bluuebunny/crossref_metadata_2025 huggingface dataset:
Identity of 4a-carbinolamine dehydratase, a component of the phenylalanine hydroxylation system, and DCoH, a transregulator of homeodomain proteins.
Authors: Citron B A , Davis M D , Milstien S , Gutierrez J , Mendel D B , Crabtree G R , Kaufman S
Published: May 2006

The principal pathway for the metabolism of phenylalanine in mammals is via conversion to tyrosine in a tetrahydrobiopterin-dependent hydroxylation reaction occurring predominantly in the liver. Recently, the proposal that certain hyperphenylalaninemic children may have a deficiency of carbinolamine dehydratase, a component of the phenylalanine hydroxylation system, has widened the interest in this area of metabolism. Upon cloning and sequencing the dehydratase, we discovered that this protein is identical to DCoH, the cofactor which regulates the dimerization of hepatic nuclear factor 1 alpha, a homeodomain transcription factor. The identity of the nuclear and cytoplasmic proteins is demonstrated by size, immunoblotting, stimulation of phenylalanine hydroxylase, and dehydratase activity. The evolution of the dual functions of regulation of phenylalanine hydroxylation activity and transcription activation in a single polypeptide is unprecedented.
Scorpion toxins targeted against the sarcoplasmic reticulum Ca(2+)-release channel of skeletal and cardiac muscle.
Authors: Valdivia H H , Kirby M S , Lederer W J , Coronado R
Published: May 2006

We report the purification of two peptides, called "imperatoxin inhibitor" and "imperatoxin activator," from the venom of the scorpion Pandinus imperator targeted against ryanodine receptor Ca(2+)-release channels. Imperatoxin inhibitor has a M(r) of approximately 10,500, inhibits [3H]ryanodine binding to skeletal and cardiac sarcoplasmic reticulum with an ED50 of approximately 10 nM, and blocks openings of skeletal and cardiac Ca(2+)-release channels incorporated into planar bilayers. In whole-cell recordings of cardiac myocytes, imperatoxin inhibitor decreased twitch amplitude and intracellular Ca2+ transients, suggesting a selective blockade of Ca2+ release from the sarcoplasmic reticulum. Imperatoxin activator has a M(r) of approximately 8700, stimulates [3H]ryanodine binding in skeletal but not cardiac sarcoplasmic reticulum with an ED50 of approximately 6 nM, and activates skeletal but not cardiac Ca(2+)-release channels. These ligands may serve to selectively "turn on" or "turn off" ryanodine receptors in fragmented systems and whole cells.
Transcription of the tumor necrosis factor alpha gene is rapidly induced by anti-immunoglobulin and blocked by cyclosporin A and FK506 in human B cells.
Authors: Goldfeld A E , Flemington E K , Boussiotis V A , Theodos C M , Titus R G , Strominger J L , Speck S H
Published: May 2006

The human tumor necrosis factor alpha (TNF-alpha) gene encodes a cytokine whose activities have been implicated in many immunopathological processes, including the activation and differentiation of lymphocytes. Originally identified as a monocyte factor, our studies and those of others have demonstrated that B and T lymphocytes produce TNF-alpha when stimulated by a variety of inducers. We report here that TNF-alpha gene transcription is rapidly and highly induced in three independently derived human Burkitt lymphoma cell lines, as well as in freshly isolated human splenic B cells, activated by antibodies to surface immunoglobulin. This burst in TNF-alpha gene transcription is associated with an induction of TNF-alpha bioactivity in the culture supernatants from stimulated splenic B cells. Moreover, induction of TNF-alpha gene transcription by anti-immunoglobulin was blocked by the immunosuppressants cyclosporin A and FK506. These studies demonstrate that TNF-alpha production is an early event in B-cell activation and they establish the efficacy of using immunosuppressants as probes in dissecting transcriptional activation pathways in human B cells.
Enzymatic catalysis and dynamics in low-water environments.
Authors: Affleck R , Xu Z F , Suzawa V , Focht K , Clark D S , Dordick J S
Published: May 2006

Enzymes suspended in organic solvents represent a versatile system for studying the involvement of water in enzyme structure and function. Addition of less than 1% (vol/vol) water to tetrahydrofuran containing 1 M 1-propanol leads to a substantial increase in the transesterification activity of subtilisin Carlsberg (from Bacillus licheniformis) that correlates with a sharp increase in the active-site polarity and a 90% decrease in the rotational correlation time (i.e., increase in mobility) of a nitroxide spin label within the active site. Water in excess of 1% has little additional effect on active-site polarity and coincides with a further increase in spin-label mobility, yet the transesterification activity decreases dramatically. Thus, transesterification activity increases and then decreases with increasing enzyme hydration and flexibility (which are presumably coupled through dielectric screening), suggesting that the conformation of partially hydrated subtilisin is different from that of the nearly dry enzyme--i.e., enzyme containing less than 9% (wt/wt) water.
Industrial ecology.
Authors: Patel C K
Published: May 2006

Industrial ecology addresses issues that will impact future production, use, and disposal technologies; proper use of the concept should reduce significantly the resources devoted to potential remediation in the future. This cradle-to-reincarnation production philosophy includes industrial processes that are environmentally sound and products that are environmentally safe during use and economically recyclable after use without adverse impact on the environment or on the net cost to society. This will require an industry-university-government round table to set the strategy and agenda for progress.
The third chromophore of DNA photolyase: Trp-277 of Escherichia coli DNA photolyase repairs thymine dimers by direct electron transfer.
Authors: Kim S T , Li Y F , Sancar A
Published: May 2006

Photolyases repair pyrimidine dimers in DNA by converting the light energy of 300- to 500-nm photons into chemical energy. Enzymes from various organisms contain two chromophore cofactors (FADH2 and either methenyltetrahydrofolate or 8-hydroxy-5-deazaflavin) that absorb the low-energy photons and initiate splitting of the cyclobutane ring by a radical mechanism. Here, we show that, in addition to these two chromophores, in the far UV range, direct excitation of one specific tryptophan residue (out of 15 total) in the polypeptide chain of Escherichia coli photolyase leads to splitting of the cyclobutane ring with high quantum yield (phi = 0.56), independent of the other chromophores. The specific tryptophan residue responsible for photosensitized repair was identified as Trp-277 by site-specific mutagenesis.
Extensive size polymorphism of the human keratin 10 chain resides in the C-terminal V2 subdomain due to variable numbers and sizes of glycine loops.
Authors: Korge B P , Gan S Q , McBride O W , Mischke D , Steinert P M
Published: May 2006

Existing data suggest that the human keratin 10 intermediate filament protein is polymorphic in amino acid sequence and in size. To precisely define the nature of the polymorphism, we have used PCR amplification and sequence analyses on DNA from several individuals including five with documented size variations of the keratin 10 protein. We found no variation in the N-terminal or rod domain sequences. However, we observed many variations in the V2 subdomain near the C terminus in glycine-rich sequences with a variation of as much as 114 base pairs (38 amino acids), but all individuals had either one or two variants. Our results show that (i) the keratin 10 system is far more polymorphic than previously realized, (ii) the polymorphism is restricted to insertions and deletions of the glycine-rich quasipeptide repeats that form the glycine-loop motif in the C-terminal domain, (iii) the polymorphism can be accounted for by simple allelic variations that segregate by normal Mendelian mechanisms, and (iv) the differently sized PCR products most likely represent different alleles of a single-copy gene per haploid genome.
J chain synthesis and secretion of hexameric IgM is differentially regulated by lipopolysaccharide and interleukin 5.
Authors: Randall T D , Parkhouse R M , Corley R B
Published: May 2006

Two functional polymeric forms of IgM can be produced by antibody-secreting B cells. Hexameric IgM lacks detectable J (joining) chain and activates complement 17-fold better than pentameric IgM, which usually contains one J chain per pentamer. Using the inducible B-cell lymphoma CH12, we determined if the synthesis of a particular polymeric form of IgM is a fixed property of B cells or can be altered. Lipopolysaccharide (LPS)-stimulated CH12 cells produced mixtures of IgM hexamers and pentamers, resulting in antibody with high complement-fixing activity. In contrast, interleukin-5-stimulated CH12 cells secreted predominantly pentameric IgM, with a correspondingly lower lytic activity. Differences in lytic activity were due only to the amount of hexameric IgM in the secreted antibody. Interleukin 5 stimulated higher production of J chain RNA and protein than LPS, while LPS induced the highest levels of the secretory form of mu protein. The amount of hexameric IgM secreted was therefore inversely proportional to the level of intracellular J chain protein in the responding B cells. We conclude that the biologic function of IgM produced by B cells differs depending on how they are stimulated and that this difference may be regulated by the relative availabilities of J chain and secretory mu proteins during IgM polymerization.
Analogues of luteinizing hormone-releasing hormone containing cytotoxic groups.
Authors: Janáky T , Juhász A , Bajusz S , Csernus V , Srkalovic G , Bokser L , Milovanovic S , Redding T W , Rékási Z , Nagy A
Published: May 2006

In an attempt to produce better cytotoxic analogues, chemotherapeutic antineoplastic radicals including an alkylating nitrogen mustard derivative of D-phenylalanine (D-melphalan), reactive cyclopropane, anthraquinone derivatives [2-(hydroxymethyl)anthraquinone and the anticancer antibiotic doxorubicin], and an antimetabolite (methotrexate) were coupled to suitably modified agonists and antagonists of luteinizing hormone-releasing hormone (LH-RH). Analogues with D-lysine6 and D-ornithine6 or N epsilon-(2,3-diaminopropionyl)-D-lysine and N delta-(2,3-diaminopropionyl)-D-ornithine were used as carriers for one or two cytotoxic moieties. The enhanced biological activities produced by the incorporation of D amino acids into position 6 of the agonistic analogues were further increased by the attachment of hydrophobic cytotoxic groups, resulting in compounds with 10-50 times higher activity than LH-RH. Most of the monosubstituted agonistic analogues showed high affinities for the membrane receptors of human breast cancer cells, while the receptor binding affinities of peptides containing two cytotoxic side chains were lower. Antagonistic carriers [Ac-D-Nal(2)1,D-Phe(4Cl)2,D-Trp3,Arg5,D-Lys6,D-Ala10] LH-RH [where Nal(2) is 3-(2-naphthyl)alanine], [Ac-D-Nal(2)1,D-Phe(4Cl)2,D-Trp3,Arg5,N epsilon-(2,3-diaminopropionyl)-D-Lys6,D-Ala10]LH-RH, and their D-Pal(3)3 homologs [Pal(3) is 3-(3-pyridyl)alanine] as well as [Ac-D-Nal(2)1,D-Phe(4Cl)2,D-Pal(3)3,Tyr5,N epsilon-(2,3-diamino-propionyl)-D-Lys6,D-Ala10]LH-RH were linked to cytotoxic compounds. The hybrid molecules inhibited ovulation in rats at doses of 10 micrograms and suppressed LH release in vitro. The receptor binding of cytotoxic analogues was decreased compared to the precursor peptides, although analogues with 2-(hydroxymethyl)anthraquinone hemiglutarate had high affinities. All of the cytotoxic analogues tested inhibited [3H]thymidine incorporation into DNA in cultures of human breast and prostate cancer cell lines. Some cytotoxic analogues also significantly suppressed the growth of mammary and prostate cancers in vivo in animal models.
Protein affinity chromatography with purified yeast DNA polymerase alpha detects proteins that bind to DNA polymerase.
Authors: Miles J , Formosa T
Published: May 2006

We have overexpressed the POL1 gene of the yeast Saccharomyces cerevisiae and purified the resulting DNA polymerase alpha polypeptide in an apparently intact form. We attached the purified DNA polymerase covalently to an agarose matrix and used this matrix to chromatograph extracts prepared from yeast cells. At least six proteins bound to the yeast DNA polymerase alpha matrix that did not bind to a control matrix. We speculate that these proteins might be DNA polymerase alpha accessory proteins. Consistent with this interpretation, one of the binding proteins, which we have named POB1 (polymerase one binding), is required for normal chromosome transmission. Mutations in this gene cause increased chromosome loss and an abnormal cell morphology, phenotypes that also occur in the presence of mutations in the yeast alpha or delta polymerase genes. These results suggest that the interactions detected by polymerase affinity chromatography are biologically relevant and may help to illuminate the architecture of the eukaryotic DNA replication machinery.
Molecular characterization of two plant flavonol sulfotransferases.
Authors: Varin L , DeLuca V , Ibrahim R K , Brisson N
Published: May 2006

cDNA clones coding for flavonol 3- and 4'-sulfotransferases (STs) were isolated by antibody screening of a cDNA expression library produced from poly(A)+ RNA extracted from terminal buds of Flaveria chloraefolia. Sequence analysis revealed full-length cDNA clones with open reading frames of 933 and 960 base pairs, which encode polypeptides containing 311 and 320 amino acids, respectively. This corresponds to a molecular mass of 36,442 Da for the 3-ST and 37,212 Da for the 4'-ST. Expression of these clones in Escherichia coli led to the synthesis of beta-galactosidase-ST fusion proteins having the same substrate and position specificities as those for the 3- and 4'-flavonol ST enzymes isolated from the plant. Comparison of the deduced amino acid sequence of the two clones revealed an overall identity of 69% in 311 amino acid residues. The two flavonol STs of F. chloraefolia also shared significant sequence similarities with steroid and aryl STs found in animal tissues and with the senescence marker protein 2 isolated from rat liver, suggesting an evolutionary link between plant and animal STs.
Telomeric arrays display high levels of heritable polymorphism among closely related plant varieties.
Authors: Broun P , Ganal M W , Tanksley S D
Published: May 2006

Tomato telomeres are composed of a terminal 7-base-pair tandem repeat and a closely liked 162-base-pair subtelomeric repeat (TGRI). Together, these repeats account for 2% of the total chromosomal DNA. Pulsed-field gel electrophoresis has been used to examine the long-range physical structure of these arrays in closely related varieties, and we report here that these arrays are undergoing heritable changes at a frequency as great as 2% per generation. Moreover, comparisons with other known hypervariable probes (e.g., human minisatellites and M13 sequences) revealed that telomeric sites are more variable than any other known region of the plant genome and can be used to distinguish closely related plant varieties (tomato and melon) that are otherwise very similar at the DNA level. The fact that the polymorphisms are inherited in a mendelian fashion suggests applications in genetic mapping of telomeres and identification of varieties.
Role of gender, season, and familiarity in discrimination of conspecific song by zebra finches (Taeniopygia guttata).
Authors: Cynx J , Nottebohm F
Published: May 2006

Operant conditioning techniques were used to assess how gender and song familiarity affect song discrimination in adult zebra finches (Taeniopygia guttata). Twenty-five males and females, divided into five cohorts, were trained to discriminate between conspecific songs at different times of year. Males discriminating between their own and another song from their own aviary reached criterion in the fewest number of trials, followed by males discriminating between two songs from their own aviary, then by males discriminating between songs they had not heard before. Females discriminating between two songs from their own aviary required more trials than males to reach criterion, but, unlike in males, song familiarity did not have a significant effect on song discrimination by females. The number of trials required to reach criterion was greater in winter than in summer, suggesting a photoperiodic effect in what has been regarded as a nonphotoperiodic species. Gender, season, and familiarity appear to affect how zebra finches discriminate between conspecific songs.
(125I)iodoazidococaine, a photoaffinity label for the haloperidol-sensitive sigma receptor.
Authors: Kahoun J R , Ruoho A E
Published: May 2006

A carrier-free radioiodinated cocaine photo-affinity label, (-)-3-(125I)iodo-4-azidococaine [(125I)IACoc], has been synthesized and used as a probe for cocaine-binding proteins. Photoaffinity labeling with 0.5 nM (125I)IACoc resulted in selective derivatization of a 26-kDa polypeptide with the pharmacology of a sigma receptor in membranes derived from whole rat brain, rat liver, and human placenta. Covalent labeling of the 26-kDa polypeptide was inhibited by 1 microM haloperidol, di(2-tolyl)guanidine (DTG), 3-(3-hydroxyphenyl)-N-(1-propyl)piperidine (3-PPP), dextromethorphan, and carbetapentane. Stereoselective protection of (125I)IACoc photolabeling by 3-PPP [(+)-3-PPP more potent than (-)-3-PPP] was observed. (125I)IACoc labeling of the 26-kDa polypeptide was also inhibited by 10 microM imipramine, amitriptyline, fluoxetine, benztropine, and tetrabenazine. The size of the (125I)I-ACoc-labeled proteins is consistent with the size of proteins photolabeled in guinea pig brain and liver membranes by using the sigma photolabel azido-[3H]DTG. Kinetic analysis of (125I)IACoc binding to rat liver microsomes revealed two sites with Kd values of 19 and 126 pM, respectively. The presence or absence of proteolytic inhibitors during membrane preparation did not alter the size of the photolabeled sigma receptor, indicating that the 26-kDa polypeptide was not derived from a larger protein. In summary, (125I)IACoc is a potent and highly specific photoaffinity label for the haloperidol-sensitive sigma receptor and will be useful for its biochemical and molecular characterization.
T-cell immunity to the joining region of p210BCR-ABL protein.
Authors: Chen W , Peace D J , Rovira D K , You S G , Cheever M A
Published: May 2006

The hallmark of chronic myelogenous leukemia is the translocation of the human c-abl protooncogene (ABL) from chromosome 9 to the specific breakpoint cluster region (bcr) of the BCR gene on chromosome 22. The t(9;22)(q34;q11) translocation results in the formation of a BCR-ABL fusion gene that encodes a 210-kDa chimeric protein with abnormal tyrosine kinase activity. The ABL and BCR genes are expressed by normal cells and thus the encoded proteins are presumably nonimmunogenic. However, the joining-region segment of the p210BCR-ABL chimeric protein is composed of unique sequences of ABL amino acids joined to BCR amino acids that are expressed only by malignant cells. The current study demonstrates that the joining region of BCR-ABL protein is immunogenic to murine T cells. Immunization of mice with synthetic peptides corresponding to the joining region elicited peptide-specific, CD4+, class II major histocompatibility complex-restricted T cells. The BCR-ABL peptide-specific T cells recognized only the combined sequence of BCR-ABL amino acids and not BCR or ABL amino acid sequences alone. Importantly, the BCR-ABL peptide-specific T cells could recognize and proliferate in response to p210BCR-ABL protein. The response of peptide-specific T cells to protein demonstrated that p210BCR-ABL can be processed by antigen-presenting cells so that the joining segment is bound to class II major histocompatibility complex molecules in a configuration similar to that of the immunizing peptide and in a concentration high enough to stimulate the antigen-specific T-cell receptor. Thus, BCR-ABL protein represents a potential tumor-specific antigen related to the transforming event and shared by many individuals with chronic myelogenous leukemia.
Mutually exclusive exon splicing of the cardiac calcium channel alpha 1 subunit gene generates developmentally regulated isoforms in the rat heart.
Authors: Diebold R J , Koch W J , Ellinor P T , Wang J J , Muthuchamy M , Wieczorek D F , Schwartz A
Published: May 2006

Several clones were isolated from a rat genomic library in order to further characterize a region of variability within the third membrane-spanning region of the fourth motif (IVS3) of the L-type voltage-dependent calcium channel. We report here that this diversity arises from alternative splicing of a primary transcript containing a single pair of adjacent exons each encoding a unique sequence for the IVS3 region. Definitive proof of a mutually exclusive splicing mechanism was obtained by genomic mapping of flanking upstream and downstream exons and by extensive sequence analysis of the relevant exon/intron boundaries. S1 nuclease protection experiments revealed that both variant forms of the IVS3 were equally expressed in newborn and fetal rat heart, whereas only a single isoform predominated in adult rat heart. The results demonstrate the existence of an important developmentally regulated switch mediated by alternatively spliced exons in cardiac tissue at a time when major changes in excitation occur.
Evidence that hematopoietic stem cells express mouse c-kit but do not depend on steel factor for their generation.
Authors: Ikuta K , Weissman I L
Published: May 2006

The interaction of the mouse c-kit receptor, designated Kit receptor, and steel factor promotes the proliferation and differentiation of hematopoietic progenitor cells. Monoclonal antibodies against the extracellular portion of the mouse Kit receptor were established. Five percent to 10% of total bone marrow cells expressed the Kit receptor, and half of them lack the expression of lineage markers. The Kit receptor was expressed on 70-80% of Thy-1.1lo Lin-Sca-1+ cells, which express Thy-1.1 antigen at a low level and constitute approximately 0.05% of adult bone marrow and fetal liver; by previous studies, these cells have been shown to be highly enriched for multipotent hematopoietic stem cells (HSCs) and are the only hematopoietic cell subset with this activity. Spleen colony formation and long-term multilineage reconstitution activities were contained in the Kit+ but not in the Kit- subpopulations of Thy-1lo Lin-Sca-1+ cells from adult bone marrow, suggesting that the Kit receptor is expressed on HSCs from the earliest stage-i.e., pluripotent HSCs. The role of steel factor in the development and self-renewal of HSCs was tested with Sl/Sl homozygote fetuses, which lack genes to encode functional steel factor. They were shown to have 30-40% of the number of HSCs on days 13-15 when compared with normal litermates. However, the absolute number of HSCs increased during fetal development in the Sl/Sl mice. The results suggest that the Kit receptor-steel factor interaction may not be essential for the initiation of hematopoiesis and the self-renewal of (at least) fetal HSCs.
Evidence for the participation of endogenous activin A/erythroid differentiation factor in the regulation of erythropoiesis.
Authors: Shiozaki M , Sakai R , Tabuchi M , Nakamura T , Sugino K , Sugino H , Eto Y
Published: May 2006

Activin A/erythroid differentiation factor (EDF) is a human protein that induces differentiation of a murine erythroleukemia cell (the Friend cell). In this study, we demonstrate that endogenous activin A/EDF activity is present in murine bone marrow and spleen. In addition, this activity is secreted by bone marrow and spleen cells in primary culture. Administration of follistatin (a specific binding protein for activin A/EDF) to mice results in a decrease of erythroid progenitors in the bone marrow and spleen. These findings support the concept that activin A/EDF and follistatin have opposing actions in the regulation of erythropoiesis.
Primary cultures of endothelial cells from the human liver sinusoid are permissive for human immunodeficiency virus type 1.
Authors: Steffan A M , Lafon M E , Gendrault J L , Schweitzer C , Royer C , Jaeck D , Arnaud J P , Schmitt M P , Aubertin A M , Kirn A
Published: May 2006

Human endothelial cells isolated from hepatic sinusoids were infected in vitro with human immunodeficiency virus type 1 (HIV-1). An early sign of infection occurring in the culture was the formation of multinucleated cells. By double-labeling immunofluorescence, 5-15% of the cells recognized as endothelial cells owing to the presence of von Willebrand factor were found to contain HIV p24 and gp120 antigens after 2 weeks. Reverse transcriptase activity was released into the medium, and different steps in the process of viral budding were observed by electron microscopy. The virus produced by the endothelial cells was found to be infectious for CEM cells, a human T-cell line. CD4 molecules are present at the surface of the endothelial cells, as demonstrated by immunogold-silver staining and backscattered electron imaging. Treatment with an anti-CD4 antibody abolished productive infection of the sinusoidal endothelial cells. The possibility that endothelial cells of the liver sinusoid are infected in vivo with HIV remains to be clearly shown.
Genetic alteration of catecholamine specificity in transgenic mice.
Authors: Kobayashi K , Sasaoka T , Morita S , Nagatsu I , Iguchi A , Kurosawa Y , Fujita K , Nomura T , Kimura M , Katsuki M
Published: May 2006

Epinephrine-producing cells are characterized by the presence of phenylethanolamine N-methyltransferase (PNMT), which catalyzes the formation of epinephrine from norepinephrine. We generated a line of transgenic mice carrying a chimeric gene containing human PNMT cDNA fused to the 4-kilobase fragment of the human dopamine beta-hydroxylase (DBH) gene promoter, to switch catecholamine phenotype in the nervous and endocrine systems. Human PNMT transcripts and immunoreactivity were mainly detected in norepinephrine neurons in brain and sympathetic ganglion as well as in norepinephrine-producing cells in adrenal medulla of transgenic mice, indicating that the human DBH gene promoter of 4 kilobases is sufficient to direct expression of the gene in norepinephrine-producing cells. Analysis of catecholamines in the various tissues showed that the expression of human PNMT in transgenic mice induced the appearance of epinephrine in sympathetic ganglion and dramatic changes in norepinephrine and epinephrine levels in brain, adrenal gland, and blood. These results indicate that the additional PNMT expression in norepinephrine-producing cells can convert these cells to the epinephrine phenotype, and suggest that norepinephrine-producing cells normally possess the basic machinery required for the synthesis of epinephrine except for PNMT. Thus it appears that the only major difference between norepinephrine- and epinephrine-producing cells is the expression of PNMT. Our transgenic animals provide an experimental model to investigate the functional differences between norepinephrine and epinephrine.
Conserved motifs in a divergent nod box of Azorhizobium caulinodans ORS571 reveal a common structure in promoters regulated by LysR-type proteins.
Authors: Goethals K , Van Montagu M , Holsters M
Published: May 2006

Nodulation of leguminous plants by Rhizobium, Bradyrhizobium, and Azorhizobium spp. is dependent on the induction by the plant host of different bacterial nodulation (nod) loci. The transcription of these nod loci is activated in the presence of plant-produced flavonoids upon binding of the NodD protein--a LysR-type activator--to specific sequences present in the nod promoters. Originally, a 47-base-pair (bp) region called the nod box was shown to be the target sequence for binding of NodD. From the comparison of the nod box sequences of (brady)rhizobia with a more divergent nod box from Azorhizobium caulinodans, we now propose a modular build-up of the nod box with the sequence A-T-C-N9-G-A-T as the binding target of the NodD protein (the NodD box). More generally, we show that LysR-type-regulated promoters contain the characteristic sequence T-N11-A as the core of an inverted repeat and propose this to be the "LysR motif" involved in specific binding to LysR-type proteins. Data obtained upon site-specific mutagenesis of this motif in the NodD box sustains this proposal. Further, we provide strong arguments that the inducer flavonoid, involved in transcriptional activation of Azorhizobium nod genes, interacts directly with the NodD protein, thereby increasing its binding affinities for the NodD box.
A newt ribozyme: a catalytic activity in search of a function.
Authors: Cremisi F , Scarabino D , Carluccio M A , Salvadori P , Barsacchi G
Published: May 2006

We analyzed the cleavage properties and the transcription regulation of the newt (Triturus vulgaris meridionalis) self-cleaving RNA. In vitro self-cleavage of model oligoribonucleotides occurs within a double hammerhead structure. In addition, an entire ribozyme molecule, as well as its catalytic domain, "trans-cleaves" in vitro appropriate oligoribonucleotide substrates. Signals encoded within the ribozyme DNA sequences regulate the ribozyme transcription, which is RNA polymerase II dependent. Finally, the deduced secondary structure of the self-cleaving RNA appears to be conserved in evolutionarily distant newt species. These features suggest that the newt ribozyme could play some role in the cell, possibly related to its cleavage properties.
Repair of DNA heteroduplexes containing small heterologous sequences in Escherichia coli.
Authors: Parker B O , Marinus M G
Published: May 2006

Plasmid heteroduplexes were constructed that contain 1, 2, 3, 4, or 5 unpaired bases within the mnt gene. These were used to assess the efficiency of repair of small heterologous sequences ("heterologies") in DNA by the Escherichia coli Dam-directed mismatch repair system. Heteroduplexes in defined states of methylation at d(GATC) sites were used to transform a repair-proficient indicator strain (which has a mnt-lac fusion coding for a nonfunctional mnt repressor) and its isogenic mutH, -L, and -S derivatives. Using this in vivo transformation system, we scored for repair on the basis of colony color: correction in favor of the strand bearing mnt+ coding information gives rise to colonies that are white, whereas correction on the opposite strand (mnt-) yields colonies that are red when grown on MacConkey agar. Failure to repair a heterology yields colonies that are both red and white ("mixed"). The correction efficiencies of two heteroduplexes, each containing a single G.T mismatch within mnt, were also monitored for purposes of comparison. Our results show that mutHLS-dependent, methyl-directed repair of heteroduplexes with 1-, 2-, and 3-base deletions is as highly efficient as the repair of G.T mismatches. Heteroduplexes with a 4-base deletion are marginally repaired and DNA with a 5-base deletion is not detectably repaired. In addition, we show that purified MutS protein from Salmonella typhimurium, which can substitute for E. coli MutS in vivo, binds to oligonucleotide duplexes containing 1, 2, 3, and 4 unpaired bases of a sequence identical with that used for the in vivo studies. Specific binding of MutS to homoduplex DNA and to DNA that had undergone a 5-base deletion was not observed.
Selective tumor uptake of a boronated porphyrin in an animal model of cerebral glioma.
Authors: Hill J S , Kahl S B , Kaye A H , Stylli S S , Koo M S , Gonzales M F , Vardaxis N J , Johnson C I
Published: May 2006

The prognosis for patients with high-grade cerebral glioma is poor. Most treatment failures are due to local recurrence of tumor, indicating that a more aggressive local therapy could be beneficial. Adjuvant treatments such as porphyrin-sensitized photodynamic therapy (PDT) or boron neutron capture therapy (BNCT) have the potential to control local recurrence. The selective tumor uptake of a boronated porphyrin was studied in CBA mice bearing an implanted intracerebral glioma. Biopsy samples of tumor, normal brain, and blood were analyzed by a fluorometric assay following intraperitoneal and intravenous administration of boronated protoporphyrin (BOPP). This compound was selectively localized to tumor at ratios as high as 400:1 relative to normal brain. Confocal laser scanning microscopy of glioma cells in vitro and in vivo showed that BOPP was localized within mitochondria and excluded from the nucleus of these cells. This discrete subcellular localization was confirmed by density gradient ultracentrifugation after homogenization of mouse tumor biopsies. The selective discrete localization of these compounds within the tumor suggests that this compound may be used as a dual PDT/BNCT sensitizer.
Identification of a Salmonella typhimurium invasion locus by selection for hyperinvasive mutants.
Authors: Lee C A , Jones B D , Falkow S
Published: May 2006

Salmonella typhimurium penetrate intestinal epithelial cells during infection. In vitro studies reveal that the availability of oxygen during bacterial growth decreases their capacity to adhere to and enter cultured epithelial cells. To identify S. typhimurium genes involved in epithelial cell entry, mutants were selected that entered HEp-2 cells when grown under repressing, aerobic culture conditions. Two types of transposons were used to generate bacterial mutations--transposons that disrupt genes (Tn10 and Tn5) and one transposon (Tn5B50) that, in addition to disrupting genes, can cause constitutive expression of genes from the neo promoter at one end of the transposon. Three classes of mutations were found that increased the ability of aerobically grown S. typhimurium to enter HEp-2 cells. One class of mutations disrupts the che operons and results in a nonchemotactic phenotype. The second class of mutations revealed that defects in rho, which encodes an essential transcription termination factor, result in hyperinvasiveness. The third class of mutations was obtained only from mutagenesis with Tn5B50, suggesting that their increased invasiveness is due to constitutive expression of a gene(s) from the exogenous neo promoter. Analysis of this third class of mutations identified a S. typhimurium locus hil (hyperinvasion locus), which is essential for bacterial entry into epithelial cells. The results suggest that hil encodes an invasion factor or an activator of invasion factor expression. hil maps between srl and mutS near minute 59.5 of the S. typhimurium chromosome, a region adjacent to other loci that have been identified as required for S. typhimurium invasiveness and virulence.
The 65-kDa subunit of human NF-kappa B functions as a potent transcriptional activator and a target for v-Rel-mediated repression.
Authors: Ballard D W , Dixon E P , Peffer N J , Bogerd H , Doerre S , Stein B , Greene W C
Published: May 2006

Molecular cloning of the polypeptide component of the Rel-related human p75 nucleoprotein complex has revealed its identity with the 65-kDa (p65) subunit of NF-kappa B. Functional analyses of chimeric proteins composed of NF-kappa B p65 C-terminal sequences linked to the DNA-binding domain of the yeast GAL4 polypeptide have indicated that the final 101 amino acids of NF-kappa B p65 comprise a potent transcriptional activation domain. Transient transfection of human T cells with an expression vector encoding NF-kappa B p65, but not NF-kappa B p50, produced marked transcriptional activation of a basal promoter containing duplicated kappa B enhancer motifs from the long terminal repeat of type 1 human immunodeficiency virus. These stimulatory effects of NF-kappa B p65 were synergistically enhanced by coexpression of NF-kappa B p50 but were completely inhibited by coexpression of the v-rel oncogene product. Together, these functional studies demonstrate that NF-kappa B p65 is a transactivating subunit of the heterodimeric NF-kappa B complex and serves as one cellular target for v-Rel-mediated transcriptional repression.
Dark induction of zeaxanthin-dependent nonphotochemical fluorescence quenching mediated by ATP.
Authors: Gilmore A M , Yamamoto H Y
Published: May 2006

Zeaxanthin-dependent nonphotochemical fluorescence quenching is a light-induced activity in plants that apparently protects against the potentially damaging effects of excess light. We report a dark-induced nonphotochemical quenching in thylakoids of Lactuca sativa L. cv. Romaine mediated by ATP. This effect is due to low lumen pH from hydrolysis-dependent proton pumping and hence required an active ATPase. The induction was optimal at 0.3 mM ATP, a physiological concentration, and occurred under conditions of little or no reverse electron flow. The properties of ATP-induced quenching were in all respects examined similar to light-induced quenching, including antimycin inhibition of quenching induction but not delta pH. We conclude that zeaxanthin-dependent quenching depends directly on lumen pH and that the role of light is indirect. Although it is known that zeaxanthin and low lumen pH are insufficient for quenching to occur, the results apparently exclude the redox state of an electron-transport carrier or formation of light-induced carotenoid triplets as a further requirement. We propose that a slow pH-dependent conformational change together with zeaxanthin cause static quenching in the pigment bed; possibly antimycin inhibits this change. Furthermore, we suggest from the ability of ATP to sustain quenching in the dark for extended periods that persistent or slowly reversible zeaxanthin quenching often observed in vivo may be due to sustained delta pH from ATP hydrolysis.
Methods and algorithms for statistical analysis of protein sequences.
Authors: Brendel V , Bucher P , Nourbakhsh I R , Blaisdell B E , Karlin S
Published: May 2006

We describe several protein sequence statistics designed to evaluate distinctive attributes of residue content and arrangement in primary structure. Considered are global compositional biases, local clustering of different residue types (e.g., charged residues, hydrophobic residues, Ser/Thr), long runs of charged or uncharged residues, periodic patterns, counts and distribution of homooligopeptides, and unusual spacings between particular residue types. The computer program SAPS (statistical analysis of protein sequences) calculates all the statistics for any individual protein sequence input and is available for the UNIX environment through electronic mail on request to V.B. (volker/genomic@stanford.edu).
An interaction between p21ras and heat shock protein hsp60, a chaperonin.
Authors: Ikawa S , Weinberg R A
Published: May 2006

Ras proteins play a crucial role in the development of neoplasia and in signal transduction in normal cells. In a search for proteins interacting with p21ras, we previously identified a protein of 60 kDa (p60) through use of a chemical cross-linker. Using information from partial amino acid sequencing of the purified protein, we isolated full-length cDNA clones encoding this 60-kDa protein. Nucleotide sequence analysis revealed that p60 is the murine heat shock protein hsp60, a chaperonin. Association of hsp60 with p21ras appears physiological, as the amount of hsp60 complexed to p21ras was similar even in cells over-expressing p21ras, and reversing the order of cross-linking and lysis of the cells, which releases large amounts of hsp60 from mitochondria, did not alter the amount of hsp60 cross-linked to p21ras.
cDNA cloning of a liver isoform of the phosphorylase kinase alpha subunit and mapping of the gene to Xp22.2-p22.1, the region of human X-linked liver glycogenosis.
Authors: Davidson J J , Ozçelik T , Hamacher C , Willems P J , Francke U , Kilimann M W
Published: May 2006

We have cloned cDNA molecules encoding another isoform of the alpha subunit of phosphorylase kinase (ATP:phosphorylase-b phosphotransferase, EC 2.7.1.38). Sequence comparison with the previously characterized muscle isoform reveals a pattern of highly conserved and variable domains and demonstrates that the isoforms are the products of distinct genes. In contrast to the muscle isoform gene, PHKA1, the gene of this additional isoform, PHKA2, is predominantly expressed in liver and other nonmuscle tissues. It was mapped to the distal short arm of the human X chromosome (Xp22.2-p22.1), the same region to which human X-linked liver glycogenosis due to phosphorylase kinase deficiency has been mapped. Thus, X-linked liver glycogenosis is probably caused by mutations affecting PHKA2.
Angiogenin supports endothelial and fibroblast cell adhesion.
Authors: Soncin F
Published: May 2006

When coated on bacteriological plastic at doses greater than or equal to 0.1 microgram/cm2, human and bovine angiogenin support calf pulmonary artery endothelial and Chinese hamster fibroblast cell adhesion and spreading, but do not affect cell adhesion when in solution. The kinetics of endothelial cell attachment to angiogenin are indistinguishable from those in the presence of gelatin. Calcium and/or magnesium ions are critical for cell adhesion or spreading onto angiogenin but protein synthesis and glycoprotein secretion are not necessary. Adhesion to angiogenin is not altered by the addition to the incubation solution of fibronectin, fibrinogen, laminin, collagen I and IV, or vitronectin. The peptide Arg-Gly-Asp-Ser inhibits endothelial cell response to angiogenin whereas the reverse peptide Ser-Asp-Gly-Arg-Gly has no effect. These findings show that angiogenin can serve as an effective substratum for cell adhesion by inducing an interaction similar to but independent from that of other extracellular matrix molecules. Induction of cell adhesion and subsequent migration may be critical steps in the process of angiogenesis.
2-Aminopurine overrides multiple cell cycle checkpoints in BHK cells.
Authors: Andreassen P R , Margolis R L
Published: May 2006

BHK cells blocked at any of several points in the cell cycle override their drug-induced arrest and proceed in the cycle when exposed concurrently to the protein kinase inhibitor 2-aminopurine (2-AP). For cells arrested at various points in interphase, 2-AP-induced cell cycle progression is made evident by arrival of the drug-treated cell population in mitosis. Cells that have escaped from mimosine G1 arrest, from hydroxyurea or aphidicolin S-phase arrest, or from VM-26-induced G2 arrest subsequently have all the hallmarks of mitosis--such as a mitotic microtubule array, nuclear envelope breakdown, and chromatin condensation. In a synchronous population, the time course of arrival in mitosis and its duration in 2-AP-treated cells that have escaped drug-induced cell cycle blocks is indistinguishable from control cells. Cells arrested in mitosis by nocodazole or taxol quickly escape mitotic arrest and enter interphase when exposed to 2-AP. 2-AP by itself does not influence the timing of cell cycle progression. We conclude that 2-AP acts to override checkpoints in every phase of the cell cycle, perhaps by inhibiting a protein kinase responsible for control of multiple cell cycle checkpoints.
Coagulation factors X, Xa, and protein S as potent mitogens of cultured aortic smooth muscle cells.
Authors: Gasic G P , Arenas C P , Gasic T B , Gasic G J
Published: May 2006

Smooth muscle cells (SMCs) in the rat carotid artery leave the quiescent state and proliferate after balloon catheter injury. The precise signals responsible for this SMC mitogenesis need to be elucidated. Although platelet-derived growth factor (PDGF), a potent SMC mitogen, is released from activated platelets, damaged endothelium, and macrophages, it cannot be solely responsible for this proliferation. In search of other SMC growth factors, we have examined several proteins of the coagulation cascade. At nanomolar concentrations, factors X, Xa, and protein S promote cultured rat aortic SMC mitosis. In contrast, factor IX is only weakly mitogenic, whereas factor VII and protein C fail to stimulate SMC division. Protein S, the most mitogenic of these coagulation cascade factors, stimulates DNA synthesis in cultured SMCs with a time course similar to that of PDGF-AA and without the delay observed for transforming growth factor beta. Antistasin and tick anticoagulant peptide, two specific factor Xa inhibitors, inhibit SMC mitogenesis due to Xa and protein S. Coagulation factors that possess mitogenic activity may contribute to intimal SMC proliferation after vascular injury as a result of angioplasty or vascular compromise during atherogenesis.
Multiple retinoid-responsive receptors in a single cell: families of retinoid "X" receptors and retinoic acid receptors in the Xenopus egg.
Authors: Blumberg B , Mangelsdorf D J , Dyck J A , Bittner D A , Evans R M , De Robertis E M
Published: May 2006

In a search for nuclear hormone receptors expressed in early development we found that Xenopus laevis eggs contain mRNAs from two retinoic acid receptor genes (xRAR alpha and xRAR gamma) and two retinoid "X" receptor genes (xRXR alpha and xRXR gamma). We also show that RXRs are members of a family of at least three genes, thus expanding the number of genes encoding retinoic acid-responsive transcription factors to six. With the exception of xRXR gamma, these maternal mRNAs are degraded before gastrulation. The RXRs isolated are differentially activated by retinoic acid and by 3,4-didehydroretinoic acid. Considered together, these four receptors provide a molecular basis for the pleiotropic effects of retinoic acid on early development, and their pattern of expression suggests a role for retinoic acid at the earliest stages of embryonic determination.
Differential accumulation of transcripts for four tomato 1-aminocyclopropane-1-carboxylate synthase homologs under various conditions.
Authors: Yip W K , Moore T , Yang S F
Published: May 2006

Degenerate oligonucleotide primers corresponding to conserved regions flanking the active-site domain of 1-aminocyclopropane-1-carboxylate (ACC) synthase (EC 4.4.1.14) were used for the polymerase chain reaction (PCR) to amplify DNA fragments from mRNA isolated from tomato fruit and tomato suspension cell culture. Antibodies raised against two conserved peptide sequences (TNPSNPLGTT and SLSKDLGLPGFRVG) were used to screen for positive colonies, after the PCR products were cloned into a Bluescript plasmid and expressed in Escherichia coli. Four distinct cDNA fragments encoding ACC synthase homologs were isolated. While pBTAS1 and pBTAS4 were obtained from fruit mRNA, cell culture mRNA yielded three sequences, pBTAS1, pBTAS2, and pBTAS3. Sequencing of these gene fragments revealed that pBTAS1 and pBTAS4 were identical to those full-length sequences previously reported by Van Der Straeten et al. [Van Der Straeten, D., Van Wiemeersch, L., Goodman, H. & Van Montague, M. (1990) Proc. Natl. Acad. Sci. USA 87, 4859-4863] and Olson et al. [Olson, D. C., White, J. A., Edelman, J., Harkin, R. N. & Kende, H. (1991) Proc. Natl. Acad. Sci. USA 88, 5340-5344] from tomato fruit, whereas pBTAS2 and pBTAS3 represent new sequences. Ribonuclease protection assays were used to examine the expression of these transcripts under three different conditions of enhanced ethylene production--namely, during fruit ripening, in response to mechanical wounding in fruit tissue, and auxin stimulation in vegetative tissue. Transcripts of pBTAS1 accumulated massively during ripening and wounding but only slightly in response to auxin treatment. Although pBTAS4 was associated with fruit ripening, it was unresponsive to auxin treatment in vegetative tissue. In contrast, the expression of pBTAS2 and pBTAS3 was greatly promoted in auxin-treated vegetative tissue but was absent from fruit tissue. While the expression of pBTAS2 was moderately dependent on wounding, pBTAS3 was unresponsive to wounding. These data support the view that ACC synthase is encoded by a multigene family and that the members are differentially expressed in response to developmental, environmental, and hormonal factors.