bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation and transmission of the Lyme disease spirochete from the southeastern United States.
Authors: Oliver J H , Chandler F W , Luttrell M P , James A M , Stallknecht D E , McGuire B S , Hutcheson H J , Cummins G A , Lane R S
Published: May 2006

The isolation of the Lyme disease spirochete (Borrelia burgdorferi) from the southeastern United States is reported. Three isolates, two from cotton mice (Peromyscus gossypinus) and one from the black-legged tick (Ixodes scapularis), were recovered from Sapelo Island, Georgia, in July and September 1991. The spirochetes were characterized by indirect fluorescent antibody assay using a battery of five monoclonal antibodies, by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) of whole cell lysates, and by the polymerase chain reaction (PCR) assay using primers for three DNA target sequences found in B. burgdorferi reference strain B-31. Transmission experiments indicate that the three Georgia isolates can infect experimentally inoculated hamsters and mice. Tick transmission of one of the isolates has been attempted so far; I. scapularis transmitted isolate SI-1 from hamsters to mice, but the lone-star tick, Amblyomma americanum, did not.
Cleavage of interleukin 1 beta (IL-1 beta) precursor to produce active IL-1 beta by a conserved extracellular cysteine protease from Streptococcus pyogenes.
Authors: Kapur V , Majesky M W , Li L L , Black R A , Musser J M
Published: May 2006

Streptococcal pyrogenic exotoxin B (SPE B), a conserved extracellular cysteine protease expressed by the human pathogenic bacterium Streptococcus pyogenes, was purified and shown to cleave inactive human interleukin 1 beta precursor (pIL-1 beta) to produce biologically active IL-1 beta. SPE B cleaves pIL-1 beta one residue amino-terminal to the site where a recently characterized endogenous human cysteine protease acts. IL-1 beta resulting from cleavage of pIL-1 beta by SPE B induced nitric oxide synthase activity in vascular smooth muscle cells and killed of the human melanoma A375 line. Two additional naturally occurring SPE B variants cleaved pIL-1 beta in a similar fashion. By demonstrating that SPE B catalyzes the formation of biologically active IL-1 beta from inactive pIL-1 beta, our data add a further dimension to an emerging theme in microbial pathogenesis that bacterial and viral virulence factors act directly on host cytokine pathways. The data also contribute to an enlarging literature demonstrating that microbial extracellular cysteine proteases are important in host-parasite interactions.
Mediation of osmoregulatory influences on neuroendocrine corticotropin-releasing factor expression by the ventral lamina terminalis.
Authors: Kovács K J , Sawchenko P E
Published: May 2006

Chronic exposure to a hyperosmolar challenge invokes coordinate, differential, and ostensibly adaptive alterations in the expression of mRNA encoding corticotropin-releasing factor (CRF) in the endocrine hypothalamus. Rats maintained on 2% (wt/vol) saline for 7 days displayed the expected reduction in CRF mRNA levels in the parvocellular neurosecretory compartment of the paraventricular nucleus, as well as a concomitant increase in CRF transcripts in oxytocin-containing magnocellular neurosecretory neurons. Also detected in salt-loaded animals was a prominent induction of the immediate-early gene product Fos in magnocellular neurosecretory cell groups and in several brain regions that are known to provide major projections to the endocrine hypothalamus. These included a triad of cell groups making up the lamina terminalis of the third ventricle, and, to a lesser extent, catecholaminergic cell groups in the caudal brain stem. Discrete transections of descending projections from structures associated with the lamina terminals, as well as excitotoxin lesions centered in one lamina terminalis-associated structure, the organum vasculosum, abolished the effects of salt loading in both the magno- and parvocellular neurosecretory systems. Knife cuts in the lamina terminalis complex that spared only projections from the organum vasculosum region or cuts that disrupted ascending catecholaminergic projections failed to modify either effect of salt loading. The results suggest the existence of a simple circuit through which osmotic influences on gene expression in the magnocellular and parvocellular neurosecretory systems are effected.
Origin of floral isolation between ornithophilous and sphingophilous plant species.
Authors: Grant V
Published: May 2006

Three plant groups in temperate western North America contain closely related ornithophilous and sphingophilous species: the Aquilegia formosa/Aquilegia caerulea group (Ranunculaceae), the Ipomopsis aggregata group (Polemoniaceae), and the Diplacus longiflorus group (Scrophulariaceae). The ornithophilous and sphingophilous species are products of allopatric speciation on the diploid level. Geographical races which are adapted to one class of pollinators in one area where these pollinators are abundant and effective and to another class of pollinators in another geographically isolated area (pollination races) represent a probable intermediate stage in the process of allopatric speciation. Mechanical and ethological isolation (collectively, floral isolation) is a byproduct of the divergence in pollination systems. Selection for reproductive isolation per se has not played any detectable role in the origin of the floral isolation in the three plant groups.
Nerve growth factor suppresses the transforming phenotype of human prolactinomas.
Authors: Missale C , Boroni F , Losa M , Giovanelli M , Zanellato A , Dal Toso R , Balsari A , Spano P
Published: May 2006

The most effective therapy of human prolactinomas is represented by dopamine D-2 receptor agonists; there is, however, a population of nonresponder patients who require surgical intervention. In the present study, we report that prolactinomas totally resistant to pharmacological therapy have a high potential of both growing in soft agar and forming tumors in nude mice and lack D-2 receptors for dopamine. These tumors express the receptors for nerve growth factor (NGF) and are sensitive to its differentiating activity. After exposure to NGF for 4 days, prolactinoma cells decreased their proliferation rate, lost their capability to form colonies in soft agar, lost their tumorigenic activity in nude mice, and reexpressed the lactotroph-specific D-2 receptor protein inhibiting prolactin release. These effects were permanent after NGF withdrawal and were reproducible in vivo in nude mice transplanted with the tumors. NGF in fact remarkably and lastingly depressed tumor growth and induced expression of D-2 receptors when injected intravenously once a day for 5 days into prolactinoma-bearing nude mice. These data suggest that NGF may induce a long-lasting switch of gene expression in human prolactinomas, modifying their transforming phenotype and reverting them to more differentiated, less malignant, dopamine-sensitive lactotroph-like cells. The possibility thus arises that short-term treatment with NGF may restore the refractory patients to conventional pharmacological therapy with D-2 agonists.
Comparative linkage maps of the rice and maize genomes.
Authors: Ahn S , Tanksley S D
Published: May 2006

Genetic linkage maps have been constructed for the rice and maize genomes on the basis of orthologous loci detected with a common set of cDNA clones. Conserved linkage groups could be identified, which together account for more than two-thirds of both genomes. In some instances, entire chromosomes or chromosome arms are nearly identical with respect to gene order and gene content. The results also reveal that most of the genes (> 72%) duplicated during ancient polyploidization are still present in the maize genome in duplicate copy. The comparative maps of rice and maize provide a basis for interpreting molecular, genetic, and breeding information between these two important species and establish a framework for ultimately connecting the genetics of all grass species.
Control analysis of the dependence of Escherichia coli physiology on the H(+)-ATPase.
Authors: Jensen P R , Michelsen O , Westerhoff H V
Published: May 2006

The H(+)-ATPase plays a central role in Escherichia coli free-energy transduction and hence in E. coli physiology. We here investigate the extent to which this enzyme also controls the growth rate, growth yield, and respiratory rate of E. coli. We modulate the expression of the atp operon and determine the effect on said properties. When quantified in terms of control coefficients, we find that, in the wild-type cell growing on glucose in minimal medium, this key enzyme (H(+)-ATPase) exerts virtually no control on growth rate (magnitude of C < 0.01), a minor positive control on growth yield (C = 0.15), and a small but negative control on respiration rate (C = -0.25). The control the enzyme exerts on the consumption rate of the carbon and free-energy substrate is negative (C = -0.15). We also studied how the control coefficients themselves vary with the expression of the atp operon. As the level of expression of the atp operon was reduced, the control exerted by the H(+)-ATPase on growth rate and growth yield increased slightly; the control on growth rate passed through a maximum (C = 0.1) and disappeared when the atp operon was not expressed at all, reflecting that with this substrate there are alternative routes for ATP synthesis. At elevated levels of the H(+)-ATPase compared to the wild type, the control exerted by the enzyme on growth rate became negative. The evolutionary context of the absence of control by the atp operon on growth rate is discussed.
Identification of a gene required for membrane protein retention in the early secretory pathway.
Authors: Nishikawa S , Nakano A
Published: May 2006

The yeast SEC12 gene product (Sec12p) is an integral membrane protein required for the protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus. Although this protein is almost exclusively localized in the ER, a significant fraction of Sec12p is modified by an enzyme that resides in the early compartment of the Golgi apparatus, suggesting that Sec12p is cycling between the ER and the early Golgi. We have taken a genetic approach to investigate the retention mechanism of Sec12p. Analysis of mutants that are defective in the retention of the Sec12-Mf alpha 1 fusion protein in the early secretory compartments has identified a gene, RER1. A recessive mutation in RER1 causes mislocalization of the authentic Sec12p as well as two different Sec12 fusion proteins to the late Golgi apparatus and even to the cell surface. However, the rer1 mutant is not defective in the retention of an ER-resident soluble protein, BiP, suggesting that soluble and membrane proteins are retained in the ER by distinct mechanisms.
Hypersensitivity to diphtheria toxin by mouse cells expressing both diphtheria toxin receptor and CD9 antigen.
Authors: Brown J G , Almond B D , Naglich J G , Eidels L
Published: May 2006

DTS-II is a highly diphtheria toxin (DT)-sensitive cell line previously isolated by transfection of wild-type DT-resistant mouse L-M(TK-) cells with the cDNA encoding a monkey Vero cell DT receptor. DTS-II cells are as toxin-sensitive as Vero cells, have approximately 3-fold more receptors than Vero cells, and have approximately 10-fold lower affinity for DT than Vero cells. We now cotransfected DTS-II cells with a plasmid containing the Vero cell cDNA coding for CD9 antigen (pCD9) and with a plasmid containing the gene for hygromycin resistance (pHyg). The stably transfected hygromycin-resistant colonies were screened for DT hypersensitivity employing a replica plate system. A DT-hypersensitive colony was isolated and purified. The purified DT-hypersensitive cells, DTS-III, (i) are approximately 10-fold more toxin-sensitive than DTS-II and Vero cells and (ii) bear approximately 10(6) DT receptors per cell (i.e., approximately 20-fold and approximately 60-fold more receptors than DTS-II and Vero cells, respectively), but their receptor affinity is still approximately 10-fold lower than that of Vero cells. Cross-linking experiments employing 125I-labeled DT demonstrated that DTS-II and DTS-III cells have essentially the same profile of DT-binding cell-surface protein(s), suggesting that CD9 antigen, although expressed on the cell surface of DTS-III cells, may not be in close proximity to the DT-binding domain of the receptor. CD9 may affect DT receptor expression by increasing receptor density at the cell surface. By employing DTS-III cells it should be possible to purify and characterize the DT cell-surface receptor protein(s).
Cloning, sequencing, and expression of the adenosylcobalamin-dependent ribonucleotide reductase from Lactobacillus leichmannii.
Authors: Booker S , Stubbe J
Published: May 2006

Ribonucleoside-triphosphate reductase (RTPR, EC 1.17.4.2) from Lactobacillus leichmannii, a monomeric adenosylcobalamin-requiring enzyme, catalyzes the conversion of nucleoside triphosphates to deoxynucleoside triphosphates. The gene for this enzyme has been cloned and sequenced. In contrast to expectations based on mechanistic considerations, there is no statistically significant sequence homology with the Escherichia coli reductase that requires a dinuclear-iron center and tyrosyl radical cofactor. The RTPR has been overexpressed and purified to homogeneity, yielding 90 mg of protein from 2.5 g of bacteria. Initial characterization of the recombinant RTPR indicates that its properties are identical to those of the RTPR isolated from L. leichmannii.
Staphylococcal enterotoxins can reactivate experimental allergic encephalomyelitis.
Authors: Schiffenbauer J , Johnson H M , Butfiloski E J , Wegrzyn L , Soos J M
Published: May 2006

Staphylococcal enterotoxins (SEs) are one member of a unique group of molecules known as superantigens. They are potent T-cell activators and stimulate a large number of T cells bearing specific T-cell-receptor beta-chain variable regions. It has been proposed that superantigens may trigger autoimmune disorders by stimulation of autoreactive T cells with restricted beta-chain variable-chain usage. We investigated the effects of SEs B and A (SEB and SEA) on the reactivation of experimental allergic encephalomyelitis, an animal model for multiple sclerosis. We report that SEB can reinduce encephalitis multiple times in PL/J mice that had previously recovered from an acute episode. SEB was also able to induce encephalitis in mice previously immunized with myelin basic protein but did not show clinical signs of disease. In addition, it was observed that T cells from PL/J mice that had been previously activated by myelin basic protein in complete Freund's adjuvant or in complete Freund's adjuvant alone were resistant to the induction of anergy by SEB. To determine whether reactivation of experimental allergic encephalomyelitis was specific for SEB, another superantigen, SEA, was employed. It was found that SEA was also able to reinduce experimental allergic encephalomyelitis in mice previously recovered from an acute episode and those that had been previously immunized with myelin basic protein but did not show clinical signs of disease. These results indicate that SEs are capable of reactivating autoreactive T cells and inducing autoimmune disease.
Kinetic characterization of a cytoplasmic myosin motor domain expressed in Dictyostelium discoideum.
Authors: Ritchie M D , Geeves M A , Woodward S K , Manstein D J
Published: May 2006

A detailed kinetic study of the interaction of a recombinant myosin head fragment (MHF) of Dictyostelium discoideum with actin and adenine nucleotides has been made by using a combination of rapid-reaction, equilibrium, and fluorescence methods. MHF is equivalent in size to a proteolytic fragment of skeletal muscle myosin, subfragment 1 (S1), the simplest unit of myosin to retain enzymatic and functional activity. The results show that qualitatively the interactions of MHF with nucleotides and actin are the same as those of S1. Both bind to rabbit actin with the same affinity, although differences in the rate constants of their interactions with nucleotides in the presence and absence of actin occur. The rate of ATP binding to MHF and the subsequent cleavage step are significantly slower than the corresponding rates with S1. The dissociation of a fluorescent analog of ADP from MHF was 5-fold faster than from S1, while its rate of binding MHF was 3-fold slower, resulting in a weaker association equilibrium constant. The ATP-induced isomerization of the actoMHF complex was 10-fold slower than for actoS1, but the binding affinities of ADP for actoMHF and actoS1 were indistinguishable. The results suggest a different degree of coupling between the nucleotide and actin binding sites of MHF and S1 which may be a common feature of nonmuscle myosins. They also provide the basis for a study of specifically modified myosins with which one can probe the sites of interaction with nucleotides or actin, as well as functional motility.
Immunochemical detection of 4-hydroxynonenal protein adducts in oxidized hepatocytes.
Authors: Uchida K , Szweda L I , Chae H Z , Stadtman E R
Published: May 2006

We report here the development of an immunochemical procedure that uses an antibody specific to the 4-hydroxynonenal (HNE) moiety for the detection of HNE-protein adducts. The HNE-specific antibody was prepared by immunizing rabbits with a HNE-keyhole limpet hemocyanin conjugate and purifying the rabbit serum on an affinity gel prepared by covalent attachment of a HNE-conjugated heptapeptide. When various preparations of glyceraldehyde-3-phosphate dehydrogenase containing 0-7.0 equivalent of HNE-histidine residues per subunit were obtained by incubating samples of glyceraldehyde-3-phosphate dehydrogenase with increased amounts of HNE and subjected to immunoblotting with the HNE-specific antibody, the intensities of the blots were directly proportional to the number of HNE-histidine adducts as measured directly by amino acid analysis. Binding of the HNE-conjugated glyceraldehyde-3-phosphate dehydrogenase to the HNE-specific antibody could be completely inhibited by HNE-N-acetylhistidine, HNE-N-acetyllysine, or HNE-glutathione, suggesting that the antigenic determinant recognized by the antibody is the HNE moiety, not the HNE-amino acid conjugates, such as HNE-histidine, HNE-lysine, and HNE-cysteine. The utility of the HNE-specific antibody was demonstrated by its ability to react selectively with a number of HNE-protein adducts in immunoblot analyses of crude homogenates of rat liver hepatocytes that had been exposed to HNE or oxidative stresses with tert-butylhydroperoxide or metal-ion-catalyzed oxidation systems.
An operational RNA code for amino acids and possible relationship to genetic code.
Authors: Schimmel P , Giegé R , Moras D , Yokoyama S
Published: May 2006

RNA helical oligonucleotides that recapitulate the acceptor stems of transfer RNAs, and that are devoid of the anticodon trinucleotides of the genetic code, are aminoacylated by aminoacyl tRNA synthetases. The specificity of aminoacylation is sequence dependent, and both specificity and efficiency are generally determined by only a few nucleotides proximal to the amino acid attachment site. This sequence/structure-dependent aminoacylation of RNA oligonucleotides constitutes an operational RNA code for amino acids. To a rough approximation, members of the two different classes of tRNA synthetases are, like tRNAs, organized into two major domains. The class-defining conserved domain containing the active site incorporates determinants for recognition of RNA mini-helix substrates. This domain may reflect the primordial synthetase, which was needed for expression of the operational RNA code. The second synthetase domain, which generally is less or not conserved, provides for interactions with the second domain of tRNA, which incorporates the anticodon. The emergence of the genetic from the operational RNA code could occur when the second domain of synthetases was added with the anticodon-containing domain of tRNAs.
The amino-terminal 200 amino acids of the plasma membrane Na+,K+-ATPase alpha subunit confer ouabain sensitivity on the sarcoplasmic reticulum Ca(2+)-ATPase.
Authors: Ishii T , Takeyasu K
Published: May 2006

Cardiac glycosides such as G-strophanthin (ouabain) bind to and inhibit the plasma membrane Na+,K(+)-ATPase but not the sarcoplasmic reticulum (SR) Ca(2+)-ATPase, whereas thapsigargin specifically blocks the SR Ca(2+)-ATPase. The chimera [n/c]CC, in which the amino-terminal amino acids Met1 to Asp162 of the SR Ca(2+)-ATPase (SERCA1) were replaced with the corresponding portion of the Na+,K(+)-ATPase alpha 1 subunit (Met1 to Asp200), retained thapsigargin- and Ca(2+)-sensitive ATPase activity, although the activity was lower than that of the wild-type SR Ca(2+)-ATPase. Moreover, this Ca(2+)-sensitive ATPase activity was inhibited by ouabain. The chimera NCC, in which Met1-Gly354 of the SR Ca(2+)-ATPase were replaced with the corresponding portion of the Na+,K(+)-ATPase, lost the thapsigargin-sensitive Ca(2+)-ATPase activity seen in CCC and [n/c]CC. [3H]Ouabain binding to [n/c]CC and NCC demonstrated that the affinity for this inhibitor seen in the wild-type chicken Na+,K(+)-ATPase was restored in these chimeric molecules. Thus, the ouabain-binding domains are distinct from the thapsigargin sites; ouabain binds to the amino-terminal portion (Met1 to Asp200) of the Na+,K(+)-ATPase alpha 1 subunit, whereas thapsigargin interacts with the regions after Asp162 of the Ca(2+)-ATPase. Moreover, the amino-terminal 200 amino acids of the Na+,K(+)-ATPase alpha 1 subunit are sufficient to exert ouabain-dependent inhibition even after incorporation into the corresponding portion of the Ca(2+)-ATPase, and the segment Ile163 to Gly354 of the SR Ca(2+)-ATPase is critical for thapsigargin- and Ca(2+)-sensitive ATPase activity.
Testis-specific expression of a metallothionein I-driven transgene correlates with undermethylation of the locus in testicular DNA.
Authors: Salehi-Ashtiani K , Widrow R J , Markert C L , Goldberg E
Published: May 2006

Mice carrying a chimeric transgene of the human testis-specific lactate dehydrogenase cDNA driven by mouse metallothionein I promoter have been reported to express the transgene in a testis-specific manner in six founder lines. To study the mechanism by which this testis-specific expression is mediated, we have examined genomic placement, expression pattern, and methylation status of the transgene. Our results indicate that transgene expression is repressed in all somatic tissues examined even when heavy metals are administered. Nuclear run-on assays indicate that failure of expression in the liver (in which the metallothionein I promoter is highly active) occurs at the transcriptional level. In contrast, the transgene mRNA is transcribed in male germ cells and is developmentally regulated during spermatogenesis. Examination of the transgene methylation status reveals that expression is inversely correlated with hypermethylation of the locus; all CpG dinucleotides examined in the promoter region were found to be fully methylated in kidney and liver but were undermethylated in testis. Since methylation of the murine metallothionein I promoter is sufficient to inhibit its activity, it is likely that suppression of the transgene in somatic tissues is mediated by methylation.
A second transcriptionally active DNA-binding site for the Wilms tumor gene product, WT1.
Authors: Wang Z Y , Qiu Q Q , Enger K T , Deuel T F
Published: May 2006

The putative Wilms tumor suppressor gene, wt1, encodes a zinc-finger protein that binds to the DNA sequence 5'-GCGGGGGCG-3'. We previously reported that WT1 has separable domains that function either to activate or suppress transcription. We now have identified a second WT1 binding sequence (5'-TCCTCCTCCTCCTCTCC-3') 3' to the transcription initiation site of the platelet-derived growth factor A-chain gene by DNase I footprinting and gel mobility shift assays. WT1 requires both 5' and 3' binding sites for transcriptional suppression; however, WT1 functions as a transcriptional activator when it binds to either the 5' or 3' site alone. This second WT1 binding sequence functions equally well as the previously identified 5'-GCGGGGGCG-3' sequence when analyzed in transient transfection assays. A core DNA sequence recognized by WT1 was defined by using related synthetic oligonucleotides. We also identified sequences similar to the WT1 binding site within the promoter regions of five other growth-related genes and demonstrated that each of these sequences also binds WT1 in gel mobility shift assays. These results thus identify a second WT1 binding site and suggest that additional growth-related genes may be transcriptionally influenced by WT1.
Purification and characterization of recombinant G16 alpha from Sf9 cells: activation of purified phospholipase C isozymes by G-protein alpha subunits.
Authors: Kozasa T , Hepler J R , Smrcka A V , Simon M I , Rhee S G , Sternweis P C , Gilman A G
Published: May 2006

A cDNA encoding G16 alpha, the alpha subunit of a heterotrimeric guanine nucleotide-binding protein, was expressed in Sf9 cells using recombinant baculovirus. G16 alpha in membrane extracts of Sf9 cells activated phospholipase C-beta 1 (PLC-beta 1) in the presence of guanosine 5'-[gamma-thio]triphosphate; the system could not be activated by Al3+, Mg2+, and F-. The G16 alpha in the cytosolic fraction of Sf9 cells did not stimulate PLC-beta 1. Concurrent expression of the G-protein beta gamma subunit complex increased the amount of G16 alpha in Sf9 cell membranes. The guanosine 5'-[gamma-thio]triphosphate-activated form of G16 alpha was purified from cholate extracts of membranes from cells expressing G16 alpha, and the G-protein beta 2 and gamma 2 subunits. G16 alpha activated PLC-beta 1, PLC-beta 2, and PLC-beta 3 in a manner essentially indistinguishable from that of Gq alpha. G16 alpha-mediated activation of PLC-beta 1 and PLC-beta 3 greatly exceeded that of PLC-beta 2. G16 alpha did not activate PLC-gamma 1 or PLC-delta 1. Thus, two distantly related members of the Gq alpha family, Gq alpha and G16 alpha, have the same ability to activate the known isoforms of PLC-beta.
catELISA: a facile general route to catalytic antibodies.
Authors: Tawfik D S , Green B S , Chap R , Sela M , Eshhar Z
Published: May 2006

The low abundance and activity of catalytic antibodies are major obstacles to their selection from the virtually unlimited repertoire of antibody binding sites. The requirement for new screening methodologies is further emphasized by the availability of combinatorial libraries, in which a functional polypeptide has to be selected out of millions of possibilities. We present a simple and sensitive screening approach (termed catELISA) based on immobilized substrates and immunodetection of the end product of the catalyzed reaction. The feasibility of catELISA is demonstrated here by the generation of potent ester-hydrolyzing antibodies by direct screening of hybridoma supernatants. We show that this approach is not only facile but general: it is not limited by type of reaction, substrate, or catalyst (enzymes, catalytic antibodies, chemical catalysts). catELISA opens a route to catalytic antibodies that replaces existing lengthy and arduous methods, thus allowing us to expand their number and improve their quality and to address questions that would otherwise be difficult to answer.
A dominant truncation allele identifies a gene, STE20, that encodes a putative protein kinase necessary for mating in Saccharomyces cerevisiae.
Authors: Ramer S W , Davis R W
Published: May 2006

This work reports the identification, characterization, and nucleotide sequence of STE20, a newly discovered gene involved in the Saccharomyces cerevisiae mating response pathway, to date one of the best understood signal transduction pathways. STE20 encodes a putative serine/threonine-specific protein kinase with a predicted molecular mass of 102 kDa. Its expression pattern is similar to that of several other protein kinases in the mating response pathway. Deletion of the kinase domain of STE20 causes sterility in both haploid mating types. This sterility can be partially suppressed by high-level production of STE12 but is not suppressible by high levels of STE4 or a dominant STE11 truncation allele. A truncation allele of STE20 was isolated that can activate the mating response pathway in the absence of exogenous mating pheromone. This allele causes dominant growth arrest that cannot be suppressed by deletions of STE4, STE5, STE7, STE11, or STE12. The allele is able to suppress the mating defect of a strain in which the STE20 kinase domain has been deleted, but not the mating defects of strains carrying mutations in STE4, STE5, STE7, STE11, or STE12.
A mutation within intron 3 of the Pax-3 gene produces aberrantly spliced mRNA transcripts in the splotch (Sp) mouse mutant.
Authors: Epstein D J , Vogan K J , Trasler D G , Gros P
Published: May 2006

The splotch (Sp) mouse mutant displays defects in neural tube closure in the form of exencephaly and spina bifida. Recently, mutations in the Pax-3 gene have been described in the radiation-induced Spr and Sp2H alleles. This led us to examine the integrity of the Pax-3 gene and its cellular mRNA transcript in the original, spontaneously arising Sp allele. A complex mutation in the Pax-3 gene including an A-->T transversion at the invariant 3' AG splice acceptor of intron 3 was identified in the Sp/Sp mutant. This genomic mutation abrogates the normal splicing of intron 3, resulting in the generation of four aberrantly spliced mRNA transcripts. Two of these Pax-3 transcripts make use of cryptic 3' splice sites within the downstream exon, generating small deletions which disrupt the reading frame of the transcripts. A third aberrant splicing event results in the deletion of exon 4, while a fourth retains intron 3. These aberrantly spliced mRNA transcripts are not expected to result in functional Pax-3 proteins and are thus responsible for the phenotype observed in the Sp mouse mutant.
The trans-activator tax of human T-cell leukemia virus type 1 (HTLV-1) interacts with cAMP-responsive element (CRE) binding and CRE modulator proteins that bind to the 21-base-pair enhancer of HTLV-1.
Authors: Suzuki T , Fujisawa J I , Toita M , Yoshida M
Published: May 2006

The trans-activator protein Tax of human T-cell leukemia virus type 1 (HTLV-1) activates the viral 21-base-pair (bp) enhancer in the long terminal repeat and has been suggested to associate indirectly with the enhancer DNA. To demonstrate this, we used DNA-affinity precipitation assay and detected the Tax protein in 21-bp DNA-protein complexes isolated from HTLV-1-infected cells. To identify cellular components in the complexes, we tested various 21-bp DNA-binding proteins by gel electrophoretic mobility-shift assay. Each binding protein gave a shifted band of each 21-bp DNA-protein complex, and exogenously added Tax protein further shifted these bands of cAMP-responsive element (CRE) binding protein (CREB) and CRE modulator but did not shift other bands. Anti-Tax antibodies blocked formation of the complex, indicating complex formations of [Tax-CREB(or CRE modulator)-21-bp DNA]. The formations of these complexes paralleled the functional activities of Tax mutants. Furthermore, the Tax-CREB complex was detected in a nuclear extract of HTLV-1-infected cells, and the Tax-CREB-21-bp-DNA complex was demonstrated as a major component of Tax complexes containing the 21-bp DNA probe. These observations indicate that Tax protein binds to CREB and CRE modulator and the complexes then bind to the 21-bp enhancer, suggesting that the complex binding to the enhancer mediates trans-activation of transcription.
Light-driven proton or chloride pumping by halorhodopsin.
Authors: Bamberg E , Tittor J , Oesterhelt D
Published: May 2006

Halorhodopsin from Halobacterium halobium was purified and reconstituted with lipids from purple membranes. The resulting protein-containing membrane sheets were adsorbed to a planar lipid membrane and photoelectric properties were analyzed. Depending on light conditions, halorhodopsin acted either as a light-driven chloride pump or as a proton pump: green light caused chloride transport and additional blue light induced proton pumping. In the living cell, both to these vectorial processes would be directed toward the cytoplasm and, compared to ion transport by bacteriorhodopsin, this is an inversed proton flow. Azide, a catalyst for reversible deprotonation of halorhodopsin, enhanced proton transport, and the deprotonated Schiff base in the 13-cis configuration (H410) was identified as the key intermediate of this alternative catalytic cycle in halorhodopsin. While chloride transport in halorhodopsin is mediated by a one-photon process, proton transport requires the absorption of two photons: one photon for formation of H410 and release of a proton, and one photon for photoisomerization of H410 and re-formation of H578 with concomitant uptake of a proton by the Schiff base.
Apolipoprotein J expression at fluid-tissue interfaces: potential role in barrier cytoprotection.
Authors: Aronow B J , Lund S D , Brown T L , Harmony J A , Witte D P
Published: May 2006

Apolipoprotein J (apoJ) is a sulfated secreted glycoprotein that exhibits ubiquitous expression, evolutionary conservation, and diverse tissue inducibility. It has been proposed to have roles in programmed cell death, sperm maturation, complement regulation, and lipid transport. To identify cell types that synthesize apoJ and to aid evaluation of its function, we screened mouse and human tissues by in situ hybridization. ApoJ was expressed at high levels in an array of specialized cell types of adult and fetal mouse tissues and in similar cell types of human tissues. Most of these cell types are highly secretory and form the cellular interfaces of many fluid compartments. This group includes epithelial boundary cells of the esophagus, biliary ducts, gallbladder, urinary bladder, ureter, kidney distal convoluted tubules, gastric glands, Brunner's glands, choroid plexus, ependyma, ocular ciliary body, endometrium, cervix, vagina, testis, epididymus, and visceral yolk sac. Several nonepithelial secretory cell types that express high levels of apoJ also line fluid compartments, such as synovial lining cells and ovarian granulosa cells. In the context of its known biochemical properties, this expression pattern suggests that localized synthesis of apoJ serves to protect a variety of secretory, mucosal, and other barrier cells from surface-active components of the extracellular environment.
Recognition of specific DNA sequences by the c-myb protooncogene product: role of three repeat units in the DNA-binding domain.
Authors: Tanikawa J , Yasukawa T , Enari M , Ogata K , Nishimura Y , Ishii S , Sarai A
Published: May 2006

The DNA-binding domain of c-Myb consists of three homologous tandem repeats of 52 amino acids. The structure of the third (C-terminal) repeat obtained by NMR analysis has a conformation related to the helix-turn-helix motif. To identify the role of each repeat in the sequence recognition of DNA, we analyzed specific interactions between c-Myb and DNA by measuring binding affinities for systematic mutants of Myb-binding DNA sites and various truncated c-Myb mutants. We found that specific interactions are localized unevenly in the AACTGAC region in the consensus binding site of c-Myb: The first adenine, third cytosine, and fifth guanine are involved in very specific interactions, in which any base substitutions reduce the binding affinity by > 500-fold. On the other hand, the interaction at the second adenine is less specific, with the affinity reduction in the range of 6- to 15-fold. The seventh cytosine involves a rather peculiar interaction, in which only guanine substitution abolishes the specific binding. The binding analyses, together with the chemical protection analyses, showed that the c-Myb fragment containing the second and third repeats covers the AACTGAC region from the major groove of DNA in such an orientation that the third repeat covers the core AAC sequence. These results suggest that the third repeat recognizes the core AAC sequence very specifically, whereas the second repeat recognizes the GAC sequence in a more redundant manner. The first (N-terminal) repeat, which covers the major groove of DNA only partially, is not significant in the sequence recognition, but it contributes to increase the stability of the Myb-DNA complex. The presence of an N-terminal acidic region upstream of the first repeat, which is important for the activation of c-myb protooncogene, was found to reduce the binding affinity by interfering with the first repeat in binding to DNA.
Synergism in replication and translation of messenger RNA in a cell-free system.
Authors: Morozov I Y , Ugarov V I , Chetverin A B , Spirin A S
Published: May 2006

Combination of the Q beta replicase reaction with the Escherichia coli cell-free translation system markedly enhances replication of a recombinant RQ-DHFR RNA consisting of the dihydrofolate reductase (DHFR) mRNA sequence inserted into RQ135(-1) RNA, an efficient naturally occurring Q beta replicase template. The enhancement is associated with a replication asymmetry previously described for the replication of Q beta phage RNA in vivo; the sense (+)-strands are produced in large excess over the antisense (-)-strands. This, in turn, results in increased synthesis of the functionally active DHFR. These effects are not observed when DHFR mRNAs or RQ135(-1) RNAs are used as templates, if the translation system is not complete, or if it is inhibited by puromycin. The coupled replication-translation of nonviral mRNA recombinants can serve as a useful model for studying the fundamental aspects of virus amplification and can be implemented for large-scale protein synthesis in vitro.
JunD mutants with spontaneously acquired transforming potential have enhanced transactivating activity in combination with Fra-2.
Authors: Kameda T , Akahori A , Sonobe M H , Suzuki T , Endo T , Iba H
Published: May 2006

Although a replication-competent retrovirus that carries junD has no transforming activity in chicken embryo fibroblasts, we have isolated mutant viruses that have spontaneously acquired transforming activity. The molecularly cloned junD genes of three such mutant viruses (T1, T2, and T3) were shown to be responsible for the cellular transformation. DNA sequence analysis indicated that a specific polynucleotide in the junD sequence was tandemly multiplied three times of five times in T1 and T2, respectively. The repeated polynucleotide encodes 16 amino acid residues that are located in a highly conserved region among Jun family proteins. The junD mutation in T3 involved an inversion, a translocation, and nucleotide substitutions that caused drastic amino acid exchanges in another well-conserved region among Jun family proteins. The transcriptional activity of these mutants was analyzed by means of transient expression experiments in F9 cells using a reporter gene containing a single AP-1 binding site. Compared with the wild-type JunD, none of them showed enhanced transactivating activity in the forms of homodimers or of heterodimers with c-Fos or Fra-1. However, they did exhibit much higher transactivating activity than the wild type when they formed heterodimers with Fra-2, indicating that the mutated regions function as transactivation domains in a partner-specific manner. Since we have previously reported that there is a basal level of Fra-2 expression in chicken embryo fibroblasts, the results may indicate that protein complexes between JunD mutants and Fra-2 play a crucial role in the cellular transforming activity.
Reproducing the three-dimensional structure of a tRNA molecule from structural constraints.
Authors: Major F , Gautheret D , Cedergren R
Published: May 2006

The three-dimensional structure of yeast tRNA(Phe) was reproduced at atomic resolution with the automated RNA modeling program MC-SYM, which is based on a constraint-satisfaction algorithm. Structural constraints used in the modeling were derived from the secondary structure, four tertiary base pairs, and other information available prior to the determination of the x-ray crystal structure of the tRNA. The program generated 26 solutions (models), all of which had the familiar "L" form of tRNA and root-mean-square deviations from the crystal structure in the range of 3.1-3.8 A. The interaction between uridine-8 and adenosine-14 was crucial in the modeling procedure, since only this among the tertiary pairs is necessary and sufficient to reproduce the L form of tRNA. Other tertiary interactions were critical in reducing the number of solutions proposed by the program.
Significant enlargement of a specific subset of CD3+CD8+ peripheral blood leukocytes mediating cytotoxic T-lymphocyte activity during human immunodeficiency virus infection.
Authors: Bensussan A , Rabian C , Schiavon V , Bengoufa D , Leca G , Boumsell L
Published: May 2006

We have obtained a monoclonal antibody termed BY55 that defines an 80-kDa cell-surface structure on a subset of circulating peripheral blood mononucleocytes. This structure, which was not detected on most cell lines or activated lymphocytes, was expressed exclusively on 15-25% of CD2+ circulating lymphocytes, including a major subset within the CD3- and the T-cell receptor gamma delta + lymphocytes and a small percentage of the CD3+CD8+ cells. Moreover, we have shown that the BY55 molecule delineated the competent killer circulating lymphocytes. In the present report, additional two- and three-color immunofluorescence studies of peripheral blood lymphocytes were done to precisely determine BY55 expression within the T-cell population. In normal individuals, peripheral blood CD3+CD8+BY55+ cells represented only 5-6% of the lymphocytes, and these cells possessed cytolytic activity. Interestingly, we found that the percentage of total BY55+ lymphocytes as well as the percentage of CD3+CD8+BY55+ was significantly increased in peripheral blood lymphocytes of human immunodeficiency virus-seropositive individuals.
Bacteriorhodopsin is involved in halobacterial photoreception.
Authors: Bibikov S I , Grishanin R N , Kaulen A D , Marwan W , Oesterhelt D , Skulachev V P
Published: May 2006

The bacterio-opsin gene was introduced into a "blind" Halobacterium salinarium mutant that (i) lacked all the four retinal proteins [bacteriorhodopsin (BR), halorhodopsin, and sensory rhodopsins (SRs) I and II] and the transducer protein for SRI and (ii) showed neither attractant response to long wavelength light nor repellent response to short wavelength light. The resulting transformed cells acquired the capability to sense light stimuli. The cells accumulated in a light spot, demonstrating the BR-mediated orientation in spatial light gradients. As in wild-type cells, a decrease in the intensity of long wavelength light caused a repellent response by inducing reversals of swimming direction, but, in contrast to wild-type cells, a decrease in the intensity of short wavelength light also repelled the cells. An increase in light intensity evoked an attractant response (i.e., a transient suppression of spontaneous reversals). Signal processing times and adaptation kinetics were similar to the SRI-mediated reactions. However, compared to SR-mediated photoresponses, higher light intensities were necessary to induce the BR-mediated responses. The light sensitivity of the transformant was increased by adding 1 mM cyanide and decreased by the addition of arginine, agents that respectively reduce and increase the light-independent generation of the electrochemical potential difference of H+ ions (delta mu H+). A decrease in irradiance to an intensity that was still high enough to saturate BR-initiated delta mu H+ changes failed to induce the repellent effect, but the addition of a protonophorous uncoupler sensitized the cell to these light stimuli. The BR D96N mutant (Asp-96 is replaced by Asn) with decreased proton pump activity showed strongly reduced BR-mediated responses. Azide, which increases this mutant's H+ pump efficiency, increased the photosensitivity of the mutant cells. Moreover, azide diminished (i) the membrane potential decreasing and (ii) repellent effects of blue light added to the orange background illumination in this mutant. We conclude that the BR-mediated photoreception is due to the light-dependent generation of delta mu H+. Our data are consistent with the assumption that the H. salinarium cell monitors the membrane energization level with a "protometer" system measuring total delta mu H+ changes or its electric potential difference component.
Peptide nucleic acid (PNA) conformation and polymorphism in PNA-DNA and PNA-RNA hybrids.
Authors: Almarsson O , Bruice T C
Published: May 2006

Two hydrogen-bonding motifs have been proposed to account for the extraordinary stability of polyamide "peptide" nucleic acid (PNA) hybrids with nucleic acids. These interresidue- and intraresidue-hydrogen-bond motifs were investigated by molecular mechanics calculations. Energy-minimized structures of Watson-Crick base-paired decameric duplexes of PNA with A-, B-, and Z-DNA and A-RNA polymorphs indicate that the inherent stability of the complementary PNA helical structures is derived from interresidue, rather than from intraresidue, hydrogen bonds in all hybrids studied. Intraresidue-hydrogen-bond lengths are consistently longer than interresidue hydrogen bonds. Helical strand stability with interresidue hydrogen bond stabilization follows the order: B-(DNA.PNA) > A-(DNA.PNA) congruent to A-RNA.PNA > Z-(DNA.PNA). In the triplex hybrids A-(RNA.PNA2) and B-(DNA.PNA2), differences between stabilities of the two decamers of thyminyl PNA with lysine amide attached to the C terminus (pnaT)10 strands are small. The Hoogsteen (pnaT)10 strands are of slightly higher potential energy than are the Watson-Crick (pnaT)10 strands. Antiparallel arrangement of PNAs in the triplex is slightly favored over the parallel arrangement based on the calculations. Examination by molecular mechanics of the PNA.DNA analogue of the NMR-derived structure for the B-double-stranded DNA dodecamer d(CG-CAAATTTGCG)2 in solution suggests that use of all bases of the genetic alphabet should be possible without loss of the specific interresidue-hydrogen-bonding pattern within the PNA strand.
Single amino acid substitutions uncouple the DNA binding and strand scission activities of Fok I endonuclease.
Authors: Waugh D S , Sauer R T
Published: May 2006

Single alanine substitution mutations at Asp-450 or Asp-467 of the type IIS restriction enzyme Fok I have no effect on the ability of the enzyme to bind strongly and selectively to its recognition site but completely eliminate its ability to cleave either strand of substrate DNA. Since wild-type Fok I shows no kinetic preference or required order of strand cleavage, these results indicate that Fok I, which evidently functions as a monomer, uses a single catalytic center to cleave both strands of DNA. In this respect, Fok I may resemble other monomeric enzymes that cleave double-stranded DNA.