in apoB mRNA. Using a cloned rat cDNA as a probe, we cloned the cDNA and genomic sequences of the gene for a human apoB mRNA editing protein. Expression of the cDNA in HepG2 cells results in editing of the intracellular apoB mRNA. By fluorescence in situ hybridization, we localized the gene for the editing protein to chromosome band 12p13.1-p13.2. By Northern blot analysis, it was shown that the human editing protein mRNA is expressed exclusively in the small intestine. The cDNA sequence predicts a translation product of 236-aa residues. By attaching an epitope tag sequence to the C terminus of the editing protein, we examined the polymerization state of the editing protein synthesized in vitro. We found that the editing protein undergoes spontaneous polymerization. The migration of the human apoB mRNA editing protein on an HPLC column and the stoichiometry of polymeric epitope-tagged to untagged protein indicate that the protein exists as a dimer. Dimerization does not require glycosylation of a consensus N-linked glycosylation sequence present in the protein and is not mediated by disulfide bridge formation. The human apoB mRNA editing protein is a cytidine deaminase showing structural homology to some known mammalian and bacteriophage deoxycytidylate deaminases. The latter enzymes exist as homopolymers. The fact that the apoB mRNA editing protein also exists as a homodimer has important implications for the mechanism of apoB mRNA editing in humans.
The LIM protein RBTN2 and the basic helix-loop-helix protein TAL1 are present in a complex in erythroid cells.
Authors: Valge-Archer V E , Osada H , Warren A J , Forster A , Li J , Baer R , Rabbitts T H
Published: May 2006

Chromosomal translocations in T-cell acute leukemias can activate genes encoding putative transcription factors such as the LIM proteins RBTN1 and RBTN2 and the DNA-binding basic helix-loop-helix transcription factor TAL1 associated with T-cell acute lymphocytic leukemia. While not expressed in normal T cells, RBTN2 and TAL1 are coexpressed in erythroid cells and are both important for erythroid differentiation. We demonstrate, using anti-RBTN2 and anti-TAL1 antisera, that the LIM protein RBTN2 is not phosphorylated and is complexed with the TAL1 phosphoprotein in the nucleus of erythroid cells. A complex containing both RBTN1 and TAL1 also occurs in a T-cell acute leukemia cell line. Since both RBTN2 and TAL1 are crucial for normal erythropoiesis, these data have important implications for transcription networks therein. Further, since both proteins can be involved in leukemogenesis, these data provide a direct link between proteins activated by chromosomal translocations in T-cell acute leukemia.
Antizyme protects against abnormal accumulation and toxicity of polyamines in ornithine decarboxylase-overproducing cells.
Authors: Suzuki T , He Y , Kashiwagi K , Murakami Y , Hayashi S , Igarashi K
Published: May 2006

Exposure of ornithine decarboxylase (ODC; L-ornithine carboxy-lyase, EC 4.1.1.17)-overproducing mouse FM3A cells to micromolar levels of spermine or spermidine caused abnormal accumulation and toxicity of polyamines. This was apparently due to the inefficiency of negative feedback control of polyamine transport by polyamines in ODC-overproducing cells. Since antizyme is the only protein thus far recognized that can interact with ODC, depletion of free antizyme was regarded as the reason for the abnormal accumulation of polyamines. Accordingly, ODC-overproducing cells were transfected with pMAMneoZ1 possessing rat antizyme cDNA under the control of a glucocorticoid-inducible promoter. In the transfected cells, the addition of dexamethasone caused an increase in the amount of antizyme with an apparent molecular mass of 27 kDa, a decrease in the amount of ODC, a decrease in the polyamine transport activity, and the recovery of growth inhibition or cell death. The results indicate that antizyme can regulate not only the amount of ODC but also the activity of polyamine transport.
Relief of p53-mediated transcriptional repression by the adenovirus E1B 19-kDa protein or the cellular Bcl-2 protein.
Authors: Shen Y , Shenk T
Published: May 2006

The p53 tumor suppressor gene product is a transcriptional regulatory protein. It activates transcription from promoters that contain a p53 DNA binding site but represses many promoters that lack its binding site. High-level expression of wild-type p53 can induce apoptosis in certain cell types, and this activity can be blocked by the adenovirus E1B 19-kDa oncoprotein or by the cellular Bcl-2 oncoprotein. Here we report that p53-mediated repression of promoters that lack a p53 binding site is abrogated by the E1B 19-kDa protein or Bcl-2 oncoprotein. In contrast, transcriptional activation by p53 still occurs in the presence of either protein. The fact that two oncoproteins capable of preventing p53-mediated apoptosis also block transcriptional repression by p53 raises the possibility that p53 might induce apoptosis, at least in part, by repressing transcription.
Human T-cell-mediated destruction of allogeneic dermal microvessels in a severe combined immunodeficient mouse.
Authors: Murray A G , Petzelbauer P , Hughes C C , Costa J , Askenase P , Pober J S
Published: May 2006

We developed a chimeric human-severe combined immunodeficient mouse model to study human allograft rejection. Mice received first partial thickness human skin grafts and then, after anastomosis of the mouse with graft human microvessels, human lymphocytes allogeneic to the skin. By 2 weeks, the skin grafts uniformly developed changes that resemble first-set skin rejection in humans. Vascular cell adhesion molecule 1 and major histocompatibility complex class II molecules were induced on the human vascular endothelium at day 6, prior to significant T-cell infiltration. Perivascular human CD4+ and CD8+ T-cell infiltrates were marked by day 11. Some T cells, adjacent to injured human vessels, expressed the cytolytic granule protein perforin. The human microvessels were destroyed by day 16 without significant damage to human keratinocytes or adjacent mouse microvessels. This small animal model may permit evaluation of potential therapeutic reagents that inhibit human T-cell-mediated injury.
Marked infidelity of human immunodeficiency virus type 1 reverse transcriptase at RNA and DNA template ends.
Authors: Patel P H , Preston B D
Published: May 2006

Human immunodeficiency virus type 1 (HIV-1) is genetically highly variable. This is attributed to the error-prone nature of HIV-1 replication and its proclivity for recombination. During replication and recombination, reverse transcriptase (RT) must polymerize DNA to the 5' ends of multiple RNA and DNA template termini while converting HIV-1 RNA to double-stranded DNA. We have determined the fidelity of HIV-1 RT in vitro during polymerization to the 5' ends of HIV-1 long terminal repeat DNA template sequences and to the end of a partial HIV-1 genomic RNA template that mimics a recombination intermediate. HIV-1 RT readily extended recessed DNA primers to form full-length blunt-end DNA-DNA and DNA-RNA duplexes. In addition, HIV-1 RT catalyzed high yields of products with one to four extra nucleotides at the 3' ends of the nascent DNAs. These products were formed processively via a nontemplated mechanism that is highly specific for the addition of purine nucleotides (A > G >> T > or = C). Thus, HIV-1 RT is extremely unfaithful at both DNA and RNA template ends, introducing errors (extra nucleotides) in one out of every two or three nascent strands processively polymerized. This error rate is 1000 times higher than for HIV-1 RT-catalyzed errors at internal template positions. Blunt-end additions were also catalyzed by other retroviral RTs at relative rates of HIV-1 approximately Moloney murine leukemia virus > avian myeloblastosis virus. These data suggest a potentially important mechanism for retroviral mutation mediated by nontemplated blunt-end addition of purines prior to forced copy-choice recombination.
Functional analysis of myosin missense mutations in familial hypertrophic cardiomyopathy.
Authors: Straceski A J , Geisterfer-Lowrance A , Seidman C E , Seidman J G , Leinwand L A
Published: May 2006

To analyze potential functional consequences of myosin heavy chain (MHC) mutations identified in patients with familial hypertrophic cardiomyopathy (FHC), we have assessed the stability of the mutant MHCs and their ability to form thick filaments. Constructs encoding wild-type rat alpha MHC and seven corresponding FHC missense mutants were transfected into COS cells. Immunoblot analysis suggested that FHC mutations do not grossly alter protein stability. Wild-type alpha MHC transfected into COS cells forms structures previously shown to be arrays of thick filaments, which also resemble myosin structures observed early in differentiation of muscle cells. Surprisingly, up to 29% of COS cells transfected with the FHC mutants failed to form filamentous structures. To assess whether this phenotype was specific for the FHC mutants and not generalizable to any myosin mutation, COS cells were transfected with a construct encoding an MHC with a 168-amino acid deletion of the hinge/rod region. This deletion construct formed filamentous structures with the same frequency as wild-type MHC. Biochemical analysis of one FHC mutant (Arg-249-->Gln) demonstrates that the structures formed by the mutant are solubilized at a lower ionic strength than those formed by wild-type MHC. We conclude that although the FHC mutant MHC is not labile, its assembly properties may be impaired.
Cellular epigenetics: topochronology of progressive "spontaneous" transformation of cells under growth constraint.
Authors: Chow M , Yao A , Rubin H
Published: May 2006

Early passages of NIH 3T3 cells yield about 10 transformed foci for every 10(5) cells seeded after the cells multiply to confluence in a standardized 2-week assay. The question arose whether more cells would give rise to foci if given more time for their development. This question could not be answered simply by extending the incubation period, since the original foci spread to cover much of the area of the culture dish. Transformed cells can also detach into the medium from the original foci to initiate new foci by reattaching at a distance. These problems were averted by growing cells in multiwell plates which in effect simulated partitioned culture dishes. All the wells in a given plate were assayed for focus formation at successive intervals up to 14 weeks. The results indicated the spatial pattern and sequence of transformation on different parts of the "partitioned" dish. The number of multiwells containing focus-forming cells increased steadily with time, indicating that all parts of a dish eventually undergo transformation. Also, most of the transformations were recorded long after confluence in the multiwells was reached. Hence such a transformation is much more likely to occur in the nondividing state rather than in the dividing state of the cells and is thus inconsistent with a mutational basis. The results suggest that "spontaneous" transformation is a population-wide, epigenetic phenomenon. This agrees with the results from clonal analysis and other studies and is well described by the concept of progressive state selection, in which "spontaneous" transformation represents a heterogeneous, adaptive response of competent cells to moderate constraints on cell growth.
Chronic active hepatitis in transgenic mice expressing interferon-gamma in the liver.
Authors: Toyonaga T , Hino O , Sugai S , Wakasugi S , Abe K , Shichiri M , Yamamura K
Published: May 2006

Interferon-gamma may play an important role in the immune response and in inflammatory diseases, including chronic active hepatitis. To understand the role of interferon-gamma in the regulation of inflammation and to establish a mouse model of chronic active hepatitis, we produced transgenic mice in which the mouse interferon-gamma gene was regulated by a liver-specific promoter, the serum amyloid P component gene promoter. Four transgenic mouse lines were generated, and two of these lines expressed mRNA of interferon-gamma in the liver. Levels of serum transaminases increased gradually as a function of age and were significantly higher than those of interferon-gamma-negative littermates after 4 weeks after birth. One transgenic mouse line showed a histology of chronic active hepatitis similar to that found in human patients, although cirrhotic changes such as fibrosis were scarce. Thus, the liver-specific production of interferon-gamma is sufficient to induce chronic inflammatory disease and this mouse is a transgenic model of chronic active hepatitis.
A rapid capsaicin-activated current in rat trigeminal ganglion neurons.
Authors: Liu L , Simon S A
Published: May 2006

A subpopulation of pain fibers are activated by capsaicin, the ingredient in red peppers that produces a burning sensation when eaten or placed on skin. Previous studies on dorsal root ganglion neurons indicated that capsaicin activates sensory nerves via a single slowly activating and inactivating inward current. In rat trigeminal neurons, we identified a second capsaicin-activated inward current. This current can be distinguished from the slow one in that it rapidly activates and inactivates, requires Ca2+ for activation, and is insensitive to the potent capsaicin agonist resiniferatoxin. The rapid current, like the slower one, is inhibited by ruthenium red and capsazepine. The two capsaicin-activated inward currents share many similarities with the two inward currents activated by lowering the pH to 6.0. These similarities include kinetics, reversal potentials, responses to Ca2+, and inhibition by ruthenium red and capsazepine. These results suggest that acidic stimuli may be an endogenous activator of capsaicin-gated currents and therefore may rationalize why pain is produced when the plasma acidity is increased, as occurs during ischemia and inflammation.
Enhancer 1 binding factor, a Ku-related protein, is a positive regulator of RNA polymerase I transcription initiation.
Authors: Hoff C M , Ghosh A K , Prabhakar B S , Jacob S T
Published: May 2006

We have previously characterized a protein, enhancer 1 binding factor (E1BF), from rat cells that can modulate RNA polymerase I-directed transcription of the rat rRNA gene in vitro. E1BF, a heterodimeric DNA binding protein composed of 72-kDa and 85-kDa subunits, is related to the human Ku autoantigen with respect to immunological and certain structural properties. To establish the direct role of E1BF in transcription, we investigated the effect of anti-Ku antibodies on RNA polymerase I-directed transcription in rat and mouse cell extracts. These antibodies, one directed against the 70-kDa Ku subunit and the other against a peptide fragment of this subunit, dissociated the E1BF heterodimer into its two subunits. The DNA-protein complex formed in the presence of the antibodies contained only the 72-kDa subunit. Preincubation of the extracts with these antibodies resulted in an almost complete inhibition of transcription. The reduced transcription was observed when either linear or circular template was used. The inhibitory effect of the antibodies was greatest when added prior to preinitiation complex formation and was minimized significantly when added after establishment of the initiation complex. The repression of rRNA gene transcription was overcome by the addition of purified E1BF. This study demonstrates that E1BF, a Ku-related protein, is required for RNA polymerase I-directed transcription, the 72-kDa subunit is the major DNA binding polypeptide, the factor acts primarily in the formation of the preinitiation complex, and heterodimerization of its two subunits is crucial for maintaining the functional integrity of the protein.
Growth hormone-releasing factor increases somatostatin release and mRNA levels in the rat periventricular nucleus via nitric oxide by activation of guanylate cyclase.
Authors: Aguila M C
Published: May 2006

Previous work has shown that growth hormone-releasing factor (GRF) stimulates cGMP production and somatostatin [somatotropin (growth hormone)-release-inhibiting factor, SRIF] release without altering cAMP accumulation by fragments of median eminence incubated in vitro. Therefore, this study was undertaken to evaluate the effect of GRF and cGMP on SRIF mRNA and SRIF release in the periventricular nuclei of male rats in vitro. SRIF mRNA levels were determined in explants of periventricular nuclei incubated for 6 hr in Waymouth's medium in the presence of various substances. Steady-state levels of SRIF mRNA were measured by an S1 nuclease protection assay using a 32P-labeled rat SRIF RNA probe. SRIF release and cGMP formation were measured at 30 min and 6 hr by RIA. SRIF mRNA levels and SRIF release were significantly (P < 0.025) increased (approximately 2-fold) by 1 microM dibutyryl cGMP, whereas sodium butyrate had no effect. This augmentation was not influenced by cycloheximide, an inhibitor of protein synthesis. Sodium nitroprusside (10 microM), an activator of the guanylate cyclase pathway via its release of nitric oxide, augmented (P < 0.001) SRIF mRNA levels and significantly increased (P < 0.05) SRIF release. GRF (1 nM) increased SRIF mRNA (P < 0.001) and stimulated the release of SRIF at 30 min (P < 0.05) and 6 hr (P < 0.01). This stimulation was abolished by 10 microM NG-monomethyl-L-arginine (L-NMMA), a specific inhibitor of nitric oxide synthase, but not by NG-monomethyl-D-arginine (D-NMMA, the inactive isomer). GRF also increased cGMP formation. This effect was completely blocked by incubation with L-NMMA but not D-NMMA. These results indicate that GRF releases nitric oxide. The nitric oxide diffuses to the adjacent SRIF neurons, where it activates guanylate cyclase, leading to increased formation of cGMP. This cGMP increases SRIF mRNA and SRIF release in the periventricular nuclei of male rats.
Pineal control of aging: effect of melatonin and pineal grafting on aging mice.
Authors: Pierpaoli W , Regelson W
Published: May 2006

Dark-cycle, night administration of the pineal hormone melatonin in drinking water to aging mice (15 months of age) prolongs survival of BALB/c females from 23.8 to 28.1 months and preserves aspects of their youthful state. Similar results were seen in New Zealand Black females beginning at 5 months and C57BL/6 males beginning at 19 months. As melatonin is produced in circadian fashion from the pineal, we grafted pineals from young 3- to 4-month-old donors into the thymus of 20-month-old syngeneic C57BL/6 male recipients, and a 12% increase in survival was induced. Prolongation of survival was also seen on pineal transplant to the thymus in C57BL/6, BALB/cJ, and hybrid female mice at 16, 19, and 22 months. In all studies, the endogenous pineal of grafted mice was left in situ. Pineal grafted aged mice display a remarkable maintenance of thymic structure and cellularity. Preservation of T-cell-mediated function, despite age, as measured by response to oxazolone is seen. Other evidence suggests that melatonin and/or pineal-related factors could produce their effects through an influence on thyroid function. These data indicate that pineal influences have a place in the physiologic regulation of aging.
Geometry of interplanar residue contacts in protein structures.
Authors: Brocchieri L , Karlin S
Published: May 2006

The relative spatial disposition of interacting side-chain planar groups (aromatic, guanidinium, amide, carboxyl, imidazole) is analyzed for 186 non-homologous well-resolved protein structures. The dihedral angle of amide or carboxyl planar groups with other planar groups accords with a random distribution of planes. By contrast, the dihedral angle of the planes between close aromatic rings or of the histidine ring interacting with aromatic residues is significantly nonrandom, showing an approximately uniform distribution. Our results indicate that edge-to-edge and edge-to-center spatial dispositions of residue planar sections are prevalent, while complete stacking configurations are uncommon. The hypothesis that electrostatic forces are a major determinant of the geometry of interactions between side-chain planar groups is discussed.
Discovery of myosin genes by physical mapping in Dictyostelium.
Authors: Titus M A , Kuspa A , Loomis W F
Published: May 2006

The diversity of the myosin family in a single organism, Dictyostelium discoideum, has been investigated by a strategy devised to rapidly identify and clone additional members of a gene family. An ordered array of yeast artificial chromosome clones that encompasses the Dictyostelium genome was probed at low stringency with conserved regions of the myosin motor domain to identify all possible myosin loci. The previously identified myosin loci (mchA, myoA-E) were detected by hybridization to the probes, as well as an additional seven previously unidentified loci (referred to as myoF-L). Clones corresponding to four of these additional loci (myoF, myoH-J) were obtained by using the isolated yeast artificial chromosomes as templates in a PCR employing degenerate primers specific for conserved regions of the myosin head. Sequence analysis and physical mapping of these clones confirm that these PCR products are derived from four previously unidentified myosin genes. Preliminary analysis of these sequences suggests that at least one of the genes (myoJ) encodes a member of a potentially different class of myosins. With the development of whole genome libraries for a variety of organisms, this approach can be used to rapidly explore the diversity of this and other gene families in a number of systems.
Inositol 1,3,4,5-tetrakisphosphate-gated channels interact with inositol 1,4,5-trisphosphate-gated channels in olfactory receptor neurons.
Authors: Fadool D A , Ache B W
Published: May 2006

Inositol 1,4,5-trisphosphate [InsP3(1,4,5)] is a major second messenger regulating Ca2+ signaling in excitable and nonexcitable cells. InsP3(1,4,5) is extensively metabolized through a network of phosphorylation and dephosphorylation steps to products with potential second messenger function. Inositol 1,3,4,5-tetrakisphosphate [InsP4(1,3,4,5)], the direct metabolite of InsP3(1,4,5), has also been associated with Ca2+ signaling, but whether InsP4(1,3,4,5) acts in combination with InsP3(1,4,5) or whether it regulates Ca2+ signaling directly and independently is unclear, particularly in neurons. We report that olfactory receptor neurons in the lobster (Panulirus argus) express an InsP4(1,3,4,5) receptor in the plasma membrane that is a functional channel. The channel differs in conductance, kinetics, and voltage sensitivity from two plasma membrane InsP3(1,4,5)-gated channels previously reported in these neurons. In close spatial proximity, the InsP4(1,3,4,5)-and InsP3(1,4,5)-gated channels interact reciprocally to alter the channels' open probabilities in what may be a novel mechanism for regulating Ca2+ entry in neurons.
Evidence for a regulatory protein involved in the increased activity of system A for neutral amino acid transport in osmotically stressed mammalian cells.
Authors: Ruiz-Montasell B , Gómez-Angelats M , Casado F J , Felipe A , McGivan J D , Pastor-Anglada M
Published: May 2006

System A for neutral amino acid transport is increased by hypertonic shock in NBL-1 cells previously induced to express system A activity by amino acid starvation. The hypertonicity-mediated effect can be blocked by cycloheximide but is insensitive to tunicamycin. The activity induced may be inactivated irreversibly by the addition of system A substrates, by a rapid mechanism insensitive to cycloheximide. In CHO-K1 cells, hypertonicity increases system A activity, as has been shown in NBL-1 cells. This effect is additive to the activity produced by derepression of system A by amino acid starvation and is insensitive to tunicamycin. Furthermore, the alanine-resistant mutant CHO-K1 alar4, which bears a mutation affecting the regulatory gene R1, involved in the derepression of system A activity after amino acid starvation, is still able to respond to the hypertonic shock by increasing system A activity to a level similar to that described in hypertonicity-induced derepressed CHO-K1 (wild type) cells. These results suggest (i) that the hypertonicity-mediated increase of system A activity occurs through a mechanism other than that involved in system A derepression and (ii) that a regulatory protein coded by an osmotically sensitive gene is responsible for further activation of preexisting A carriers.