bluuebunny/crossref_metadata_2025 huggingface dataset:
Stimulation of B-cell proliferation by membrane-associated molecules from activated T cells.
Authors: Brian A A
Published: May 2006

Activation of B cells to proliferation and antibody secretion is dependent on soluble lymphokines secreted by activated T cells. Activation of T cells results from physical contact between B cells and T cells through binding of the T-cell antigen receptor to a complex of antigen and class II major histocompatibility complex (MHC) molecules. To determine whether this interaction also contributes to B-cell activation by mechanisms other than those mediated by soluble T cell-derived lymphokines, I examined the ability of isolated T-cell plasma membranes to stimulate proliferation in cultures of unfractionated B cells. Membranes prepared from a cloned antigen-specific helper T-cell line induced substantial proliferation provided that the T cells had been mitogen-activated before isolation of membranes. Membranes from splenic Con A-treated blasts also stimulated B-cell proliferation, suggesting that this activity may be a common property of some subsets of activated T cells. Induction of B-cell proliferation was not found to be antigen-dependent or MHC-restricted, indicating no significant contribution by the T-cell receptor for antigen. The presence of interleukins 4 and 5 in membrane fractions was indicated by proliferation of lymphokine-sensitive cell lines, although culture supernatants from mitogen-activated T cells proved to be far more potent sources of these activities. The combined effect of membranes and lymphokine-containing culture supernatants in B-cell cultures was greater than their added effects in separate cultures. This observation suggests that lymphokines or molecules with mitogenic activity for B cells other than those found in abundance in culture supernatants may be present on activated T-cell membranes.
Reduced growth-factor requirement of keloid-derived fibroblasts may account for tumor growth.
Authors: Russell S B , Trupin K M , Rodríguez-Eaton S , Russell J D , Trupin J S
Published: May 2006

Keloids are benign dermal tumors that form during an abnormal wound-healing process in genetically susceptible individuals. Although growth of normal and keloid cells did not differ in medium containing 10% (vol/vol) fetal bovine serum, keloid cultures grew to significantly higher densities than normal cells in medium containing 5% (vol/vol) plasma or 1% fetal bovine serum. Conditioned medium from keloid cultures did not stimulate growth of normal cells in plasma nor did it contain detectable platelet-derived growth factor or epidermal growth factor. Keloid fibroblasts responded differently than normal adult fibroblasts to transforming growth factor beta. Whereas transforming growth factor beta reduced growth stimulation by epidermal growth factor in cells from normal adult skin or scars, it enhanced the activity of epidermal growth factor in cells from keloids. Normal and keloid fibroblasts also responded differently to hydrocortisone: growth was stimulated in normal adult cells and unaffected or inhibited in keloid cells. Fetal fibroblasts resembled keloid cells in their ability to grow in plasma and in their response to hydrocortisone. The ability of keloid fibroblasts to grow to higher cell densities in low-serum medium than cells from normal adult skin or from normal early or mature scars suggests that a reduced dependence on serum growth factors may account for their prolonged growth in vivo. Similarities between keloid and fetal cells suggest that keloids may result from the untimely expression of a growth-control mechanism that is developmentally regulated.
Isolation and structure of corticostatin peptides from rabbit fetal and adult lung.
Authors: Zhu Q Z , Hu J , Mulay S , Esch F , Shimasaki S , Solomon S
Published: May 2006

A 34-amino acid peptide and three other structurally related peptides were isolated from rabbit fetal and adult lung. These cationic arginine- and cysteine-rich peptides inhibit corticotropin (ACTH)-stimulated rat adrenal cell corticosterone production. The peptide was called corticostatin (CSI). CSI was purified by reverse-phase HPLC and was shown to be homogenous from its amino acid analysis. Its sequence was determined on a gas-phase sequenator. The structure of CSI is Gly-Ile-Cys-Ala-Cys-Arg-Arg-Arg-Phe-Cys-Pro-Asn-Ser-Glu-Arg-Phe-Ser-Gly- Tyr-Cys - Arg-Val-Asn-Gly-Ala-Arg-Tyr-Val-Arg-Cys-Cys-Ser-Arg-Arg. CSI was found to markedly inhibit ACTH-stimulated corticosterone production by rat adrenal cells in vitro but did not affect basal levels. CSI did not affect the stimulation of aldosterone synthesis by angiotensin II in rat zona glomerulosa cells but it did suppress ACTH-stimulated aldosterone synthesis in whole adrenal cells, demonstrating that CSI is a specific inhibitor of ACTH-stimulated corticosteroid synthesis. The minimum effective concentration of CSI inhibiting ACTH-stimulated (33 pM) corticosterone production was 5 nM (20 ng/ml), the ED50 (50% effective dose) was 25 nM and steroidogenesis was completely inhibited at concentrations greater than 500 nM (2 micrograms/ml).
Gene structure for the alpha 1 chain of a human short-chain collagen (type XIII) with alternatively spliced transcripts and translation termination codon at the 5' end of the last exon.
Authors: Tikka L , Pihlajaniemi T , Henttu P , Prockop D J , Tryggvason K
Published: May 2006

Two overlapping human genomic clones that encode a short-chain collagen, designated alpha 1(XIII), were isolated by using recently described cDNA clones. Characterization of the cosmid clones that span approximately equal to 65,000 base pairs (bp) of the 3' end of the gene established several unusual features of this collagen gene. The last exon encodes solely the 3' untranslated region and it begins with a complete stop codon. The 10 adjacent exons vary in size from 27 to 87 bp and two of them are 54 bp. Therefore, the alpha 1-chain gene of type XIII collagen has some features found in genes for fibrillar collagens but other features that are distinctly different. Previous analysis of overlapping cDNA clones and nuclease S1 mapping of mRNAs indicated one alternative splicing site causing a deletion of 36 bp from the mature mRNA. The present study showed that the 36 bp is contained within the gene as a single exon and also that the gene has a 45-bp -Gly-Xaa-Xaa- repeat coding exon not found in the cDNA clones previously characterized. Nuclease S1 mapping experiments indicated that this 45-bp exon is found in normal human skin fibroblast mRNAs. Accordingly, the data demonstrate that there is alternative splicing of at least two exons of the type alpha 1(XIII)-chain gene.
Subunit S1 of pertussis toxin: mapping of the regions essential for ADP-ribosyltransferase activity.
Authors: Pizza M , Bartoloni A , Prugnola A , Silvestri S , Rappuoli R
Published: May 2006

The toxicity of pertussis toxin is mediated by the ADP-ribosyltransferase activity of subunit S1. To understand the structure-function relationship of subunit S1 and guide the construction of nontoxic molecules suitable for vaccines, we constructed and expressed in Escherichia coli a series of amino-terminal and carboxyl-terminal deletion mutants as well as a number of molecules containing amino acid substitutions. The shortest peptide still retaining enzymatic activity contains amino acids 2-179. Within this region we identified three mutants in which amino acid substitutions abolish the enzymatic activity. Mutation of amino acids 8 and 9 or 50 and 53, located within the region of the S1 subunit of pertussis toxin homologous to cholera toxin, causes loss of enzymatic activity. Outside this homology region, substitution of Glu-129 with glycine or aspartic acid also eliminates the enzymatic activity of the S1 subunit. In this respect, Glu-129 resembles the glutamic acid that is crucial for the catalytic activity of diphtheria and Pseudomonas toxins. Once introduced into the Bordetella pertussis chromosome, the above mutations should lead to the synthesis of nontoxic pertussis toxin molecules suitable for vaccine production.
Two developmental genes encoding sigma factor homologs are arranged in tandem in Bacillus subtilis.
Authors: Masuda E S , Anaguchi H , Yamada K , Kobayashi Y
Published: May 2006

The sporulation-essential gene spoIIG of the Gram-positive bacterium Bacillus subtilis encodes the sporulation-specific sigma factor sigma 29(sigma E). We report here the initial characterization of a gene, referred to as ORF3, located immediately downstream of the spoIIG gene. The results indicate that ORF3 encodes a sigma homolog, whose expression is highly regulated during development. Analysis of the ORF3 nucleotide sequence reveals an open reading frame encoding a polypeptide of 260 amino acid residues (molecular mass of 30.1 kDa). Its predicted amino acid sequence shows significant similarity to that of other RNA polymerase sigma factor sequences. S1 nuclease mapping experiments indicate that ORF3 is initially cotranscribed with spoIIG from about 1 to 4 hr into the sporulation process and that later on ORF3 is transcribed independently from a new site located between spoIIG and ORF3. The role of ORF3 was investigated by constructing a deletion mutation in its structural gene. The mutant exhibits normal growth but is unable to produce heat-resistant spores. We propose that the ORF3 gene product is a sigma factor or a related peptide essential for sporulation at a late stage of development.
Allelic variants of acetylcholinesterase: genetic evidence that all acetylcholinesterase forms in avian nerves and muscles are encoded by a single gene.
Authors: Rotundo R L , Gomez A M , Fernandez-Valle C , Randall W R
Published: May 2006

Two acetylcholinesterase (AcChoEase) polypeptide chains, alpha and beta, are expressed in avian nerves and muscles with apparent molecular masses of 110 and 100 kDa, respectively. We now show that individual quails express alpha, beta, or both AcChoEase polypeptide chains. By mating studies we show that the two AcChoEase polypeptides are autosomal and segregate as codominant alleles in classical Mendelian fashion. Biochemical studies of the two allelic AcChoEase polypeptides indicate that they have the same turnover number, have the same Km for acetylcholine, are immunoprecipitated to the same extent with a monoclonal anti-AcChoEase antibody, and can assemble with equal efficiency into multimeric forms. Thus there are no obvious functional differences between the two alleles. In heterozygotes, the rates of synthesis of the two polypeptides are identical, suggesting that there are no differences in expression of these two genes. Within an individual, nerves and muscles always express the same AcChoEase forms isolated from muscle indicates that all AcChoEase forms are comprised of the same allelic polypeptide chains. In contrast to the nicotinic acetylcholine receptors that appear to be encoded by complex multigene families, our studies on AcChoEase show that all forms of this important synaptic component in electrically excitable cells are encoded by a single gene. Thus differences in assembly and localization of the multiple synaptic forms of AcChoEase must arise through posttranscriptional events, posttranslational modifications of a similar AcChoEase polypeptide chain or both.
Mapping of brain areas containing RNA homologous to cDNAs encoding the alpha and beta subunits of the rat GABAA gamma-aminobutyrate receptor.
Authors: Séquier J M , Richards J G , Malherbe P , Price G W , Mathews S , Möhler H
Published: May 2006

An in situ hybridization technique was used to determine the distribution in rat brain of RNA homologous to cDNA clones encoding the alpha and beta subunits of the rat brain GABAA gamma-aminobutyrate receptor. The subunit proteins were mapped in adjacent sections autoradiographically and immunohistochemically. Many brain areas containing high densities of GABAA receptors showed strong hybridization signals with both the alpha- and the beta-subunit antisense RNA probe--e.g., cerebral cortex, hippocampus, and cerebellum. On a cellular level, a dense dendritic localization of GABAA receptors was correlated with a strong hybridization in the corresponding somata--e.g., in mitral cells of the olfactory bulb, pyramidal cells of hippocampus, granule cells of the dentate gyrus, and Purkinje and granule cells of the cerebellum. In some brain areas--e.g., substantia nigra--the intensity of the hybridization signal with the beta-subunit probe was much weaker than that with the alpha-subunit probe, whereas the inverse ratio of hybridization intensity was found in others--e.g., in bed nucleus. This regional heterogeneity in the hybridization pattern may reflect regional differences in RNA stability, transcription rate, or subunit composition. The results open the way for studies on the regulation of GABAA-receptor gene expression in normal and pathological brain in situ.
Functional reconstitution of a proton-translocating system responsive to fusicoccin.
Authors: Aducci P , Ballio A , Blein J P , Fullone M R , Rossignol M , Scalla R
Published: May 2006

Crude fusicoccin binding proteins and a partially purified plasma membrane H+-transporting ATPase (EC 3.6.1.34), both solubilized from maize tissues, were simultaneously inserted into liposomes by the freeze-thaw method. ATP-driven intravesicular acidification in the proteoliposomes, measured by the fluorescence quenching of the dye 9-amino-6-chloro-2-methoxyacridine, markedly increased upon addition of fusicoccin to the reconstituted system. This effect could not be observed when binding sites and ATPase preparations were separately reconstituted into the proteoliposomes, thus demonstrating that fusicoccin binding to its receptor is a prerequisite for ATPase stimulation.
Role of disulfide bridges in determining the biological activity of interleukin 3.
Authors: Clark-Lewis I , Hood L E , Kent S B
Published: May 2006

Total chemical synthesis of analog proteins was used to examine the requirement for specific disulfide bridges for the biological activity of interleukin 3 (IL-3), a growth factor that stimulates multiple lineages of hemopoietic cells. Four structural analogs of the mature, 140 amino acid murine IL-3 molecule were synthesized in which specific cysteine residues were replaced by alanines. In a quantitative IL-3 assay, based on [3H]thymidine incorporation into factor-dependent cells, the IL-3 analog with alanines substituted for all four cysteines--i.e., [Ala17,79,80,140]IL-3--had 1/500th as much activity as the molecule synthesized according to the native sequence. The two analogs [Cys17,79,Ala80,140]IL-3 and [Cys17,140,Ala79,80]IL-3 had similarly low activity, whereas the [Cys17,80,Ala79,140]IL-3 analog had 2000-fold higher activity than these three analogs, and 3-fold higher than the molecule with the native sequence. This shows that in IL-3 a single disulfide bridge, between cysteines 17 and 80, is required for biological activity that approximates physiological levels. This disulfide probably stabilizes the tertiary structure of the protein to give a conformation that is optimal for function.
Ethylene-regulated expression of a tomato fruit ripening gene encoding a proteinase inhibitor I with a glutamic residue at the reactive site.
Authors: Margossian L J , Federman A D , Giovannoni J J , Fischer R L
Published: May 2006

We report the isolation from tomato (Lycopersicon esculentum) of an ethylene-responsive member of the proteinase inhibitor gene family. DNA sequence analysis of a full-length cDNA clone indicates that the ethylene-responsive gene is distantly related to the tomato proteinase inhibitor I gene, having 53% sequence identity. The predicted amino acid sequence reveals 47% and 45% sequence identity with the tomato and potato proteinase inhibitor I polypeptides, respectively. Additionally, the ethylene-responsive inhibitor has evolved a completely different pattern of gene expression and inhibitory specificity than other members of the inhibitor I family. Gel blot hybridization experiments show that, unlike the tomato proteinase inhibitor I gene, it is not induced in wounded leaves. In contrast, it is activated by the plant hormone ethylene in leaves and during fruit ripening. Furthermore, the ethylene-responsive inhibitor exhibits a novel reactive site, having glutamic acid as the P1 residue. This suggests that the ethylene-responsive proteinase inhibitor does not react with chymotrypsin, as does proteinase inhibitor I, but that it reacts with proteolytic enzymes that cleave at glutamic residues, such as the Staphylococcus aureus V8 proteinase, for which no inhibitors are known. Finally, isolation and analysis of a genomic clone reveals that the ethylene-responsive proteinase inhibitor gene is tightly linked to another, yet unidentified, coordinately expressed gene. We discuss these results with regard to the function and evolution of proteinase inhibitor genes in tomato.
Gene expression in the embryonic nervous system of Xenopus laevis.
Authors: Richter K , Grunz H , Dawid I B
Published: May 2006

Development of the nervous system in the amphibian embryo is initiated during gastrulation by an inductive interaction between chordamesoderm and dorsal ectoderm. The induced ectoderm forms the neural plate while uninduced ectoderm generates epidermis. We screened for genes activated during gastrulation and expressed specifically in the nervous system of Xenopus laevis in the expectation that clones representing such genes will constitute useful markers for the study of early neurogenesis. Probes were prepared from adult brain RNA by subtraction with RNA from ovary and from different combinations of adult kidney, muscle, and skin; cDNA libraries prepared from early to late neurula embryo RNA were screened with these probes. Six clones were chosen for further study. Three of these clones are not represented in the maternal RNA population but are activated at the late gastrula stage; the other three increase from a maternal base. Expression of five of the genes is restricted to the neural plate during embryogenesis, and all six are restricted to the central nervous system in premetamorphic tadpoles and adults. One of the clones encodes an apparently neurospecific isoform of beta-tubulin; the identity of the other clones is unknown. Expression of all six genes is suppressed in axis-deficient embryos that lack dorsal structures including the brain.
Developmental changes in fibroblast growth factor in the chicken embryo limb bud.
Authors: Munaim S I , Klagsbrun M , Toole B P
Published: May 2006

Cell proliferation is a major event during early limb development. Significant levels of growth factor activity, as measured by stimulation of DNA synthesis in mouse BALB/c 3T3 cells, were found in extracts of chicken embryo limb buds at early stages of development. Extracts from stage-18 limbs (3 days of incubation) were 2 to 3 times more potent than were extracts from older stages, namely 22-24 (4 days), 26 (5 days), and 28 (6 days). Basic fibroblast growth factor (bFGF) was measured specifically using an RIA, and the amounts of factor obtained corresponded to the activities measured by the 3T3 cell-growth assay. In addition, most growth factor in the extracts bound with high affinity to heparin-Sepharose columns. Western (immunologic) blotting and immunoprecipitation with an antibody specific for bFGF revealed a protein of identical size to bFGF--i.e., 18 kDa, in the extracts. Thus, a growth factor with the properties of bFGF is present in the early limb, and the level of this factor is highest when proliferation is a predominant cellular event in the developing limb. These and other data suggest that fibroblast growth factor is a key regulatory factor in embryonic growth and morphogenesis.
Up-regulation of interleukin 4/B-cell stimulatory factor 1 receptor expression.
Authors: Ohara J , Paul W E
Published: May 2006

The expression of interleukin 4 (IL-4) receptors on resting T and B lymphocytes was enhanced 4- to 8-fold by IL-4 stimulation of these cells. Other agents such as lipopolysaccharide and anti-IgM for B cells and concanavalin A for T cells also caused increased IL-4 receptor expression, although to a somewhat smaller degree than IL-4. Using a newly developed flow cytometric analysis based on the binding of biotinylated IL-4 and phycoerythrin-streptavidin, it was observed that receptor up-regulation in a T-cell population treated with IL-4 was a feature of the majority of the T cells. Analysis of IL-4 by cross-linkage of 125I-labeled IL-4 to IL-4 receptor with disuccinimidyl suberate indicated that the IL-4-IL-4 receptor complex was the same size in the resting and up-regulated cells, implying that the same receptor species found in resting cells was up-regulated in response to IL-4.
Enhanced thrombin generation and platelet binding on herpes simplex virus-infected endothelium.
Authors: Visser M R , Tracy P B , Vercellotti G M , Goodman J L , White J G , Jacob H S
Published: May 2006

Atherosclerotic lesions have been reported to contain herpes simplex virus 1 (HSV-1) genomic material. This, and other previous evidence, suggests that latent viral infection may be an atherogenic trigger. Moreover, active HSV-1 lesions manifest marked fibrin deposition in microvessels. In this report we show that very early infection of human endothelial cells with HSV-1 appears to alter surface conformation as detected by merocyanine 540 staining. Concomitantly, the efficiency of prothrombinase complex assembly increases, resulting in a 2- to 3-fold accelerated rate of thrombin generation on the cell surface. Increased thrombin generation is probably doubly procoagulant, since we also demonstrate that thrombin-induced platelet accumulation on HSV-infected endothelium (50.7 +/- 9.3%) is increased compared to uninfected endothelium (9.5 +/- 2.1%; P less than 0.002). Associated with HSV infection, prostacyclin secretion in response to thrombin is diminished by a factor of 20, probably explaining the enhanced platelet attachment. We conclude that HSV infection shifts endothelial cell properties from anticoagulant to procoagulant, both by promoting prothrombinase complex formation and function and by increasing platelet binding, well before cell disruption takes place. Virus-induced changes in the endothelial plasma membrane and diminished prostacyclin secretion are suggested as the pathways for this pathophysiologic mechanism, which may be germane to atherosclerotic thrombosis as well as HSV-mediated tissue necrosis.
Expression of the human growth hormone variant gene in cultured fibroblasts and transgenic mice.
Authors: Selden R F , Wagner T E , Blethen S , Yun J S , Rowe M E , Goodman H M
Published: May 2006

The nucleotide sequence of the human growth hormone variant gene, one of the five members of the growth hormone gene family, predicts that it encodes a growth hormone-like protein. As a first step in determining whether this gene is functional in humans, we have expressed a mouse metallothionein I/human growth hormone variant fusion gene in mouse L cells and in transgenic mice. The growth hormone variant protein expressed in transiently transfected L cells is distinct from growth hormone itself with respect to reactivity with anti-growth hormone monoclonal antibodies, behavior during column chromatography, and isoelectric point. Transgenic mice expressing the growth hormone variant protein are 1.4- to 1.9-fold larger than nontransgenic controls, suggesting that the protein has growth-promoting properties.
Identification of a common chemical signal regulating the induction of enzymes that protect against chemical carcinogenesis.
Authors: Talalay P , De Long M J , Prochaska H J
Published: May 2006

Carcinogenesis is blocked by an extraordinary variety of agents belonging to many different classes--e.g., phenolic antioxidants, azo dyes, polycyclic aromatics, flavonoids, coumarins, cinnamates, indoles, isothiocyanates, 1,2-dithiol-3-thiones, and thiocarbamates. The only known common property of these anticarcinogens is their ability to elevate in animal cells the activities of enzymes that inactivate the reactive electrophilic forms of carcinogens. Structure-activity studies on the induction of quinone reductase [NAD(P)H:(quinone-acceptor) oxidoreductase, EC 1.6.99.2] and glutathione S-transferases have revealed that many anti-carcinogenic enzyme inducers contain a distinctive and hitherto unrecognized chemical feature (or acquire this feature after metabolism) that regulates the synthesis of these protective enzymes. The inducers are Michael reaction acceptors characterized by olefinic (or acetylenic) bonds that are rendered electrophilic (positively charged) by conjugation with electron-withdrawing substrates. The potency of inducers parallels their efficiency in Michael reactions. Many inducers are also substrates for glutathione S-transferases, which is further evidence for their electrophilicity. These generalizations have not only provided mechanistic insight into the perplexing question of how such seemingly unrelated anticarcinogens induce chemoprotective enzymes, but also have led to the prediction of the structures of inducers with potential chemoprotective activity.
Coexistence and gene expression of phenylethanolamine N-methyltransferase, tyrosine hydroxylase, and neuropeptide tyrosine in the rat and bovine adrenal gland: effects of reserpine.
Authors: Schalling M , Dagerlind A , Brené S , Hallman H , Djurfeldt M , Persson H , Terenius L , Goldstein M , Schlesinger D , Hökfelt T
Published: May 2006

Expression and regulation of the catecholamine-synthesizing enzymes phenylethanolamine N-methyltransferase (PNMTase; S-adenosyl-L-methionine:phenylethanolamine N-methyltransferase, EC 2.1.1.28) and tyrosine hydroxylase [TyrOHase; tyrosine 3-monooxygenase, L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2] and the coexisting neuropeptide tyrosine (NPY) were studied in rat and bovine adrenal medulla. By using both immunohistochemistry and in situ hybridization, PNMTase- and NPY-positive cells exhibited a close overlap in bovine medulla and were preferentially localized in the outer two-thirds of the medulla. Although TyrOHase and its mRNA were observed in virtually all medullary gland cells, TyrOHase mRNA levels were much higher in the PNMTase- and NPY-positive cells. After administration of the catecholamine-depleting drug reserpine to rats, a brief increase, followed by a dramatic decrease, in the level of PNMTase mRNA was observed in the adrenal medulla. In contrast, mRNA for both TyrOHase and NPY only exhibited an increase, whereby the TyrOHase mRNA peak preceded that of NPY mRNA. Different regulatory mechanisms may thus operate for these three compounds coexisting in the adrenal medulla.
Expression of diphtheria toxin fragment A and hormone-toxin fusion proteins in toxin-resistant yeast mutants.
Authors: Perentesis J P , Genbauffe F S , Veldman S A , Galeotti C L , Livingston D M , Bodley J W , Murphy J R
Published: May 2006

Mutants of the eukaryote Saccharomyces cerevisiae, previously selected for resistance to diphtheria toxin, were investigated for their suitability as hosts for the expression of tox-related proteins. The structural gene for the toxin, encoding the fragment A catalytic domain, was modified for efficient intracellular expression in eukaryotes and placed downstream of the yeast GAL1 promoter element in a plasmid. Transformed mutant yeast grown in galactose, which induces that promoter, were viable and contained active fragment A. In contrast, sensitive, wild-type cells harboring this plasmid grew normally under repressing conditions but were killed when the GAL1 promoter was induced. Additional constructions were also prepared that included sequences encoding either the lymphocyte growth factor interleukin 2 or alpha-melanocyte-stimulating hormone along with the lipid-associating domains of fragment B and the leader peptide of the Kluyveromyces lactis killer toxin. Resistant mutant strains transformed with these plasmids efficiently expressed and secreted the expected chimeric toxins.
Allosteric equilibria in the binding of fibrinogen to platelets.
Authors: De Cristofaro R , Landolfi R , De Candia E , Castagnola M , Di Cera E , Wyman J
Published: May 2006

The binding of fibrinogen to platelets occurs according to the law of mass action. The platelet receptor binds reversibly a single fibrinogen molecule and undergoes a conformational transition between two allosteric states, T and R, that differ in their affinity for fibrinogen. The equilibrium between the two forms is shifted by ADP toward the R (high-affinity) state, thus promoting the aggregation process. This model opens the way to consideration of allosteric modulation of the binding of fibrinogen to its platelet receptor.
In vitro-derived leukemic erythroid cell lines induced by a raf- and myc-containing retrovirus differentiate in response to erythropoietin.
Authors: Klinken S P , Nicola N A , Johnson G R
Published: May 2006

In vitro infection of murine fetal liver cells with a retrovirus containing v-raf and v-myc oncogenes has produced continuous lines of immature erythroid cells that are leukemogenic. These cells synthesized a factor that stimulated their growth in vitro before autonomous variants emerged. Approximately 1000 high-affinity erythropoietin receptors could be detected per cell, and the hormone induced terminal differentiation in these cells. The lines were generated at an extremely low frequency (approximately 1 in 10(7) cells), suggesting that the combination of raf and myc is insufficient to develop erythroid cell lines and that additional events are necessary for transformation.
Intramolecular recombination of chloroplast genome mediated by short direct-repeat sequences in wheat species.
Authors: Ogihara Y , Terachi T , Sasakuma T
Published: May 2006

Structural alterations of the chloroplast genome tend to occur at "hot spots" on the physical map. To clarify the mechanism of mutation of chloroplast genome structure in higher plants, we determined the nucleotide sequence of the hot-spot region of chloroplast DNAs related to length mutations (deletions/insertions) in Triticum (wheat) and Aegilops. From a comparison of this region in wheat with the corresponding region of tobacco or liverwort, it is evident that one of the open reading frames in tobacco (ORF512) has been replaced in wheat by the rpl23 gene, which is a member of the ribosomal protein gene operon. In the deleted positions and in the original genome of Triticum and Aegilops, consensus sequences forming short direct repeats were found, indicating that these deletions were a result of intramolecular recombination mediated by these short direct-repeat sequences. By two independent recombination events in the Aegilops crassa type of chloroplast genome, which is shared by Triticum monococcum, Ae. bicornis, Ae. sharonensis, Ae. comosa, and Ae. mutica, the novel chloroplast DNA sequences of T. aestivum and Ae. squarrosa were generated. This finding indicates the existence of illegitimate recombination in the chloroplast genome and presents a mechanism for producing genetic diversity of that genome.
Identification and analysis of the major M2 autoantigens in primary biliary cirrhosis.
Authors: Fussey S P , Guest J R , James O F , Bassendine M F , Yeaman S J
Published: May 2006

Primary biliary cirrhosis (PBC) is a chronic cholestatic liver disease characterized by the presence of antimitochondrial antibodies in the serum. It is possible that the PBC-specific immunoreactive trypsin-sensitive antigens on the inner mitochondrial membrane, termed M2, are important in the pathogenesis of this autoimmune disease. We have previously shown that a major M2"a" antigen is the E2 component of the pyruvate dehydrogenase multienzyme complex located within mitochondria. Analysis of the primary structure of the E2 components of all three 2-oxo acid dehydrogenase complexes reveals a high degree of homology with a similar highly segmented structure including lipoyl domains, E3-binding domains, C-terminal catalytic domains, and interdomain linker sequences. Immunoblotting of PBC patients' sera against purified E2 protein from 2-oxoglutarate dehydrogenase complex and branched-chain 2-oxo acid dehydrogenase complex reveals that these polypeptides are also autoantigens in this disease. Sera from 29 of 40 (72.5%) PBC patients gave a positive response against bovine 2-oxoglutarate dehydrogenase complex E2 and from 25 of 40 (62.5%) PBC patients gave a positive response against bovine branched-chain 2-oxo acid dehydrogenase complex E2. All 40 PBC patients (100%) have autoantibodies directed against at least one of the E2 components of the family of 2-oxo acid dehydrogenase complexes. Identification of these M2 mitochondrial autoantigens and detailed knowledge of their structure will allow important questions concerning this autoimmune disease to be addressed.
Ex vivo model of an immobilized-enzyme reactor.
Authors: Bernstein H , Langer R
Published: May 2006

Immobilized-enzyme reactors are beginning to be studied for a variety of therapeutic applications. To facilitate the design of these devices for different clinical situations and a diverse patient population, mathematical models may be valuable. An immobilized-heparinase (EC 4.2.2.7) reactor was selected as a model system. The device removes heparin from blood that has been anticoagulated to prevent thrombus formation. Heparinase was immobilized to cross-linked agarose particles. A mathematical model was developed to describe the clearance of heparin by the reactor ex vivo and compared to experimental clearances measured in sheep. The model accounted for enzymatic degradation as well as the binding of heparin and its breakdown products to antithrombin. The device was modeled as a steady-state continuously stirred tank reactor. Molar conservation equations within the agarose particles accounted for simultaneous diffusion and chemical reaction. The model had no adjustable parameters and was able to predict the clearance of heparin within 5-25% for three different animals and 12 different perfusions.
His-426 of the Pseudomonas aeruginosa exotoxin A is required for ADP-ribosylation of elongation factor II.
Authors: Wozniak D J , Hsu L Y , Galloway D R
Published: May 2006

Exotoxin A (ETA) is recognized as the most toxic product associated with the opportunistic pathogen Pseudomonas aeruginosa. Identification of the amino acids in the polypeptide sequence that are required for toxin activity is critical for vaccine development. By defining the nucleotide sequence of the structural gene of a mutant that encodes an enzymatically inactive ETA (CRM 66), we identified an essential amino acid (His-426), which is involved in the ADP-ribosyltransferase activity associated with functional ETA. A monoclonal antibody that inhibits ETA enzymatic activity in vitro fails to react with ETA variants that have a His 426----Tyr substitution. Several mono-ADP-ribosylating toxins, including diphtheria and pertussis toxins, within the primary amino acid sequences carry a histidine residue that is conserved in spacing and in location with respect to other critical residues. Analysis of the three-dimensional structure of ETA revealed that His-426 is not associated with the proposed NAD+ binding site. These findings should be useful for the design and construction of toxin vaccines.
Different structures in the amino-terminal domain of the ornithine transcarbamylase leader peptide are involved in mitochondrial import and carboxyl-terminal cleavage.
Authors: Kraus J P , Novotný J , Kalousek F , Swaroop M , Rosenberg L E
Published: May 2006

The cytoplasmic precursor of mitochondrial ornithine transcarbamylase (carbamoyl-phosphate:L-ornithine carbamoyltransferase, EC 2.1.3.2) contains an amino-terminal leader peptide of 32 amino acids. Secondary structure and helical-wheel analyses predict that the extreme amino-terminal domain (residues 1-15) forms an alpha-helix. To test this thesis, leucine residues at positions 2, 5, 8, and 9 were systematically replaced by either helix-breaking glycine residues or by helix-preserving alanine residues. Triple substitutions of glycine for leucine in positions 2, 5, and 9 or 5, 8, and 9 abolished the uptake of the rat precursor by intact mitochondria, whereas similar alanine substitutions had much less effect. Theoretical computations predicted that the decreased helical stability of the Gly-5,8,9 substitution could be significantly increased by replacing a serine in position with phenylalanine. The introduction of Phe-3, indeed, restored the mitochondrial uptake of the mutant precursor. These results lend strong support to the hypothesis that an alpha-helix is present at the leader's amino terminus during the import of the precursor by mitochondria. Although the precursors with the triply-substituted leaders were impaired with respect to import, they were still cleaved readily by a protease found in a mitochondrial matrix fraction. Substitution of glycine or alanine for all four leucine residues, however, rendered the leader uncleavable at the carboxyl-terminal cleavage site. These results suggest that the structure of the amino-terminal domain is important for recognition of the carboxyl-terminal cleavage sites by the matrix proteases.
Cocrystal structure of an editing complex of Klenow fragment with DNA.
Authors: Freemont P S , Friedman J M , Beese L S , Sanderson M R , Steitz T A
Published: May 2006

High-resolution crystal structures of editing complexes of both duplex and single-stranded DNA bound to Escherichia coli DNA polymerase I large fragment (Klenow fragment) show four nucleotides of single-stranded DNA bound to the 3'-5' exonuclease active site and extending toward the polymerase active site. Melting of the duplex DNA by the protein is stabilized by hydrophobic interactions between Phe-473, Leu-361, and His-666 and the last three bases at the 3' terminus. Two divalent metal ions interacting with the phosphodiester to be hydrolyzed are proposed to catalyze the exonuclease reaction by a mechanism that may be related to mechanisms of other enzymes that catalyze phospho-group transfer including RNA enzymes. We suggest that the editing active site competes with the polymerase active site some 30 A away for the newly formed 3' terminus. Since a 3' terminal mismatched base pair favors the melting of duplex DNA, its binding and excision at the editing exonuclease site that binds single-stranded DNA is enhanced.
Probing the Escherichia coli glnALG upstream activation mechanism in vivo.
Authors: Sasse-Dwight S , Gralla J D
Published: May 2006

In vivo "footprints" of the glnA regulatory region under activating conditions demonstrate that the three most upstream activator sequences bind the protein NRI in the cell. Together, protections at these sites span six of seven consecutive major grooves and lie on the same helix face. E sigma 54 protects two major grooves of DNA approximately 60 base pairs downstream at the glnAp2 promoter and primarily on the opposite helix face. Experiments using potassium permanganate to probe open complex formation in vivo demonstrate that NRI is absolutely required for E sigma 54 to open the promoter DNA. Together, the dimethyl sulfate and permanganate studies verify [Reitzer, L. J., Bueno, R., Cheng, W. D., Abrams, S. A., Rothstein, D. M., Hunt, T. P., Tyler, B. & Magasanik, B. (1987) J. Bacteriol. 169, 4279-4284] that E sigma 54 occupies the glnAp2 promoter in a closed complex in vivo even in the presence of excess nitrogen and the absence of NRI. Furthermore, the slow step in transcriptional activation is shown to be an NRI-dependent conformational change in the downstream promoter DNA, which results in DNA melting. These observations place interesting restrictions on models describing the mechanism by which NRI activates transcription from glnAp2 at a distance.
Characterization of a cDNA clone encoding the calmodulin-binding domain of mouse brain calcineurin.
Authors: Kincaid R L , Nightingale M S , Martin B M
Published: May 2006

A cDNA clone corresponding to a portion of the catalytic subunit of calmodulin (CaM)-dependent phosphoprotein phosphatase (calcineurin) was isolated from a murine brain library by expression vector immunoscreening. A beta-galactosidase fusion protein that reacted on Western blots with anti-calcineurin antibodies and biotinylated CaM was purified in preparative amounts using CaM-Sepharose affinity chromatography. Partial digestion of the hybrid protein with Staphylococcus aureus V-8 protease produced several immunoreactive peptides that appeared identical to fragments generated from authentic brain calcineurin. The 1111-base-pair (bp) EcoRI insert contained an open reading frame encoding a protein of 35 kDa followed by a 190-bp 3' noncoding region; seven peptides obtained by partial amino acid sequencing of the bovine brain enzyme were found in the deduced sequence. A domain approximately 12 kDa from the carboxyl terminus was deduced to be the CaM-binding site based on consensus structural features and a sequence of seven amino acids highly related to smooth muscle myosin light-chain kinase. Two regions with identity to protein phosphatases 1 and 2A were found in the amino half of the cloned sequence; however, the intervening sequence contained apparent insertions, suggesting splicing of subdomains. Thus, the structure of calcineurin is chimeric, consisting of conserved catalytic elements and a regulatory CaM-binding domain.
Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer.
Authors: Frohman M A , Dush M K , Martin G R
Published: May 2006

We have devised a simple and efficient cDNA cloning strategy that overcomes many of the difficulties encountered in obtaining full-length cDNA clones of low-abundance mRNAs. In essence, cDNAs are generated by using the DNA polymerase chain reaction technique to amplify copies of the region between a single point in the transcript and the 3' or 5' end. The minimum information required for this amplification is a single short stretch of sequence within the mRNA to be cloned. Since the cDNAs can be produced in one day, examined by Southern blotting the next, and readily cloned, large numbers of full-length cDNA clones of rare transcripts can be rapidly produced. Moreover, separation of amplified cDNAs by gel electrophoresis allows precise selection by size prior to cloning and thus facilitates the isolation of cDNAs representing variant mRNAs, such as those produced by alternative splicing or by the use of alternative promoters. The efficacy of this method was demonstrated by isolating cDNA clones of mRNA from int-2, a mouse gene that expresses four different transcripts at low abundance, the longest of which is approximately 2.9 kilobases. After less than 0.05% of the cDNAs produced had been screened, 29 independent int-2 clones were isolated. Sequence analysis demonstrated that the 3' and 5' ends of all four int-2 mRNAs were accurately represented by these clones.
Transformation of the cryobehavior of rye protoplasts by modification of the plasma membrane lipid composition
Authors: Steponkus Peter L. , Uemura Matsuo , Balsamo Ronald A. , Arvinte Tudor , Lynch Daniel V.
Published: May 2006

The freezing tolerance of protoplasts isolated from nonacclimated rye leaves (
Secale cereale
L. cv Puma) was significantly altered by using a pH-induced protoplastliposome fusion technique to modify the lipid composition of the plasma membrane. The increase in freezing tolerance was elicited by fusion with liposomes composed of either the total phospholipid fraction isolated from the plasma membrane of cold-acclimated leaves or single mono- or diunsaturated species of phosphatidylcholine (PtdCho). Of the PtdCho species tested, dilinoleoylphosphatidylcholine ([Lin
2
]PtdCho) and dilinolenoylphosphatidylcholine ([Lnn
2
]PtdCho) liposomes were the most effective; 1-palmitoyl-2-oleoylphosphatidylcholine, 1-palmitoyl-2-linoleoylphosphatidylcholine, or dioleoylphosphatidylcholine liposomes were somewhat less effective; dimyristoylphosphatidylcholine or dipalmitoylphosphatidylcholine liposomes had no effect. The increased freezing tolerance was the result of a transformation in the cryobehavior of the plasma membrane during freeze-induced osmotic contraction. In control nonacclimated protoplasts, osmotic contraction resulted in endocytotic vesiculation of the plasma membrane which was irreversible and resulted in lysis during osmotic expansion after melting of the suspending medium. In nonacclimated protoplasts fused with mono- or diunsaturated species of PtdCho, osmotic contraction resulted in the reversible formation of exocytotic extrusions of the plasma membrane—as normally occurs in protoplasts isolated from cold-acclimated leaves (acclimated protoplasts). In scanning electron micrographs, the morphology of the extrusions of nonacclimated protoplasts fused with [Lin
2
]PtdCho was virtually indistinguishable from that of the extrusions formed in acclimated protoplasts. These studies provide direct evidence that changes in the lipid composition of the plasma membrane are causally related to one facet of the cold-acclimation process.
Release of IgD-binding factor by T cells under the influence of interleukin 2, interleukin 4, or cross-linked IgD.
Authors: Amin A R , Coico R F , Finkelman F , Siskind G W , Thorbecke G J
Published: May 2006

Helper T cells with receptors specific for IgD have immunoaugmenting properties. We have now detected soluble IgD-binding factor in cell supernatants immobilized on nitrocellulose paper by their ability to bind 125I-labeled IgD. IgD-binding factor is released by normal splenic T cells stimulated with recombinant interleukin 2, recombinant interleukin 4, or crosslinked IgD in amounts paralleling the induction of IgD receptors on the cells. IgD receptors are constitutively produced by antigen-specific helper T-cell hybridomas 2H10 and A3.4C6. Incubation of these hybridoma cells with recombinant interleukin 2 increases release of IgD-binding factor while reducing expression of IgD receptors. Specificity of the binding factor for IgD is established by (i) competitive inhibition; (ii) the ability of the binding factor to bind radiolabeled IgD and not monoclonal IgE, IgG2a, or polyclonal IgG; and (iii) the removal of the binding factor on passage through an IgD-Sepharose column and recovery in a subsequent acid eluate.
Epstein-Barr virus (EBV) infection of murine L cells expressing recombinant human EBV/C3d receptor.
Authors: Ahearn J M , Hayward S D , Hickey J C , Fearon D T
Published: May 2006

The normal host range of Epstein-Barr virus (EBV) is limited to primate B lymphocytes and certain epithelial cells that express the C3d/EBV receptor [complement receptor 2 (CR2, CD21)]. In the present study, expansion of the tissue tropism of EBV has been accomplished by stably transfecting the murine fibroblast L cell line with pMT.CR2. neo.1, a eukaryotic expression vector promoting the transcription of a complementary DNA insert encoding human CR2. High CR2-expressing transfected L cells were selected by fluorescence-activated cell sorting. The recombinant CR2 was shown to have the same molecular weight as wild-type CR2 from Raji cells and to mediate the binding by the transfectants of particles bearing the iC3b and C3d fragments of the third component of complement. All CR2-expressing L cells, but not nontransfected controls, also bound EBV, as assessed by indirect immunofluorescence. After a 60-hr culture, approximately 0.5% of the CR2-expressing cells preincubated with EBV demonstrated immunofluorescent staining of EBV nuclear antigen with serum from a patient with nasopharyngeal carcinoma. No fluorescent staining of cells was seen with monoclonal antibodies to the early antigen complex or to gp350/220, indicating that the infection was predominantly latent. Infected cells cultured for up to 4 weeks remained EBV nuclear antigen-positive. The capacity of recombinant human CR2 to confer on murine L cells susceptibility to stable latent infection by EBV indicates that this receptor is a primary determinant of the tissue tropism of EBV and may facilitate studies of cell-specific factors that regulate the viral growth cycle.