bluuebunny/crossref_metadata_2025 huggingface dataset:
Protonation states of membrane-embedded carboxylic acid groups in rhodopsin and metarhodopsin II: a Fourier-transform infrared spectroscopy study of site-directed mutants.
Authors: Fahmy K , Jäger F , Beck M , Zvyaga T A , Sakmar T P , Siebert F
Published: May 2006

A method was developed to measure Fourier-transform infrared (FTIR) difference spectra of detergent-solubilized rhodopsin expressed in COS cells. Experiments were performed on native bovine rhodopsin, rhodopsin expressed in COS cells, and three expressed rhodopsin mutants with amino acid replacements of membrane-embedded carboxylic acid groups: Asp-83-->Asn (D83N), Glu-122-->Gln (E122Q), and the double mutant D83N/E122Q. Each of the mutant opsins bound 11-cis-retinal to yield a visible light-absorbing pigment. Upon illumination, each of the mutant pigments formed a metarhodopsin II-like species with maximal absorption at 380 nm that was able to activate guanine nucleotide exchange by transducin. Rhodopsin versus metarhodopsin II-like photoproduct FTIR-difference spectra were recorded for each sample. The COS-cell rhodopsin and mutant difference spectra showed close correspondence to that of rhodopsin from disc membranes. Difference bands (rhodopsin/metarhodopsin II) at 1767/1750 cm-1 and at 1734/1745 cm-1 were absent from the spectra of mutants D83N and E122Q, respectively. Both bands were absent from the spectrum of the double mutant D83N/E122Q. These results show that Asp-83 and Glu-122 are protonated both in rhodopsin and in metarhodopsin II, in agreement with the isotope effects observed in spectra measured in 2H2O. A photoproduct band at 1712 cm-1 was not affected by either single or double replacements at positions 83 and 122. We deduce that the 1712 cm-1 band arises from the protonation of Glu-113 in metarhodopsin II.
Repair by human cell extracts of single (6-4) and cyclobutane thymine-thymine photoproducts in DNA.
Authors: Szymkowski D E , Lawrence C W , Wood R D
Published: May 2006

One cis-syn cyclobutane thymine dimer or one (6-4) thymine-thymine photoproduct was built into an identical sequence of a closed-circular M13 duplex DNA, and nucleotide excision repair synthesis carried out by human cell extracts in the area containing each lesion was determined. Extracts from normal cells repaired the (6-4) photoproduct with a patch size of approximately 20-30 nucleotides, but repair was at least 10-fold lower at the cyclobutane dimer. The (6-4) lesion was repaired with comparable efficiency to a single acetylamino-fluorene-guanine adduct in a similar location. Extract from nucleotide excision repair-deficient xeroderma pigmentosum group A cells could not remove any of these adducts but could complete repair of the lesions after incision with Escherichia coli UvrABC proteins. This direct comparison of repair of two UV photoproducts, in an in vitro system where chromatin assembly and transcription are absent, suggests that the more rapid repair of the (6-4) lesion observed in the mammalian cell genome overall is due in part to a significant difference in the ability of the repair complex to locate and incise these lesions in DNA.
Gene targeting of X chromosome-linked chronic granulomatous disease locus in a human myeloid leukemia cell line and rescue by expression of recombinant gp91phox.
Authors: Zhen L , King A A , Xiao Y , Chanock S J , Orkin S H , Dinauer M C
Published: May 2006

The X chromosome-linked chronic granulomatous disease (X-CGD) locus, which encodes the gp91phox subunit of the phagocyte respiratory-burst oxidase cytochrome b, was disrupted by homologous recombination in the PLB-985 human myeloid cell line to develop an in vitro model of X-CGD. Superoxide formation was absent in targeted cells after differentiation to granulocytes but was rescued by stable transfection and expression of wild-type gp91phox cDNA. The targeted cell line should be useful in experiments aimed at defining functional regions within gp91phox by expression of mutant gp91phox cDNAs, complementing studies of naturally occurring mutations in X-CGD. In addition, the mutant line provides a model system in which to establish an experimental basis for the treatment of X-CGD patients with gene replacement therapy. Rescued clones containing even modest amounts of recombinant gp91phox had respiratory-burst activity comparable to the wild-type PLB-985 line, suggesting that functional correction of X-CGD neutrophils may not require high-level expression of gp91phox.
Genetic evidence for the roles of the bud-site-selection genes BUD5 and BUD2 in control of the Rsr1p (Bud1p) GTPase in yeast.
Authors: Bender A
Published: May 2006

Yeast cells normally display either an axial (for MATa or MAT alpha cells) or bipolar (for MATa/alpha cells) pattern of bud-site selection. The RSR1 gene, which was previously identified as a multicopy suppressor of Ts- mutations in the bud-emergence gene CDC24, encodes a GTPase of the Ras family that is required for both budding patterns. Mutations in Rsr1p that presumably block its ability to bind or hydrolyze GTP cause a randomized budding phenotype, suggesting that regulators of Rsr1p will prove to be required for proper bud positioning. The BUD5 gene product is required for proper bud-site selection and contains similarity to GDP-dissociation stimulators (GDS) for Ras-type proteins, suggesting that Bud5p may be a GDS for Rsr1p. Here I report that BUD5 is required for wild-type RSR1, but not for mutationally activated rsr1val12, to serve as a multicopy suppressor of cdc24, indicating that Bud5p functions as a GDS for Rsr1p in vivo. To identify the GAP (GTPase-activating protein) for Rsr1p, a genetic selection was designed based on the observation that mutationally activated rsr1val12, but not wild-type RSR1, can serve as a multicopy suppressor of yeast RAS2(Ts) mutants. Mutants were selected that allowed wild-type RSR1 to act as a multicopy suppressor of RAS2(Ts). Two such mutations proved to be in the BUD2 gene, suggesting that Bud2p functions as a GAP for Rsr1p in vivo.
Transforming growth factor beta 1 (TGF-beta 1) controls expression of major histocompatibility genes in the postnatal mouse: aberrant histocompatibility antigen expression in the pathogenesis of the TGF-beta 1 null mouse phenotype.
Authors: Geiser A G , Letterio J J , Kulkarni A B , Karlsson S , Roberts A B , Sporn M B
Published: May 2006

The phenotype of the transforming growth factor beta 1 (TGF-beta 1) null mouse has been previously described and is characterized by inflammatory infiltrates in multiple organs leading to a wasting syndrome and death as early as 3 weeks after birth. Since this phenotype occurs in the absence of any detectable pathogen, potential autoimmune disease mechanisms were investigated. We examined major histocompatibility complex (MHC) mRNA expression in tissues of the TGF-beta 1 null mouse and found levels of both the class I and class II MHC mRNA elevated compared to normal or TGF-beta 1 heterozygous littermates. This elevated expression was seen prior to any evidence of inflammatory infiltrates, suggesting a causal relationship between increased MHC expression and activation of immune cell populations. Cell surface expression of MHC molecules was detected by immunohistochemistry and correlated well with mRNA levels. Expression of mRNA for interferon gamma and its receptor was unchanged at the ages when increased MHC expression became apparent. Down-regulation of class I MHC expression by TGF-beta 1 was also demonstrated in vitro in fibroblasts isolated from TGF-beta 1 null mice. These findings suggest that one natural function of TGF-beta 1 is to control expression of both MHC classes. Altered regulation of MHC expression may be a critical step leading to the multifocal inflammation and wasting syndrome seen in the TGF-beta 1 null mouse. These results suggest potential applications for TGF-beta in the management of autoimmune disease, allograft rejection, and other problems associated with altered MHC expression.
Punctuated equilibrium and positive Darwinian evolution in vesicular stomatitis virus.
Authors: Nichol S T , Rowe J E , Fitch W M
Published: May 2006

RNA viruses possess the potential for rapid evolution and serve as excellent models to test evolutionary theory. Molecular phylogenetic analysis of the P gene for a larger number of diverse natural isolates of vesicular stomatitis virus reveals no evidence for a molecular clock but instead shows a stepwise evolutionary pattern unlike that ever seen before. Each step out from the tree's ancestral root to terminal branch tips correlates not with time of virus isolation but with a south-to-north geographical progression from Panama to the United States. The grossly unequal rates of change within this single species imply an underlying mechanism at odds with the prevailing notion that neutral changes are the dominating feature of molecular evolution. This is also a demonstration of punctuated equilibrium at the molecular level.
Functional expression and photoaffinity labeling of a cloned P2U purinergic receptor.
Authors: Erb L , Lustig K D , Sullivan D M , Turner J T , Weisman G A
Published: May 2006

ATP and UTP can function as extracellular signaling molecules by activating plasma membrane receptors termed P2 purinergic receptors. In the present study a P2U receptor cDNA has been expressed in K-562 human leukemia cells, one of the few available mammalian cell lines that lacks an endogenous P2U receptor. In stably transfected cells, low micromolar concentrations of ATP or UTP activated the receptor, resulting in the mobilization of intracellular calcium but not the influx of extracellular calcium. A photoaffinity agonist of the P2U receptor, 3'-O-(4-benzoylbenzoyl)adenosine 5'-[alpha-32P]triphosphate ([alpha-32P]BzATP), photolabeled several proteins in plasma membranes from the stable transfectant or from untransfected K-562 cells. The photolabeling of a 53-kDa protein was significantly greater in plasma membranes from the stable transfectant than from untransfected cells. A mutant receptor containing six consecutive histidine residues at its carboxyl terminus was constructed and used to verify that this 53-kDa protein was the P2U receptor. In plasma membranes from cells expressing the histidine-tagged P2U receptor, but not from cells expressing the wild-type receptor, a single [alpha-32P]BzATP-labeled protein with a molecular mass of 53 kDa was retained on a Ni(2+)-charged Sepharose column, which binds many proteins containing a polyhistidine tag. Photolabeling of the 53-kDa protein by [alpha-32P]BzATP was inhibited by ATP but not by UTP, raising the possibility that the P2U receptor may have distinct binding sites for each nucleotide.
A conserved helix motif complements the protein kinase core.
Authors: Veron M , Radzio-Andzelm E , Tsigelny I , Ten Eyck L F , Taylor S S
Published: May 2006

Residues 40-300 of the mammalian catalytic (C) subunit of cAMP-dependent protein kinase define a conserved bilobal catalytic core shared by all eukaryotic protein kinases. Contiguous to the core is an extended amphipathic alpha-helix (A helix). Trp30, a prominent feature of this helix, fills a deep hydrophobic pocket between the two lobes on the surface opposite to the active site. The C subunit in Dictyostelium discoideum shows sequence conservation of residues 40-350 with the mouse enzyme but contains an N-terminal extension of 332 residues. A sequence corresponding to the A helix contiguous to the core is absent. However, we have now identified a remote A-helix motif (residues 77-98). When the core of the Dictyostelium C subunit was modeled, based on the mouse C subunit, complementarity between this putative A helix and the surface of the core was found to be conserved. Analysis of other protein kinases reveals that the A-helix motif is not restricted to cAMP-dependent protein kinase. In the Src-related family of protein kinases, for example, an A helix is very likely contiguous to the core, thus serving as a linker between the conserved catalytic core and the Src homology 2 domain. We predict that an A-helix motif complementary to the core will be a conserved feature of most eukaryotic protein kinases.
The phylogenetically predicted base-pairing interaction between alpha and alpha' is required for group II splicing in vitro.
Authors: Harris-Kerr C L , Zhang M , Peebles C L
Published: May 2006

The correct folding of group II introns apparently depends on multiple tertiary base-pairing interactions. Understanding the relationship between spliceosome and group II splicing systems ultimately requires a three-dimensional model for both structures. In turn, successful modeling depends at least in part on identifying tertiary base pairings. Sequence elements alpha and alpha' are partners in a potential interaction of approximately 6 base pairs that can be identified within domain 1 of most group II introns. In comparisons between related introns, alpha and alpha' maintain their potential for Watson-Crick base pairing, even though their primary sequences can vary [Michel, F., Umesono, K. & Ozeki, H. (1989) Gene 82, 5-30]. Substitutions were constructed at alpha and alpha' for a block of 6 bases each in the group II intron a5 gamma, the last intron of the COXI gene from the mitochondrial DNA of Saccharomyces cerevisiae. Each substitution was defective for self-splicing, while the compensatory double derivative was restored to active splicing. The alpha-alpha' interaction is required for the first step of splicing--that is, recognition of the 5' splice junction and transesterification with the branch site--since the derivative transcripts displayed little or no activity. The compensatory double derivative produced lariat introns and spliced exons with normal structures, showing that splicing activity and precise recognition were restored. We conclude that the alpha-alpha' base pairing is necessary for efficient self-splicing by intron a5 gamma under several conditions. This result also provides an additional constraint for any three-dimensional model of group II intron structure.
The human myelin basic protein gene is included within a 179-kilobase transcription unit: expression in the immune and central nervous systems.
Authors: Pribyl T M , Campagnoni C W , Kampf K , Kashima T , Handley V W , McMahon J , Campagnoni A T
Published: May 2006

Two human Golli (for gene expressed in the oligodendrocyte lineage)-MBP (for myelin basic protein) cDNAs have been isolated from a human oligodendroglioma cell line. Analysis of these cDNAs has enabled us to determine the entire structure of the human Golli-MBP gene. The Golli-MBP gene, which encompasses the MBP transcription unit, is approximately 179 kb in length and consists of 10 exons, seven of which constitute the MBP gene. The human Golli-MBP gene contains two transcription start sites, each of which gives rise to a family of alternatively spliced transcripts. At least two Golli-MBP transcripts, containing the first three exons of the gene and one or more MBP exons, are produced from the first transcription start site. The second family of transcripts contains only MBP exons and produces the well-known MBPs. In humans, RNA blot analysis revealed that Golli-MBP transcripts were expressed in fetal thymus, spleen, and human B-cell and macrophage cell lines, as well as in fetal spinal cord. These findings clearly link the expression of exons encoding the autoimmunogen/encephalitogen MBP in the central nervous system to cells and tissues of the immune system through normal expression of the Golli-MBP gene. They also establish that this genetic locus, which includes the MBP gene, is conserved among species, providing further evidence that the MBP transcription unit is an integral part of the Golli transcription unit and suggest that this structural arrangement is important for the genetic function and/or regulation of these genes.
Overexpression of human loricrin in transgenic mice produces a normal phenotype.
Authors: Yoneda K , Steinert P M
Published: May 2006

The cornified cell envelope (CE) of terminally differentiating stratified squamous epithelial cells is a complex multiprotein assembly about 15 nm thick of which loricrin is a major component. We have produced transgenic mice bearing the human loricrin transgene in order to study the role of loricrin in CE assembly, structure, and function. By analyses of RNA and protein, we show that the human transgene is expressed in mouse epithelial tissues in an appropriate developmental manner but at an overall level about twice that of endogenous mouse loricrin. Thus the 20-kbp construct used contains all necessary regulatory elements. By immunogold electron microscopy, all of the expressed protein is incorporated into the CE. That no alternations were noted indicates that overproduction of the loricrin component of the CE does not affect the flexible structure or function of the epithelial tissues. Furthermore, these data imply that loricrin may be the last protein to be deposited onto, and thus lines, the intracellular surface of the CE, where it may be accessible to interact with the subjacent keratin intermediate-filament network.
The Escherichia coli hflA locus encodes a putative GTP-binding protein and two membrane proteins, one of which contains a protease-like domain.
Authors: Noble J A , Innis M A , Koonin E V , Rudd K E , Banuett F , Herskowitz I
Published: May 2006

The hflA (high frequency of lysogenization) locus of Escherichia coli governs the lysis-lysogeny decision of bacteriophage lambda by controlling stability of the phage cII protein. hflA contains three genes, hflX, hflK, and hflC, encoding polypeptides of 50, 46, and 37 kDa, respectively. We have determined the nucleotide sequence of 3843 base pairs containing hflA and have found three large open reading frames corresponding to hflX, hflK, and hflC. HflX contains the three sequence motifs typical of GTP-binding proteins and appears to be a member of a distinct family of putative GTPases. HflC and HflK appear to be integral membrane proteins which show some similarity to each other and to a human membrane protein. The C-terminal region of HflC contains a domain resembling the catalytic domain of ClpP, a bacterial ATP-dependent protease. We hypothesize that HflK and HflC constitute a distinct membrane-bound protease whose activity may be modulated by HflX GTPase.
Barrier properties of testis microvessels.
Authors: Holash J A , Harik S I , Perry G , Stewart P A
Published: May 2006

The blood-testis barrier is believed to be constituted by tight junctions between Sertoli cells in seminiferous tubules and possibly by myoid cells that encircle these tubules. We now show that testis microvessels are endowed with several markers of barrier properties of brain microvessels, such as the glucose transporter, P-glycoprotein, and gamma-glutamyl transpeptidase. Quantitative EM studies show that the endothelium in testis, as in brain, is continuous and has long junctional profiles and few vesicles. However, a small proportion of testis capillaries have expansions in their junctional clefts suggestive of patent paracellular channels, which may explain their higher permeability. Because barrier features are thought to be induced and/or maintained in brain microvessels by astrocytes, we assessed whether astrocyte-like cells exist in the testis. We found that the intertubular Leydig cells, adjacent to microvessels, express the astrocyte markers: glial fibrillary acidic protein, glutamine synthetase, and S-100 protein. We suggest that the testis endothelium contributes to the blood-testis barrier and that these endothelial barrier features are influenced by Leydig cells. We believe that the endothelial and the epithelial (Sertoli) components of the blood-testis barrier are "in series" and complement each other in achieving a stable milieu for spermatogenesis.
Transgenic sorghum plants via microprojectile bombardment.
Authors: Casas A M , Kononowicz A K , Zehr U B , Tomes D T , Axtell J D , Butler L G , Bressan R A , Hasegawa P M
Published: May 2006

Transgenic sorghum plants have been obtained after microprojectile bombardment of immature zygotic embryos of a drought-resistant sorghum cultivar, P898012. DNA delivery parameters were optimized based on transient expression of R and C1 maize anthocyanin regulatory elements in scutellar cells. The protocol for obtaining transgenic plants consists of the delivery of the bar gene to immature zygotic embryos and the imposition of bialaphos selection pressure at various stages during culture, from induction of somatic embryogenesis to rooting of regenerated plantlets. One in about every 350 embryos produced embryogenic tissues that survived bialaphos treatment; six transformed callus lines were obtained from three of the eight sorghum cultivars used in this research. Transgenic (T0) plants were obtained from cultivar P898012 (two independent transformation events). The presence of the bar and uidA genes in the T0 plants was confirmed by Southern blot analysis of genomic DNA. Phosphinothricin acetyltransferase activity was detected in extracts of the T0 plants. These plants were resistant to local application of the herbicide Ignite/Basta, and the resistance was inherited in T1 plants as a single dominant locus.
A human mitochondrial ATP-dependent protease that is highly homologous to bacterial Lon protease.
Authors: Wang N , Gottesman S , Willingham M C , Gottesman M M , Maurizi M R
Published: May 2006

We have cloned a human ATP-dependent protease that is highly homologous to members of the bacterial Lon protease family. The cloned gene encodes a protein of 963 amino acids with a calculated molecular mass of 106 kDa, slightly higher than that observed by Western blotting the protein from human tissues and cell lines (100 kDa). A single species of mRNA was found for this Lon protease in all human tissues examined. The protease is encoded in the nucleus, and the amino-terminal portion of the protein sequence contains a potential mitochondrial targeting presequence. Immunofluorescence microscopy suggested a predominantly mitochondrial localization for the Lon protease in cultured human cells. A truncated LON gene, in which translation was initiated at Met118 of the coding sequence, was expressed in Escherichia coli and produced a protease that degraded alpha-casein in vitro in an ATP-dependent manner and had other properties similar to E. coli Lon protease.
Anti-pig IgM antibodies in human serum react predominantly with Gal(alpha 1-3)Gal epitopes.
Authors: Sandrin M S , Vaughan H A , Dabkowski P L , McKenzie I F
Published: May 2006

A major problem with pig-to-human-tissue xenograft studies is that humans have natural antibodies to pig cells; these antibodies would cause hyperacute rejection if pig tissues were xenografted to humans. Here we show that most of human IgM antibodies present in the serum of healthy donors and reactive with pig cells react with galactose in an (alpha 1-3) linkage with galactose--i.e., Gal(alpha 1-3)Gal. Absorption studies demonstrated that the antibodies detected the same or similar epitopes on the surface of pig erythrocytes, blood and splenic lymphocytes, and aortic endothelial cells (EC). The antibodies were sensitive to 2-mercaptoethanol (2ME) treatment, did not bind to protein A or G, and were present in the high molecular weight fraction of serum; they are clearly IgM antibodies. Further, the antibodies did not react with human ABO blood group substances and are not related to human blood group A or B, which carry a terminal galactose. The reaction of human serum with pig erythrocytes was specifically inhibited by mono- and disaccharides: D-galactose, melibiose, stachyose, methyl-alpha-D-galactopyranoside, and D-galactosamine but not by D-glucose or methyl-beta-D-galactopyranoside; demonstrating that the reaction is with galactose in an alpha and not a beta linkage. A cDNA clone encoding the murine alpha-1,3-galactosyltransferase (which transfers a terminal galactose residue with an (alpha 1-3) linkage to a subterminal galactose) was isolated by polymerase chain reaction (PCR), cloned, and transfected into COS cells, which are of Old World monkey origin and, like humans, do not express Gal(alpha 1-3)Gal. After transfection, COS cells became strongly reactive with human serum and with IB4 lectin [which reacts only with Gal(alpha 1-3)Gal]; this reactivity could be removed by absorption with pig erythrocytes. As most of the antibody reacting with pig cells can be removed by absorption with either melibiose or Gal(alpha 1-3)Gal+ COS cells, most of these react with Gal(alpha 1-3)Gal. These findings provide the basis for genetic manipulation of the pig alpha-1,3-galactosyltransferase for future transplantation studies.
Induction of long-term facilitation in Aplysia sensory neurons by local application of serotonin to remote synapses.
Authors: Clark G A , Kandel E R
Published: May 2006

Long-term synaptic facilitation at the connections of Aplysia sensory neurons onto their target cells involves alterations in gene expression. How then are the relevant cellular signals for the induction and expression of long-term synaptic changes conveyed between the nucleus and remote synaptic terminals? We have explored this question using a set of remote, peripheral terminals of siphon sensory cells, which are approximately 3 cm from the sensory cell body in the abdominal ganglion. We found that these remote synapses, like the proximal synapses previously studied in dissociated cell culture, can exhibit long-term facilitation 24 hr after cell-wide serotonin application. Furthermore, serotonin applications restricted to the remote synaptic terminals nevertheless produced long-term facilitation, indicating that signals generated in synaptic regions can trigger the long-term process, perhaps via retrograde signals to the nucleus to modify gene expression, followed by anterograde signals back to the terminal. Serotonin applications restricted to the cell body and proximal synapses of the sensory neuron also produced long-term facilitation at remote synapses, although to a lesser extent, suggesting that long-term facilitation is expressed cell-wide, but that superimposed on this cell-wide facilitation there appears to be a component that is synapse-specific.
Nonequilibrium air clathrate hydrates in Antarctic ice: a paleopiezomdter for polar ice caps.
Authors: Craig H , Shoji H , Langway C C
Published: May 2006

"Craigite," the mixed-air clathrate hydrate found in polar ice caps below the depth of air-bubble stability, is a clathrate mixed crystal of approximate composition (N2O2).6H2O. Recent observations on the Byrd Station Antarctic core show that the air hydrate is present at a depth of 727 m, well above the predicted depth for the onset of hydrate stability. We propose that the air hydrate occurs some 100 m above the equilibrium phase boundary at Byrd Station because of "piezometry"--i.e., that the anomalous depth of hydrate occurrence is a relic of a previous greater equilibrium depth along the flow trajectory, followed by vertical advection of ice through the local phase-boundary depth. Flowline trajectories in the ice based on numerical models show that the required vertical displacement does indeed occur just upstream of Byrd Station. Air-hydrate piezometry can thus be used as a general parameter to study the details of ice flow in polar ice caps and the metastable persistence of the clathrate phase in regions of upwelling blue ice.
Inhibition of granulocyte differentiation by G1 cyclins D2 and D3 but not D1.
Authors: Kato J Y , Sherr C J
Published: May 2006

Growth factor-induced signals govern the expression of three D-type cyclins, which, in turn, function as regulatory subunits of cyclin-dependent kinases (cdks) to control cell cycle transitions during the late G1 interval. 32D myeloid cells, which self-renew as uncommitted precursors in interleukin 3 (IL-3), express cyclins D2 and D3 (but not D1) in complexes with cdk4 and cdk2. When transferred to granulocyte colony-stimulating factor (G-CSF), 32D cells stop dividing and terminally differentiate to mature neutrophils. Cyclin D and cdk4 expression ceased as cells underwent growth arrest in G-CSF, but cdk2 levels were sustained. 32D cells engineered to ectopically express D-type cyclins exhibited contracted G1 intervals with a compensatory lengthening of S phase but remained IL-3 dependent for cell growth; those overexpressing cyclins D2 and D3 (but not D1) were unable to differentiate and died in G-CSF. Cyclin D2 mutants, which cannot efficiently bind to, or functionally interact with, the retinoblastoma protein (pRb) or its relatives (p107) did not block differentiation. Conversely, the introduction of a catalytically inactive cdk4 mutant into cells overexpressing cyclin D2 restored their G-CSF response. The persistence of cdk2 and its predilection to functionally interact with cyclins D2 and D3 rather than D1 might explain the specificity of the differentiation blockade.
BC200 RNA: a neural RNA polymerase III product encoded by a monomeric Alu element.
Authors: Martignetti J A , Brosius J
Published: May 2006

We demonstrate that the BC200 RNA gene, which encodes a neural small cytoplasmic RNA, is a member of the most prodigious family of interspersed repetitive DNA and that its product represents an example of a primate tissue-specific RNA polymerase III transcript. The BC200 RNA gene is an early monomeric member and one of the few postulated transcriptionally active Alu sequences in this family of nearly half a million retropositionally amplified elements dispersed throughout the human genome. Furthermore, the isolation of two pseudogenes, BC200 beta and BC200 gamma, demonstrates the gene's transpositional ability. Interestingly, the BC200 beta pseudogene may have been generated by a conversion-like event after the human/chimpanzee divergence, resulting in an exchange of the left arm of a dimeric Alu element with the BC200 RNA coding sequence. Our data on conserved features of the active BC200 alpha gene suggest that its RNA product has been "exapted" into a function of the primate brain and provides a selective advantage to the species.
Functional expression of P-glycoprotein encoded by the mouse mdr3 gene in yeast cells.
Authors: Ruetz S , Raymond M , Gros P
Published: May 2006

We have expressed P-glycoprotein (P-gp) encoded by the mouse mdr3 gene in the yeast Saccharomyces cerevisiae and have developed an experimental protocol to isolate and purify inside-out plasma membrane vesicles (IOVs) from these cells. Biochemical characterization of IOVs from control and P-gp-expressing cells isolated by this procedure show that they are greatly enriched for plasma membrane markers, are tightly sealed, and are competent for D-glucose transport. P-gp expression in these vesicles results in the appearance of a specific ATP-dependent and temperature-sensitive transport of the drugs colchicine and vinblastine that is osmotically sensitive. P-gp-mediated drug transport into these IOVs is inhibited by a known P-gp modulator, verapamil, and can be abrogated by prior incubation of the IOVs with an anti-P-gp antibody. A Ser-939-->Phe mutation within the predicted transmembrane domain 11 of P-gp, which is known to modulate its function in mammalian cells, drastically reduces drug transport in IOVs obtained from yeast cells expressing the mutant protein. The successful demonstration of active drug transport into IOVs from P-gp-expressing yeast cells indicates that P-gp can mediate both chemotherapeutic drugs and a-pheromone transport in yeast cells.
Catalytic contribution of flap-substrate hydrogen bonds in "HIV-1 protease" explored by chemical synthesis.
Authors: Baca M , Kent S B
Published: May 2006

An analogue of "HIV-1 protease" was designed in which the ability to donate important water-mediated hydrogen bonds to substrate was precisely and directly deleted. Chemical ligation of unprotected peptide segments was used to synthesize this "backbone-engineered" enzyme. The functionally relevant amide -CONH- linkage between residues Gly49-Ile50 in each flap of the enzyme was replaced by an isosteric thioester -COS- bond. The backbone-engineered enzyme had normal substrate specificity and affinity (Km). However, the catalytic activity (kcat) was reduced approximately 3000-fold compared to the native amide bond-containing enzyme. Inhibition by the reduced peptide bond substrate analogue MVT-101 was unaffected compared with native enzyme. By contrast, the normally tight-binding hydroxyethylamine inhibitor JG-365 bound to the backbone-engineered enzyme with an approximately 2500-fold reduction in affinity. The reduced catalytic activity of the -Gly49-psi(COS)-Ile50-backbone-engineered enzyme analogue provides direct experimental evidence to support the suggestion that backbone hydrogen bonds from the enzyme flaps to the substrate are important for the catalytic function of the HIV-1 protease.
A translation-attenuating intraleader open reading frame is selected on coronavirus mRNAs during persistent infection.
Authors: Hofmann M A , Senanayake S D , Brian D A
Published: May 2006

Short open reading frames within the 5' leader of some eukaryotic mRNAs are known to regulate the rate of translation initiation on the downstream open reading frame. By employing the polymerase chain reaction, we learned that the 5'-terminal 5 nt on the common leader sequence of bovine coronavirus subgenomic mRNAs were heterogeneous and hypervariable throughout early infection in cell culture and that as a persistent infection became established, termini giving rise to a common 33-nt intraleader open reading frame were selected. Since the common leader is derived from the genomic 5' end during transcription, a common focus of origin for the heterogeneity is expected. The intraleader open reading frame was shown by in vitro translation studies to attenuate translation of downstream open reading frames in a cloned bovine coronavirus mRNA molecule. Selection of an intraleader open reading frame resulting in a general attenuation of mRNA translation and a consequent attenuation of virus replication may, therefore, be a mechanism by which coronaviruses and possibly other RNA viruses with a similar transcriptional strategy maintain a persistent infection.
The effect of sequences with high AU content on mRNA stability in tobacco.
Authors: Ohme-Takagi M , Taylor C B , Newman T C , Green P J
Published: May 2006

Little is known about the mechanisms that target transcripts for rapid degradation in plants. In mammalian cells, sequences with a high AU content and multiple AUUUA motifs have been shown to cause mRNA instability when present in the 3' untranslated regions of several transcripts. This precedent, coupled with the poor accumulation of AU-rich foreign transcripts in plants (e.g., BT-toxin mRNAs), prompted us to test whether AU sequences could destabilize transcripts in tobacco. To address this question, we made a set of constructs containing sequences with high AU content inserted into the 3' untranslated regions of reporter genes. The stability of the corresponding transcripts was then assayed in stably transformed cell lines of tobacco. These experiments showed that a 60-base sequence containing 11 copies of the AUUUA motif (AUUUA repeat) markedly destabilized a beta-glucuronidase reporter transcript compared to a no-insert control or a 60-base spacer sequence (GC control). Another sequence with an identical A+U content had little effect. The same results were obtained when each sequence was assayed within the 3' untranslated region of a beta-globin reporter transcript. In regenerated transgenic plants, the AUUUA repeat decreased the accumulation of the beta-globin transcript by approximately 14-fold, compared to the GC control. Taken together, our results indicate that the AUUUA repeat is recognized as an instability determinant in plant cells and that the effect is due to the sequence of the element, not simply to the high AU content.
A gene encoding a protein related to eukaryotic protein kinases from the filamentous heterocystous cyanobacterium Anabaena PCC 7120.
Authors: Zhang C C
Published: May 2006

Protein kinases play essential roles in the development of eukaryotic cells. These enzymes display various degrees of sequence similarity in their catalytic domains. This conservation has allowed the identification of protein kinases in a variety of organisms, including the Gram-negative bacterium Myxococcus xanthus. In this study, sequences related to those encoding eukaryotic protein kinases were amplified by PCR from DNA of Anabaena PCC 7120, a filamentous cyanobacterium that differentiates cells specifically for nitrogen fixation, called heterocysts, under conditions of combined nitrogen limitation. Results from Southern hybridization and sequencing of PCR products suggest the presence of a family of similar protein kinases in this strain. One of the corresponding genes (pknA) was isolated from a gene library. The N-terminal region of its amino acid sequence shows significant similarity to the catalytic domains of eukaryotic-type protein kinases. Expression of this gene was found to be developmentally regulated. Inactivation of pknA led to colonies that appeared light green and rough in the absence of combined nitrogen. Mutant filaments produce fewer heterocysts than wild-type ones. These results suggest that pknA is required for both normal cellular growth and differentiation of Anabaena PCC 7120.
Mutational analysis of the helper component-proteinase gene of a potyvirus: effects of amino acid substitutions, deletions, and gene replacement on virulence and aphid transmissibility.
Authors: Atreya C D , Pirone T P
Published: May 2006

We have previously provided evidence that amino acid substitutions within the N-terminal portion of the helper component-proteinase (HC-Pro) from tobacco vein mottling virus (TVMV), in particular at Lys-307, not only affect the aphid transmission activity of HC-Pro but also have a significant effect on TVMV virulence. In the present study amino acids which differ in their charge properties were substituted at position 307. A highly basic residue was required to retain helper component activity and virulence. Deletion and insertion mutagenesis in the 5' terminus of the HC-Pro gene suggested that this RNA domain may be an essential element for TVMV infectivity. Replacement of the TVMV HC-Pro gene with that from another potyvirus, zucchini yellow mosaic virus, maintained infectivity and aphid transmissibility of the chimeric virus, although symptoms were attenuated. Our results suggest that, in addition to its importance in aphid transmission, the HC-Pro gene may be of general importance in regulating virulence of potyviruses, possibly by interaction of these sequences with the host.
Immunocytochemical localization of the plasma membrane calcium pump, calbindin-D28k, and parvalbumin in Purkinje cells of avian and mammalian cerebellum.
Authors: de Talamoni N , Smith C A , Wasserman R H , Beltramino C , Fullmer C S , Penniston J T
Published: May 2006

A monoclonal antibody produced against the human erythrocyte plasma membrane calcium pump (PMCA) was shown to react immunohistochemically with an epitope of the PMCA in avian and mammalian cerebellum. Western blot analysis of purified synaptosomes and homogenates from avian cerebellum revealed major immunoreactive proteins with molecular masses (130 kDa and 138 kDa) similar to those of purified erythrocyte PMCA. Dual-imaging confocal immunofluorescence microscopy of avian cerebellum showed that the PMCA antibody stained the periphery of the soma whereas calbindin-D28k was located in the cytosol. PMCA heavily stained the more distal dendrites of the Purkinje cells and, within the resolution of the fluorescence procedure, colocalized with calbindin-D28k. By using alkaline phosphatase-conjugated second antibody, PMCA was again localized to the peripheral soma, to a segmental pattern in dendrites, and to presumed spiny elements. The soma periphery and dendrites of Purkinje cells of the rat cerebellum were also prominently stained with anti-PMCA antibody and compared to parvalbumin localization. Dendritic depolarization and dendritic spiking behavior are significant Ca(2+)-dependent events of Purkinje cells. The rapid decline of intracellular free Ca2+ after the rapid rise time of Ca2+ transients is considered to be due to sequestration by Ca2+ buffers, uptake by intracellular stores, and Ca2+ extrusion mechanisms, the latter a function of PMCA now shown immunohistochemically to be a prominent feature of Purkinje cell dendrites.
Isolation of cDNAs for perilipins A and B: sequence and expression of lipid droplet-associated proteins of adipocytes.
Authors: Greenberg A S , Egan J J , Wek S A , Moos M C , Londos C , Kimmel A R
Published: May 2006

The major cAMP-dependent protein kinase (A-kinase) substrate in adipocytes is perilipin, a protein found exclusively at the surface of the lipid storage droplets. Using anti-perilipin serum, we have isolated two related classes of full-length coding cDNAs, designated perilipin A and B, from a rat adipocyte cDNA expression library. The two cDNAs derive from two mRNA species that arise by differential splicing. The mRNAs are predicted to encode perilipins A and B, proteins of 517 aa (56,870 Da) and 422 aa (46,420 Da), respectively, which share a common 406-aa N-terminal sequence. The predicted perilipin A contains peptides present in proteolytic digests of the purified 62-kDa form of perilipin from rat adipocytes, as well as the requisite consensus A-kinase phosphorylation sites. Like perilipin A, the B form is expressed in adipocytes and is associated with lipid storage droplets. Modeling of predicted secondary structures fails to reveal an underlying basis for the tenacious association of perilipins with lipid droplets. These proteins exhibit a significant sequence relationship (approximately 65% similarity through 105 aa) with only one other known protein, the adipocyte differentiation-related protein (ADRP). Like the perilipins, ADRP appears to be adipocyte-specific, which suggests that they interact in a related intracellular pathway. The molecular probes for perilipins A and B described here will permit detailed analyses of their functional role(s) in lipid metabolism.
Coexisting stable conformations of gaseous protein ions.
Authors: Suckau D , Shi Y , Beu S C , Senko M W , Quinn J P , Wampler F M , McLafferty F W
Published: May 2006

For further insight into the role of solvent in protein conformer stabilization, the structural and dynamic properties of protein ions in vacuo have been probed by hydrogen-deuterium exchange in a Fourier-transform mass spectrometer. Multiply charged ions generated by electrospray ionization of five proteins show exchange reactions with 2H2O at 10(-7) torr (1 torr = 133.3 Pa) exhibiting pseudo-first-order kinetics. Gas-phase compactness of the S-S cross-linked RNase A relative to denatured S-derivatized RNase A is indicated by exchange of 35 and 135 hydrogen atoms, respectively. For pure cytochrome c ions, the existence of at least three distinct gaseous conformers is indicated by the substantially different values--52, 113, and 74--of reactive H atoms; the observation of these same values for ions of a number--2, 7, and 5, respectively--of different charge states indicates conformational insensitivity to coulombic forces. For each of these conformers, the compactness in vacuo indicated by these values corresponds directly to that of a known conformer structure in the solution from which the conformer ions are produced by electrospray. S-derivatized RNase A ions also exist as at least two gaseous conformers exchanging 50-140 H atoms. Gaseous conformer ions are isometrically stable for hours; removal of solvent greatly increases conformational rigidity. More specific ion-molecule reactions could provide further details of conformer structures.
Tumor necrosis factor alpha induces the expression of transforming growth factor alpha and the epidermal growth factor receptor in human pancreatic cancer cells.
Authors: Schmiegel W , Roeder C , Schmielau J , Rodeck U , Kalthoff H
Published: May 2006

Recombinant human tumor necrosis factor (TNF)-alpha increased the expression of epidermal growth factor receptor (EGFR) mRNA and protein in all of six human pancreatic carcinoma cell lines tested. In addition, TNF-alpha increased the expression of an EGFR ligand, transforming growth factor (TGF)-alpha, at the mRNA and protein level in all cell lines. Increased expression of EGFR protein was associated with elevated steady-state EGFR mRNA levels. Nuclear run-on analysis showed that increase in EGFR mRNA was due to an increased rate of transcription. Induction of EGFR mRNA expression by TNF-alpha was abrogated by cycloheximide but occurred independently of TNF-alpha-induced production of TGF-alpha protein. Protein kinase A or Gi-type guanine nucleotide-binding proteins were not involved in this process as assessed by using appropriate stimulators and inhibitors of these signal transduction pathways. By contrast, staurosporine, an inhibitor of protein kinase C, partially inhibited, and 4-bromophenacyl bromide, a phospholipase inhibitor, completely inhibited TNF-alpha-dependent EGFR mRNA expression. The phospholipase C-specific inhibitor tricyclodecan-9-yl xanthogenate did not alter TNF-alpha-dependent EGFR mRNA expression, suggesting that phospholipase A2 is involved in the modulation of EGFR expression by TNF-alpha. The simultaneous induction of a ligand/receptor system by TNF-alpha suggests that this cytokine modulates autocrine growth-regulatory pathways in pancreatic cancer cells.
Pattern of aromatic and hydrophobic amino acids critical for one of two subdomains of the VP16 transcriptional activator.
Authors: Regier J L , Shen F , Triezenberg S J
Published: May 2006

Structural features of the transcriptional activation domain of the herpes simplex virion protein VP16 were examined by oligonucleotide-directed mutagenesis. Extensive mutagenesis at position 442 of the truncated VP16 activation domain (delta 456), normally occupied by a phenylalanine residue, demonstrated the importance of an aromatic amino acid at that position. On the basis of an alignment of the VP16 sequence surrounding Phe-442 and the sequences of other transcriptional activation domains, we subjected leucine residues at positions 439 and 444 of VP16 to mutagenesis. Results from these experiments suggest that bulky hydrophobic residues flanking Phe-442 also contribute significantly to the function of the truncated VP16 activation domain. Restoration of amino acids 457-490 to various Phe-442 mutants partially restored activity. Although the pattern of amino acids surrounding Phe-473 resembles that surrounding Phe-442, mutations of Phe-473 did not dramatically affect activity; in fact, Phe-475 appears more sensitive to mutations than does Phe-473. We infer that the two regions of VP16 (amino acids 413-456 and 457-490) possess unique structural features, although neither is likely to be an amphipathic alpha-helix or an "acidic blob." These results, considered with previous in vitro activation and inhibition studies, suggest that the two subdomains of VP16 affect transcription by different mechanisms.