bluuebunny/crossref_metadata_2025 huggingface dataset:
Mitosis-specific phosphorylation of polyomavirus middle-sized tumor antigen and its role during cell transformation.
Authors: Pérez L , Paasinen A , Schnierle B , Käch S , Senften M , Ballmer-Hofer K
Published: May 2006

Transformation of cells in culture by polyomavirus is mediated by one of its early gene products, middle-sized tumor antigen (MTAg). This protein forms multiple complexes with cellular enzymes such as tyrosine kinases (pp60c-src), a phosphatidylinositol 3-kinase, and phosphatase 2A. Association with MTAg leads to the activation of pp60c-src through interference with phosphorylation at Tyr-527, a site negatively regulating src kinase activity. MTAg abrogates mitosis-specific activation of pp60c-src, resulting in constitutive high kinase activity of the enzyme throughout all phases of the cell cycle. Here we report that MTAg is transiently modified during mitosis, resulting in an increase in its apparent molecular size on SDS/acrylamide gels. Similarly, MTAg isolated from interphase cells and phosphorylated by the cell cycle-regulated serine/threonine kinase p34cdc2 in vitro has increased molecular mass. The large molecular mass form of the protein can be converted to the authentic 56-kDa form upon dephosphorylation by potato acid phosphatase. Two putative phosphorylation sites for a cdc2-like kinase were identified as Thr-160 and -291, respectively. Conversion of Thr-160 to Ala resulted in a transformation-defective mutant protein that was still capable of associating with pp60c-src, phosphatidylinositol 3-kinase, and phosphatase 2A, while the corresponding mutant in position 291 was wild type with respect to all parameters measured so far. These data suggest that phosphorylation by p34cdc2 or a related cell cycle-regulated kinase modulates the interaction of MTAg with cellular targets that are crucial for cell transformation.
Csx: a murine homeobox-containing gene specifically expressed in the developing heart.
Authors: Komuro I , Izumo S
Published: May 2006

The molecular control of the differentiation process depends in part on lineage-restricted transcription factors that regulate expression of tissue-specific genes. Although significant progress has been made in molecular understanding of skeletal muscle differentiation, no information is available concerning the genes involved in development of the heart, the first organ to form in vertebrate embryos. Many vertebrate homeobox-containing genes have been shown to be expressed in broad regions of the mouse embryo, but no expression of a homeobox gene has been found in the most anterior region of the early embryo, the heart primordium. We report here on the cloning of a murine homeobox cDNA, Csx (cardiac-specific homeobox). The Csx homeodomain sequence is divergent from those of the Hox class genes but is related to that of Drosophila msh-2 (NK-4), which plays a key role in Drosophila heart formation. Csx is conserved in evolution and Csx homologs exist in all vertebrates examined. Transcripts of Csx are detected from the presomite stage (7.5 days postcoitum), when mesoderm differentiates into promyocardium. Csx expression is restricted in the myocardial cells from 8.5 days postcoitum through adult. Csx is not expressed in skeletal or smooth muscle or any other tissues examined. Expression of Csx precedes that of cardiac-specific genes in embryonic stem cells differentiating into beating myocardial cells in vitro. Although physiological function of Csx is yet to be determined, the temporal and spacial pattern of Csx expression raises a possibility that Csx may play a critical role in the differentiation of cardiac cells.
CCAAT/enhancer-binding protein mRNA is translated into multiple proteins with different transcription activation potentials.
Authors: Ossipow V , Descombes P , Schibler U
Published: May 2006

The CCAAT/enhancer-binding protein (C/EBP) alpha is a leucine zipper protein that is preferentially expressed in certain cell types, such as adipocytes and hepatocytes. Here we show that C/EBP alpha mRNA is translated into two major proteins, C/EBP-42 and C/EBP-30, that differ in their content of N-terminal amino acid sequences. These results are best explained by a ribosome-scanning mechanism in which a fraction of ribosomes ignore the first two AUGs and initiate translation at an AUG located 351 nt downstream of the first one. Because C/EBP-30, the translation product initiated at the third AUG, is devoid of the potent transcription-activation domain contained in C/EBP-42, the former protein stimulates transcription from the mouse albumin promoter much less efficiently than the latter. The gene encoding the liver-enriched transcriptional-activator protein LAP (C/EBP-beta) has also been shown to issue two proteins, LAP and the liver-enriched transcriptional-inhibitory protein LIP, with different transcription-activation potentials. The production of multiple proteins from a single mRNA is not only shared between different C/EBP family members but also appears to be conserved in vertebrate evolution.
Polarization of yeast cells in spatial gradients of alpha mating factor.
Authors: Segall J E
Published: May 2006

The process of cell fusion during mating of the yeast Saccharomyces cerevisiae is mediated by factors secreted by the mating partners. Spatial gradients of one of these mating factors, alpha-factor, polarized the growth of projections by MATa cells. The site of previous budding did not affect the direction of polarization, and subsequent budding was also polarized if mating factor was removed. Orientation occurred in the presence of nocodazole, suggesting that microtubules were not critical. At extremely low concentrations of alpha-factor, sst2-mutants (which in genetic studies do not discriminate between partners producing different amounts of alpha-factor) were able to polarize their projections. The sensitivity of this spatial sensing mechanism in wild-type cells is such that differences in receptor occupancy estimated to be about 1% are sufficient for orientation.
Isoprenylation of the plant molecular chaperone ANJ1 facilitates membrane association and function at high temperature.
Authors: Zhu J K , Bressan R A , Hasegawa P M
Published: May 2006

We demonstrate that ANJ1, a higher plant homolog of the bacterial molecular chaperone DnaJ, is a substrate in vitro for protein farnesyl- and geranylgeranyl-transferase activities present in cell extracts of the plant Atriplex nummularia and yeast Saccharomyces cerevisiae. Isoprenylation did not occur when cysteine was replaced by serine in the CAQQ motif at the carboxyl terminus of ANJ1, indicating that this sequence functions as a CaaX consensus sequence for polyisoprenylation (where C is cysteine, a is an aliphatic residue, and X is any amino acid residue). Substitution of leucine for the terminal glutamine did not result in the expected geranylgeranylation as occurs with mammalian proteins containing a carboxyl-terminal leucine. Unlike the wild-type ANJ1, neither of the proteins containing these amino acid substitutions could functionally complement the yeast temperature-sensitive mutant mas5. Farnesylation enhanced the association of ANJ1 with A. nummularia microsomal membranes. Electrophoretic mobility of ANJ1 from the plant indicated that the protein is isoprenylated in vivo.
Giardiavirus double-stranded RNA genome encodes a capsid polypeptide and a gag-pol-like fusion protein by a translation frameshift.
Authors: Wang A L , Yang H M , Shen K A , Wang C C
Published: May 2006

Giardiavirus is a small, nonenveloped virus comprising a monopartite double-stranded RNA genome, a major protein of 100 kDa, and a less abundant polypeptide of 190 kDa. It can be isolated from the culture supernatant of Giardia lamblia, a parasitic flagellate in human and other mammals, and efficiently infects other virus-free G. lamblia. A single-stranded copy of the viral RNA can be electroporated into uninfected G. lamblia cells to complete the viral replication cycle. Giardiavirus genomic cDNA of 6100 nt was constructed and its sequence revealed the presence of two large open reading frames that are separated by a -1 frameshift and share an overlap of 220 nt. The 3' open reading frame contains all consensus RNA-dependent RNA polymerase sequence motifs. A heptamer-pseudoknot structure similar to those found at ribosomal slippage sites in retroviruses and yeast killer virus was identified within this overlap. Immunostudies using antisera against synthesized peptides from four regions in the two open reading frames indicated that the 100- and 190-kDa viral proteins share a common domain in the amino-terminal region. But the 190-kDa protein makes a -1 switch of its reading frame beyond the presumed slippage heptamer and is therefore a -1 frameshift fusion protein similar to the gag-pol fusion protein found in retroviruses.
A model of human immunodeficiency virus encephalitis in scid mice.
Authors: Tyor W R , Power C , Gendelman H E , Markham R B
Published: May 2006

Human immunodeficiency virus (HIV)-associated dementia complex is a common and devastating manifestation of the late phases of HIV infection. The pathogenesis of dementia complex is poorly understood and effective treatments have not been developed, in part because of the lack of an appropriate animal model. Mice with severe combined immunodeficiency (scid mice), which accept xenografts without rejection, were intracerebrally inoculated with human peripheral blood mononuclear cells and HIV. One to 4 weeks after inoculation, the brains of these mice contained human macrophages (some of which were HIV p24 antigen positive), occasional multinucleated cells, and striking gliosis by immunocytochemical staining. Human macrophages also were frequently positive for tumor necrosis factor type alpha and occasionally for interleukin 1 and VLA-4. Cultures of these brains for HIV were positive. Generally, human macrophages were not present in the brains of control mice, nor was significant gliosis, and HIV was not recovered from mice that received HIV only intracerebrally. Pathologically, this model of HIV encephalitis in scid mice resembles HIV encephalitis in humans and the data suggest that the activation of macrophages by infection with HIV results in their accumulation and persistence in brain and in the development of gliosis. This model of HIV encephalitis should provide insights into the pathogenesis and treatment of this disorder.
A model for tumor suppression using H-1 parvovirus.
Authors: Telerman A , Tuynder M , Dupressoir T , Robaye B , Sigaux F , Shaulian E , Oren M , Rommelaere J , Amson R
Published: May 2006

A model system is proposed to investigate, at the molecular level, the pathways of tumor suppression. As a tool for the selection of cells with a suppressed phenotype, we used the H-1 parvovirus that preferentially kills various neoplastic cells. From the human K562 leukemia cells, we isolated a clone, KS, that is resistant to the cytopathic effect of the H-1 virus and displays a suppressed malignant phenotype. The suppressed malignancy and the cellular resistance to H-1 killing appear to depend on the activity of wild-type p53. Whereas the KS cells express wild-type p53, the protein is undetectable in the parental K562 cells. Experiments with p53 mutants suggest that wild-type p53, in its functionally intact state, contributes to the resistance against the cytopathic effect of H-1 parvovirus.
Molecular cloning and characterization of rho, a ras-related small GTP-binding protein from the garden pea.
Authors: Yang Z , Watson J C
Published: May 2006

The rho proteins, members of the ras superfamily of small GTP-binding proteins, play a central role in the modulation of cellular functions involving the actin cytoskeleton such as in the establishment of cell polarity and morphology. As a first step in elucidating signal transduction pathways leading to processes mediated by the actin cytoskeleton in plants, we initiated cloning and characterization of rho proteins from pea. One rho-related, partial cDNA clone of 167 bp was isolated utilizing a polymerase chain reaction-based cloning strategy, using degenerate primers that correspond to conserved domains within the rho proteins. A full-length cDNA was isolated by screening a pea cDNA library using the 167-bp cDNA as a probe. The Rho1Ps cDNA contains an open reading frame encoding a polypeptide (Rho1Ps) of 197 amino acids that shows 45-64% sequence identity to members of the rho family and about 30% identity to other members of the ras superfamily. In addition to the nucleotide-binding and GTPase domains, Rho1Ps shares conserved residues and motifs unique to the rho proteins. Purified Rho1Ps protein expressed in Escherichia coli retains specific GTP-binding activity. These data indicate that Rho1Ps encodes a small GTP-binding protein of the rho family. The Rho1Ps transcript is expressed in all organs of pea seedlings, being more abundant in root tips and apical buds. DNA gel blot analyses show that the rho proteins in pea are encoded by a multigene family.
Weighting in sequence space: a comparison of methods in terms of generalized sequences.
Authors: Vingron M , Sibbald P R
Published: May 2006

Four methods for weighting aligned biological sequences have recently appeared that differ mathematically, philosophically, and in their results. Thus, while there is consensus about the need to weight sequences, the method to use is contentious. A geometric analysis based on a continuous sequence space is presented that provides a common framework in which to compare the methods. It is concluded that there are two "best" methods. When the sequences are known to be phylogenetically related and a tree can be generated without introducing excessive stress into the data, the method of Altschul et al. [Altschul, S. F., Carroll, R. J. & Lipman, D. J. (1989) J. Mol. Biol. 207, 647-653] is appropriate. When the sequences are not known to be phylogenetically related or a tree cannot be produced without unduly distorting the distances between the sequences, a modification of the method of Sibbald and Argos [Sibbald, P. R. & Argos, P. (1990) J. Mol. Biol. 216, 813-818] is preferable.
Chimeric-transgenic mice represent a powerful tool for studying how the proliferation and differentiation programs of intestinal epithelial cell lineages are regulated.
Authors: Hermiston M L , Green R P , Gordon J I
Published: May 2006

An in vivo system has been developed for examining the effects of wild-type or mutant proteins on cell fate determination in the mouse intestinal epithelium or on the proliferation and differentiation programs of its component epithelial lineages. This system takes advantage of the fact that at the conclusion of gut morphogenesis, each intestinal crypt is composed of a monoclonal population of cells descended from a single active multipotent stem cell, each villus is supplied by several monoclonal crypts, and the four principal cell types of the intestinal epithelium differentiate during a rapid, geographically well-organized migration along the crypt-to-villus axis. Embryonic stem (ES) cells (129/Sv origin) are initially transfected with recombinant DNAs consisting of a reporter of interest linked to transcriptional regulatory elements that control the cell lineage-specific, differentiation-dependent, and axial patterns of expression of fatty acid binding protein genes in the gut. Stably transfected ES cells are subsequently introduced into host C57BL/6 blastocysts to generate chimeric-transgenic mice. At the borders of ES cell-derived and host blastocyst-derived epithelium, intestinal villi are found that are supplied by both ES cell- and host blastocyst-derived crypts. These villi can be rapidly identified in fixed whole-mount preparations of intestine using the alpha-L-fucose-specific Ulex europaeus agglutinin type I (UEA-I) lectin. They appear striped because UEA-I recognizes a cell-surface carbohydrate polymorphism between the inbred strains used to generate the chimeric animals. The strength of this system derives from the fact that two gut epithelial populations can be compared and contrasted that occupy virtually identical positions along the crypt-to-villus and duodenal-to-colonic axes within the same animal and differ only by the presence or absence of a single gene product. The band of blastocyst-derived epithelium in these striped, polyclonal villi can be used as an internal control to assess the biological effect of the transfected gene product produced in the adjacent stripe of ES-derived cells. The system can be used for either gain-of-function or loss-of-function experiments.
Cell lineage-specific and differentiation-dependent patterns of CCAAT/enhancer binding protein alpha expression in the gut epithelium of normal and transgenic mice.
Authors: Chandrasekaran C , Gordon J I
Published: May 2006

The proliferation and differentiation programs of gut epithelial cells are expressed rapidly and perpetually along an anatomically well defined pathway. The mouse intestine thus provides an excellent in vivo model system to define the contributions of CCAAT enhancer binding protein alpha (C/EBP alpha) and related bZIP proteins to these processes. Immunocytochemical studies revealed that C/EBP alpha is produced in villus-associated enterocytes located in the duodenum and jejunum of adult mice. The protein is located in the cytoplasmic and nuclear compartments of these cells. C/EBP alpha is not detectable in proliferating and nonproliferating epithelial cells situated in small intestinal crypts nor is it evident in any gut epithelial cell lineage located in the ileum and colon. The related C/EBP beta and C/EBP delta proteins are not detectable by sensitive immunocytochemical methods in epithelial cells distributed along the duodenal-to-colonic axis. Developmental surveys indicate that C/EBP alpha is confined to postmitotic, villus-associated epithelial cells during conversion of the polyclonal intervillus epithelium to monoclonal crypts. Analyses of intestinal isografts reveal that these developmental stage-specific, lineage-specific, differentiation-dependent, and regional patterns of C/EBP alpha expression can be established and maintained in the absence of exposure to luminal contents. Transgenic mice containing nucleotides -1178 to +28 of the rat intestinal fatty acid binding protein gene (I-FABP-1178 to +28) linked to the simian virus 40 large tumor antigen (T antigen) gene express T antigen in villus-associated enterocytes. This results in reentry of enterocytes into the cell cycle and a silencing of C/EBP alpha expression without an apparent effect on the accumulation of several markers of this lineage's terminal differentiation program or on gut morphogenesis. These findings indicate that there is a relationship between expression of C/EBP alpha in enterocytes and their exit from the cell cycle and suggest that I-FABP-1178 to +28/simian virus 40 T antigen transgenic mice could provide a screening assay for examining the role of C/EBP alpha in regulating the activity of genes known to be transcribed during differentiation of this gut epithelial cell lineage.
Aging and resistance to oxidative damage in Caenorhabditis elegans.
Authors: Larsen P L
Published: May 2006

The dauer larva state and the age-1 mutation, both of which extend life-span in Caenorhabditis elegans, were tested for hyperresistance to cellular damage that may be relevant to aging. The age-1 strain TJ401 displayed hyperresistance to oxidative stress relative to its parental strain. The activities of two enzymes that protect cells from oxidative damage, superoxide dismutase (SOD) and catalase, showed an age-dependent increase in mutant animals, which was not seen in the parental strain. These increases in activities paralleled the time course of the hyperresistance. The results are consistent with the age-1 gene product functioning as a negative regulator of SOD and catalase activities. In wild-type and age-1 dauer larvae, elevated levels of SOD activity, but not of catalase activity, were present when compared with young adults. The common increase in SOD activity prompted cloning the C. elegans Cu/Zn SOD gene. Its position on the physical map of the genome was in the region to which the age-1 gene has been genetically mapped, but it is unlikely that a mutation at the SOD locus confers the Age phenotype. Results support the free radical theory of aging by suggesting that the increased resistance to oxidative stress may be among the causes of increased longevity in both strain TJ401 and in the dauer larva.
Skeletal troponin C reduces contractile sensitivity to acidosis in cardiac myocytes from transgenic mice.
Authors: Metzger J M , Parmacek M S , Barr E , Pasyk K , Lin W I , Cochrane K L , Field L J , Leiden J M
Published: May 2006

Depressed contractile function plays a primary role in the pathophysiology of acute myocardial ischemia. Intracellular acidification is an important factor underlying the inhibition of force production in the ischemic myocardium. The effect of acidosis to depress contractility is markedly greater in cardiac as compared to skeletal muscle; however, the molecular basis of this difference in sensitivity to acidosis is not clearly understood. In this report, we describe transgenic mice that express the fast skeletal isoform of troponin C (sTnC) in cardiac muscle. In permeabilized single cardiac myocytes the shift in the midpoint of the tension-pCa relationship (i.e., pCa50, where pCa is -log[Ca2+]) due to lowering pH from 7.00 to 6.20 was 1.27 +/- 0.03 (n = 7) pCa units in control cardiac TnC (cTnC) expressing myocytes and 0.96 +/- 0.04 (n = 11) pCa unit in transgenic cardiac myocytes (P < 0.001). The effect of pH to alter maximum Ca(2+)-activated tension was unchanged by TnC isoforms in these cardiac myocytes. In a reciprocal experiment, contractile sensitivity to acidosis was increased in fast skeletal muscle fibers following extraction of endogenous sTnC and reconstitution with purified cTnC in vitro. Our findings demonstrate that TnC plays an important role in determining the profound sensitivity of cardiac muscle to acidosis and identify cTnC as a target for therapeutic interventions designed to modify ischemia-induced myocardial contractile dysfunction.
The human gastrin/cholecystokinin type B receptor gene: alternative splice donor site in exon 4 generates two variant mRNAs.
Authors: Song I , Brown D R , Wiltshire R N , Gantz I , Trent J M , Yamada T
Published: May 2006

Gastrin and its carboxyl-terminal homolog cholecystokinin (CCK) exert a variety of biological actions in the brain and gastrointestinal tract that are mediated in part through one or more G protein-coupled receptors which exhibit similar affinity for both peptides. Genomic clones encoding a human gastrin/CCKB receptor were isolated by screening a human EMBL phage library with a partial-length DNA fragment which was based on the nucleotide sequence of the canine gastrin receptor. The gene contained a 1356-bp open reading frame consisting of five exons interrupted by 4 introns and was assigned to human chromosome 11p15.4. A region of exon 4, which encodes a portion of the putative third intracellular loop, appears to be alternatively spliced to yield two different mRNAs, one containing (452 amino acids; long isoform) and the other lacking (447 amino acids; short isoform) the pentapeptide sequence Gly-Gly-Ala-Gly-Pro. The two receptor isoforms may contribute to functional differences in gastrin- and CCK-mediated signal transduction.
Transport of glutathione diethyl ester into human cells.
Authors: Levy E J , Anderson M E , Meister A
Published: May 2006

Glutathione monoesters in which the carboxyl group of the glycine residue is esterified were previously found, in contrast to glutathione itself, to be effectively transported into various types of cells and to be converted intracellularly into glutathione. Glutathione monoesters are thus useful for prevention of oxidative stress, certain toxicities, and for treatment of glutathione deficiency. Glutathione diethyl ester is rapidly split to the glutathione monoethyl ester by mouse plasma glutathione diester alpha-esterase activity. Thus, as expected, glutathione mono- and diesters have similar effects on cellular glutathione levels in mice. However, human plasma lacks glutathione diester alpha-esterase; thus, it became of interest to compare the transport properties of glutathione mono- and diesters in human cells. We found that human cells (erythrocytes, peripheral blood mononuclear cells, fibroblasts, ovarian tumor cells, and purified T cells) transport glutathione diethyl ester much more effectively than the corresponding monoethyl (glycyl) ester. Human cells rapidly convert glutathione diethyl ester to the monoester, whose intracellular levels rise to levels that are significantly higher than levels found after application of the monoester to the cells. High levels of the monoester provide the cells with a means of producing glutathione over a period of time. We conclude that glutathione diethyl ester is highly effective as a delivery agent for glutathione monoester, and thus for glutathione, in human cells and therefore could serve to decrease oxidative stress and toxicity. Hamster (and certain other animals) also lack plasma glutathione diester alpha-esterase and therefore would be suitable animal models. Previously reported toxicity of certain glutathione ester preparations appears to reflect the presence of impurities rather than effects of the esters.
Homologous recombination in the nuclear genome of Chlamydomonas reinhardtii.
Authors: Sodeinde O A , Kindle K L
Published: May 2006

Nuclear transformation of the unicellular green alga Chlamydomonas reinhardtii has thus far been characterized by integration of the introduced DNA into nonhomologous sites. In this study, the occurrence of homologous recombination events during transformation was investigated with the intent of developing strategies for gene targeting and gene disruption. Homologous recombination was monitored by using nonfunctional 5' and 3' deletion derivatives of the wild-type C. reinhardtii nit1 gene (encodes nitrate reductase) as selectable markers (p5' delta and p3' delta respectively) and the low reverting nit1-305 strain as the transformation recipient. After introduction of the DNA into the cell, intermolecular recombination between p5' delta and p3' delta occurs at a high frequency to restore a functional nit1 gene, indicating the presence of homologous recombination machinery in mitotic cells. Gene-targeting events at the nit1 locus were selected by restoring nit1-305 cells to prototrophy after transformation with only p5' delta and were confirmed by analysis of genomic DNA. By comparing the number of transformants obtained after transformation with p5' delta to the number obtained after transformation with a functional nit1 gene, the frequency of homologous-to-random integration events ranged between 1:1000 after glass bead-mediated transformation and 1:24 after bombardment with DNA-coated tungsten microprojectiles.
A yeast expression system for human galactose-1-phosphate uridylyltransferase.
Authors: Fridovich-Keil J L , Jinks-Robertson S
Published: May 2006

Galactose-1-phosphate uridylyltransferase (GALT) (UTP: alpha-D-hexose-1-phosphate uridylyltransferase, EC 2.7.7.10) is an essential enzyme of the Leloir pathway of galactose metabolism. Mutations in human GALT are associated with the potentially lethal disorder galactosemia, which affects 1 in 30,000-60,000 live-born infants. Although a number of base substitutions have been identified in the GALT alleles of galactosemia patients, the detailed biochemical impact of these mutations on GALT enzymatic activity remains obscure. Similarly, little is known about the sequence/structure/function relationships for wild-type human GALT. As a first step toward addressing these questions, we have developed a yeast-based expression system for the human enzyme. The wild-type human GALT coding sequence has been introduced into a strain of Saccharomyces cerevisiae that carries a disruption of the GALT-encoding GAL7 gene and, therefore, expresses no endogenous GALT. Transformants were tested for restoration of GALT activity both indirectly, by cell growth on galactose, and directly, by analysis of enzyme activity in cell extracts. The results of both tests were striking; wild-type human GALT functioned in yeast almost as well as the endogenous enzyme. In contrast, cells transformed with either human or yeast GALT sequences engineered to carry a common human GALT mutation, Q188R (changing Gln188 to Arg), exhibited essentially no detectable GALT activity and failed to grow on galactose. Lymphoblasts from patients homozygous for the Q188R mutation similarly exhibited essentially no detectable GALT activity in parallel assays. The results reported here establish the utility of the yeast-based expression system for human GALT and set the stage for more detailed studies of this important enzyme and its role in galactosemia.
Phylogenic and ontogenic expression of hepatocellular bile acid transport.
Authors: Boyer J L , Hagenbuch B , Ananthanarayanan M , Suchy F , Stieger B , Meier P J
Published: May 2006

The phylogenic and ontogenic expression of mRNA for the Na+/bile acid cotransporter was determined by Northern analysis utilizing a full-length cDNA probe recently cloned from rat liver. mRNA was detected in several mammalian species, including rat, mouse, and man, but could not be found in livers from nonmammalian species, including chicken, turtle, frog, and small skate. When expression of the bile acid transporter in developing rat liver was studied, mRNA was detected between 18 and 21 days of gestation, at the time when Na(+)-dependent bile acid transport is first detected. Two hepatoma cell lines (HTC and HepG2), the latter of which is known to have lost the Na+/bile acid cotransport system, also did not express mRNA for this transporter. Finally, when mRNA from the lower vertebrate (the small skate) was injected into Xenopus oocytes, only a sodium-independent, chloride-dependent transport system for bile acids was expressed, confirming the integrity of the mRNA and consistent with prior functional studies of bile acid transport in this species. These findings establish that the Na+/bile acid cotransport mRNA is first transcribed in mammalian species, a process that is recapitulated late during mammalian fetal development in rat liver, and that this mRNA is lost in dedifferentiated hepatocytes. In contrast, the mRNA for a multispecific Na+/independent organic anion transport system is transcribed earlier in vertebrate evolution.
Conserved nucleotide sequences in the open reading frame and 3' untranslated region of selenoprotein P mRNA.
Authors: Hill K E , Lloyd R S , Burk R F
Published: May 2006

Rat liver selenoprotein P contains 10 selenocysteine residues in its primary structure (deduced). It is the only selenoprotein characterized to date that has more than one selenocysteine residue. Selenoprotein P cDNA has been cloned from human liver and heart cDNA libraries and sequenced. The open reading frames are identical and contain a signal peptide, indicating that the protein is secreted by both organs and is therefore not exclusively produced in the liver. Ten selenocysteine residues (deduced) are present. Comparison of the open reading frame of the human cDNA with the rat cDNA reveals a 69% identity of the nucleotide sequence and 72% identity of the deduced amino acid sequence. Two regions in the 3' untranslated portion have high conservation between human and rat. Each of these regions contains a predicted stable stem-loop structure similar to the single stem-loop structures reported in 3' untranslated regions of type I iodothyronine 5'-deiodinase and glutathione peroxidase. The stem-loop structure of type I iodothyronine 5'-deiodinase has been shown to be necessary for incorporation of the selenocysteine residue at the UGA codon. Because only two stem-loop structures are present in the 3' untranslated region of selenoprotein P mRNA, it can be concluded that a separate stem-loop structure is not required for each selenocysteine residue.
Crystal structure of lignin peroxidase.
Authors: Edwards S L , Raag R , Wariishi H , Gold M H , Poulos T L
Published: May 2006

The crystal structure of lignin peroxidase (LiP) from the basidiomycete Phanerochaete chrysosporium has been determined to 2.6 A resolution by usine multiple isomorphous replacement methods and simulated annealing refinement. Of the 343 residues, residues 3-335 have been accounted for in the electron density map, including four disulfide bonds. The overall three-dimensional structure is very similar to the only other peroxidase in this group for which a high-resolution crystal structure is available, cytochrome c peroxidase, despite the fact that the sequence identity is only approximately 20%, LiP has four disulfide bonds, while cytochrome c peroxidase has none, and LiP is larger (343 vs. 294 residues). The basic helical fold and connectivity defined by 11 helical segments with the heme sandwiched between the distal and proximal helices found in cytochrome c peroxidase is maintained in LiP. Both enzymes have a histidine as a proximal heme ligand, which is hydrogen bonded to a buried aspartic acid side chain. The distal or peroxide binding pocket also is similar, including the distal arginine and histidine. The most striking difference is that, whereas cytochrome c peroxidase has tryptophans contacting the distal and proximal heme surfaces, LiP has phenylalanines. This in part explains why, in the reaction with peroxides, cytochrome c peroxidase forms an amino acid-centered free radical, whereas LiP forms a porphyrin pi cation radical.
Abnormal activation of H+ conductance in NADPH oxidase-defective neutrophils.
Authors: Nanda A , Grinstein S , Curnutte J T
Published: May 2006

To combat bacterial infection, phagocytes generate superoxide (O2-) and other microbicidal oxygen radicals. NADPH oxidase, the enzyme responsible for O2- synthesis, is deficient in chronic granulomatous disease (CGD) patients. Although O2- generation is accompanied by a large burst of metabolic acid production, intracellular pH (pHi) remains near neutrality due to the concomitant stimulation of H+ extrusion. Three major pathways contribute to pHi regulation in activated phagocytes: Na+/H+ exchange, vacuolar-type H+ pumps, and a H+ conductance. The present study analyzed the relationship between activation of the NADPH oxidase and stimulation of the H+ extrusion mechanisms in human blood neutrophils. Phorbol ester-induced activation of Na+/H+ exchange and H+ pumping occurred normally in cells from CGD patients. Unlike normal individuals, however, CGD patients were unable to activate the H+ conductive pathway. Thus, activation of the H+ conductance appears to be contingent on the assembly of a functional NADPH oxidase. These findings imply a dual role of the NADPH oxidase in O2- synthesis and in the regulation of pHi. The oxidase (or some components thereof) may itself undertake H+ translocation or, alternatively, may signal the activation of a separate H+ conducting entity.
Solubilized proteins from carrot (Daucus carota L.) membranes bind calcium channel blockers and form calcium-permeable ion channels.
Authors: Thuleau P , Graziana A , Ranjeva R , Schroeder J I
Published: May 2006

Calcium channels have been suggested to play a major role in the initiation of a large number of signal transduction processes in higher plant cells. However, molecular components of higher plant Ca2+ channels remain unidentified to date. Calcium channel blockers of the phenylalkylamine family and bepridil specifically inhibit Ca2+ influx into carrot (Daucus carota L.) cells. By using a phenylalkylamine azido derivative, a 75-kDa carrot membrane protein has been previously identified. Here we have partially purified this Ca2+ channel blocker-binding protein by lectin-affinity and ion-exchange chromatographies. The protein fraction containing the 75-kDa binding protein was incorporated into giant liposomes. Single-channel patch-clamp studies on these proteoliposomes showed the presence of Ca2+-permeable channel currents. These Ca2+-permeable channels were not stable. Recordings after durations of 2-10 min showed the appearance of nonselective ion channels with a permeability to calcium and chloride ions. These nonselective Ca2+-permeable ion channels, in contrast, were stable and were recorded for extended durations. The addition of the Ca2+ channel-blocker bepridil (10 M) led to the inhibition of these nonselective Ca2+-permeable channels by reducing the probability of channel opening. These results suggest that the 75-kDa Ca2+ channel blocker-binding protein from carrot cells plays a role in channel sensitivity to Ca2+ channel inhibitors and may constitute one of the components of Ca2+ channels in higher plants.
Cloning, sequence determination, and regulation of the ribonucleotide reductase subunits from Plasmodium falciparum: a target for antimalarial therapy.
Authors: Rubin H , Salem J S , Li L S , Yang F D , Mama S , Wang Z M , Fisher A , Hamann C S , Cooperman B S
Published: May 2006

Malaria remains a leading cause of morbidity and mortality worldwide, accounting for more than one million deaths annually. We have focused on the reduction of ribonucleotides to 2'-deoxyribonucleotides, catalyzed by ribonucleotide reductase, which represents the rate-determining step in DNA replication as a target for antimalarial agents. We report the full-length DNA sequence corresponding to the large (PfR1) and small (PfR2) subunits of Plasmodium falciparum ribonucleotide reductase. The small subunit (PfR2) contains the major catalytic motif consisting of a tyrosyl radical and a dinuclear Fe site. Whereas PfR2 shares 59% amino acid identity with human R2, a striking sequence divergence between human R2 and PfR2 at the C terminus may provide a selective target for inhibition of the malarial enzyme. A synthetic oligopeptide corresponding to the C-terminal 7 residues of PfR2 inhibits mammalian ribonucleotide reductase at concentrations approximately 10-fold higher than that predicted to inhibit malarial R2. The gene encoding the large subunit (PfR1) contains a single intron. The cysteines thought to be involved in the reduction mechanism are conserved. In contrast to mammalian ribonucleotide reductase, the genes for PfR1 and PfR2 are located on the same chromosome and the accumulation of mRNAs for the two subunits follow different temporal patterns during the cell cycle.
A hypothesis for the HLA-B27 immune dysregulation in spondyloarthropathy: contributions from enteric organisms, B27 structure, peptides bound by B27, and convergent evolution.
Authors: Scofield R H , Warren W L , Koelsch G , Harley J B
Published: May 2006

Several human rheumatic diseases occur predominantly in persons who carry the histocompatibility (HLA) class I allele B27. They have also been related to Gram-negative enteric microorganisms. In addition, the recent recovery of peptides bound to B27 has allowed an understanding of the structural requirements for their binding. Using the accumulated data base of protein sequences, we have tested a series of hypotheses. First, we have asked whether the primary amino acid sequence of the hypervariable regions of HLA-B27 shares short sequences with the proteins of Gram-negative enteric bacteria. The data demonstrate that, unique among the HLA-B molecules, the hypervariable regions of HLA-B27 unexpectedly share short peptide sequences with proteins from these bacteria. Second, we have asked whether the enteric proteins tend to satisfy the structural requirements for peptide binding to B27 in those regions of the sequence shared with B27. This hypothesis also tends to be true, especially in an allelically variable part of the B27 sequence which is predicted to bind B27 if it were to be presented as a free peptide. We conclude that HLA-B27 and enteric Gram-negative bacteria have undergone a previously unappreciated form of convergent evolution which may be important in the process leading to these rheumatic diseases. Moreover, the regions of the enteric bacterial proteins which are contiguous with the short sequences shared with B27 tend to have structures which are also predicted to bind B27. These observations suggest a mechanism for autoimmunity and lead to the prediction that the B27-associated diseases are mediated by a subset of T-cell receptors, B27, and the peptides bound by B27.
High-mobility-group 1 protein mediates DNA bending as determined by ring closures.
Authors: Pil P M , Chow C S , Lippard S J
Published: May 2006

High-mobility-group 1 protein (HMG1) is an abundant eukaryotic DNA-binding protein, the cellular role of which remains ill-defined. To test the ability of HMG1 itself to mediate curvature in double-stranded DNA, we examined its effect on the phage T4 DNA ligase-dependent cyclization of short DNA fragments. HMG1 caused circle formation for fragments > or = 87 bp. Fragments of 123, 100, 92, and 87 bp did not cyclize in the absence of protein but formed covalently closed circular monomers efficiently in the presence of HMG1, indicating that the protein is capable of introducing bends into the duplex. The bending activity was maintained by a 79-amino acid polypeptide corresponding to a single HMG-box domain of HMG1. The binding affinity for the DNA minicircle was greater than for the corresponding linear fragment. These findings indicate that the role of HMG1 could involve both structure-specific recognition of prebent DNA and distortion of the DNA helix by bending and that the HMG-box domain may actually be responsible for this activity.
Genetic mapping of tumor susceptibility genes involved in mouse plasmacytomagenesis.
Authors: Mock B A , Krall M M , Dosik J K
Published: May 2006

Plasmacytomas (PCTs) were induced in 47% of BALB/cAnPt mice by the intraperitoneal injection of pristane, in 2% of (BALB/c x DBA/2N)F1, and in 11% of 773 BALB/cAnPt x (BALB/cAnPt x DBA/2N)F1 N2 backcross mice. This result indicates a multigenic mode of inheritance for PCT susceptibility. To locate genes controlling this complex genetic trait, tumor susceptibility in backcross progeny generated from BALB/c and DBA/2N (resistant) mice was correlated with alleles of 83 marker loci. The genotypes of the PCT-susceptible progeny displayed an excess homozygosity for BALB/c alleles within a 32-centimorgan stretch of mouse chromosome 4 (> 95% probability of linkage) with minimal recombination (12%) near Gt10. Another susceptibility gene on mouse chromosome 1 may be linked to Fcgr2 (90% probability of linkage); there were excess heterozygotes for Fcgr2 among the susceptible progeny and excess homozygotes among the resistant progeny. Regions of mouse chromosomes 4 and 1 that are correlated with PCT susceptibility share extensive linkage homology with regions of human chromosome 1 that have been associated with cytogenetic abnormalities in multiple myeloma and lymphoid, breast, and endocrine tumors.
Mitochondrial DNA sequence evolution in the Arctoidea.
Authors: Zhang Y P , Ryder O A
Published: May 2006

Some taxa in the superfamily Arctoidea, such as the giant panda and the lesser panda, have presented puzzles to taxonomists. In the present study, approximately 397 bases of the cytochrome b gene, 364 bases of the 12S rRNA gene, and 74 bases of the tRNA(Thr) and tRNA(Pro) genes from the giant panda, lesser panda, kinkajou, raccoon, coatimundi, and all species of the Ursidae were sequenced. The high transition/transversion ratios in cytochrome b and RNA genes prior to saturation suggest that the presumed transition bias may represent a trend for some mammalian lineages rather than strictly a primate phenomenon. Transversions in the 12S rRNA gene accumulate in arctoids at about half the rate reported for artiodactyls. Different arctoid lineages evolve at different rates: the kinkajou, a procyonid, evolves the fastest, 1.7-1.9 times faster than the slowest lineage that comprises the spectacled and polar bears. Generation-time effect can only partially explain the different rates of nucleotide substitution in arctoids. Our results based on parsimony analysis show that the giant panda is more closely related to bears than to the lesser panda; the lesser panda is neither closely related to bears nor to the New World procyonids. The kinkajou, raccoon, and coatimundi diverged from each other very early, even though they group together. The polar bear is closely related to the spectacled bear, and they began to diverge from a common mitochondrial ancestor approximately 2 million years ago. Relationships of the remaining five bear species are derived.
A gene affecting Wallerian nerve degeneration maps distally on mouse chromosome 4.
Authors: Lyon M F , Ogunkolade B W , Brown M C , Atherton D J , Perry V H
Published: May 2006

When a nerve axon is cut or crushed, the nerve fibers in the distal part of the axon, separated from the cell body, undergo a form of spontaneous degeneration, known as Wallerian degeneration. A substrain of the mouse inbred strain C57BL, known as C57BL/Ola, carries a mutant form of a gene involved in Wallerian degeneration in the peripheral and central nervous systems, and in retrograde degeneration of retinal ganglion cells. Wallerian degeneration in this substrain is abnormally slow. Previously the defect had been shown to be due to an autosomal dominant gene. The locus has been given the name and symbol Wallerian degeneration Wld, with the mutant allele Wlds (Wallerian degeneration-slow). The Wld locus has now been mapped, by using conventional and molecular markers, to the distal end of chromosome 4, near the locus of pronatriodilatin (Pnd). The order of loci (with recombination distances in centimorgans, cM) is cen-D4Mit11-8.9 +/- 1.7 cM-Fuca-2.5 +/- 0.93 cM-Akp-2-3.2 +/- 1.1 cM-D4Mit48-3.5 +/- 1.1 cM-(Wld, Pnd, D4Mit49)-0.71 +/- 0.50 cM-(Eno-1, D4Mit33)-1.4 +/- 0.70 cM-D4Mit42-2.5 +/- 0.93 cM-D4Smh6b. The information on the position of the Wld locus should be valuable in further characterization of this gene involved in nerve degeneration and regeneration.
Modulation of transmission gain by protons at the photoreceptor output synapse.
Authors: Barnes S , Merchant V , Mahmud F
Published: May 2006

Synaptic transmission of the light response from photoreceptors to second-order cells of the retina was studied with the whole-cell patch-clamp technique in tiger salamander (Ambystoma tigrinum) retinal slices. Synaptic strength is modulated by extracellular pH in a striking manner: Light-sensitive postsynaptic currents in horizontal and bipolar cells were found to be exponential functions of pH, exhibiting an e-fold increase per 0.23 pH unit over the pH range from 7 to 8. Calcium channel currents in isolated photoreceptors were measured and also exhibited proton sensitivity. External alkalinization from pH 7 to 8 shifted the voltage dependence of channel activation negative by 12 mV. A model of the synaptic transfer function suggested that presynaptic Ca channels could be the primary sites of proton action. Increased Ca influx and transmitter release brought about by alkalinization give rise to larger postsynaptic currents. These results suggest that activity-dependent interstitial pH changes known to occur in the retina, while not alleviating signal clipping at this synapse, may provide an adaptative mechanism controlling gain at the photoreceptor output synapse.
Serine-173 of the Epstein-Barr virus ZEBRA protein is required for DNA binding and is a target for casein kinase II phosphorylation.
Authors: Kolman J L , Taylor N , Marshak D R , Miller G
Published: May 2006

An Epstein-Barr virus-encoded protein, ZEBRA, mediates the switch from latency to the viral lytic life cycle. ZEBRA's domain structure and DNA binding specificity resemble that of cellular transcriptional activators such as c-Fos/c-Jun. We show that ZEBRA, like c-Jun, is phosphorylated by casein kinase II (CKII). The principal site of phosphorylation is serine-173 (S173), five amino acids upstream of the basic DNA recognition domain. CKII phosphorylation abrogated ZEBRA's capacity to bind its target DNA sequences. S173 is a functional component of ZEBRA's DNA binding domain, since mutation of S173 to alanine (S173A) reduced DNA binding in vitro to 10% of wild-type levels. Transcriptional activation of a native viral promoter in vivo by mutant S173A was also reduced markedly. Reversible phosphorylation of S173 is likely to be an important means of regulating ZEBRA's activity in vivo.