bluuebunny/crossref_metadata_2025 huggingface dataset:
Exclusion of male mitochondria and plastids during syngamy in barley as a basis for maternal inheritance
Authors: Mogensen H. Lloyd
Published: May 2006

It is known from genetic analyses that maternal inheritance of cytoplasmic organelles is the rule among plants and animals. Although recognized as one of several possible mechanisms for strictly maternal cytoplasmic inheritance, exclusion of sperm cytoplasm at the time of gametic fusion has remained poorly documented for the flowering plants. In the present investigation, enucleated, cytoplasmic bodies approximately the size of intact, prefusion sperm cells have been observed within degenerated synergids and adjacent to recently fertilized egg cells. A complete series of ultrathin sections (68 sections) through such a cytoplasmic body revealed 59 mitochondria, 3 plastids, 7 dictyosomes, and a large vacuole with no limiting membrane. This structure is interpreted as the entire male cytoplasm that was left outside the egg during fusion between egg and sperm. The observation of only one cytoplasmic body per embryo sac may indicate a preliminary fusion between sperm cells or, more likely, the existence of a fundamentally different mechanism of fertilization between the second sperm and the central cell.
Nerve growth factor mRNA and protein in the testis and epididymis of mouse and rat.
Authors: Ayer-LeLievre C , Olson L , Ebendal T , Hallböök F , Persson H
Published: May 2006

In situ hybridization using beta-nerve growth factor (NGF) DNA probes was used to demonstrate NGF mRNA in spermatocytes and early spermatids of adult mouse. NGF mRNA-containing cells were also identified in the epithelium of convoluted ducts in mouse corpus epididymidis. Blot-hybridization analysis of RNA prepared from mouse testis and epididymis as well as from rat epididymis confirmed the presence of a 1.3-kilobase (kb) NGF mRNA in these tissues. In the rat testis, however, only a 1.5-kb NGF mRNA was found, corresponding in size to a minor NGF mRNA detected in the rat brain, heart, and epididymis. By using affinity-purified anti-NGF antibodies, NGF-like immunoreactivity was observed in germ cells of rat and mouse testis and in the lumen of epididymis. Extracts of both mouse epididymis and testis stimulated fiber outgrowth in cultured sympathetic ganglia, and the effect was blocked by antibodies to mouse NGF. A two-site enzyme immunoassay showed the presence of 10 and 70 ng of NGF per g of tissue in the mouse testis and epididymis, respectively. Furthermore, RNA blot analysis showed the presence of mRNA for the NGF receptor in mouse testis. These results suggest a nonneurotrophic role for NGF in the male reproductive system, possibly in survival maturation and/or motility of spermatozoa.
Allorecognition of purified major histocompatibility complex glycoproteins by cytotoxic T lymphocytes.
Authors: Elliott T J , Eisen H N
Published: May 2006

To investigate how T cells recognize allogeneic class I proteins encoded by the major histocompatibility complex (MHC), we examined the human cytotoxic T lymphocytes (CTL) elicited in a mixed lymphocyte reaction against a lymphoblastoid B-cell line (JY) whose MHC-class I proteins are HLA-A2 and -B7. By panning the responding T cells on plates that were coated with purified HLA-A2, an essentially pure population of CD8+ anti-HLA-A2 CTL was isolated in a single step and established as a cell line designated A2p. In addition to lysing HLA-A2+ target cells, the A2p cells lysed HLA-A2- cells, including mouse cells (P815), when purified native HLA-A2 was attached to them, but not when denatured HLA-A2 was attached. Thus, contrary to the general rule that T cells recognize sequential antigenic determinants in denatured protein antigens, the alloreactive CTLs appear to recognize determinants that depend upon the native configuration of HLA-A2; however, the possibility that these T cells recognize a peptide adduct persistently associated with purified, soluble HLA-A2 has not been ruled out.
Localization of the main immunogenic region of human muscle acetylcholine receptor to residues 67-76 of the alpha subunit.
Authors: Tzartos S J , Kokla A , Walgrave S L , Conti-Tronconi B M
Published: May 2006

The majority of antibodies to the acetylcholine receptor (AcChoR), both in the human disease myasthenia gravis and in its experimental models, are directed against an extracellular area of the AcChoR alpha subunit called the main immunogenic region (MIR). We have studied the binding of anti-AcChoR monoclonal antibodies (mAbs) to 26 synthetic peptides corresponding to the hydrophilic parts of the human AcChoR alpha subunit. The binding sites for eight anti-MIR mAbs and for eight anti-alpha-subunit, non-anti-MIR mAbs were localized. Anti-MIR mAbs bound to one peptide corresponding to residues 63-80 of the human alpha subunit. A second panel of peptides corresponding to the various parts of the alpha-subunit segment 63-80 was synthesized. Anti-MIR antibodies bound to a peptide that contained the alpha-subunit sequence 67-76. Thus, a main constituent loop of the MIR is localized between residues 67 and 76 of the alpha subunit.
Expression of the differentiation-induced gene for fatty acid-binding protein is activated by glucocorticoid and cAMP.
Authors: Cook J S , Lucas J J , Sibley E , Bolanowski M A , Christy R J , Kelly T J , Lane M D
Published: May 2006

We have isolated and characterized a fragment of the gene encoding adipose fatty acid-binding protein (gene 422) from a 3T3-L1 adipocyte genomic library. The 5'-flanking sequence of the 422 gene contains potential regulatory regions for adipose-specific expression. At position -120 there is a fat-specific element that occurs in several genes expressed as preadipocytes differentiate, and at position -393 there is a glucocorticoid regulatory element core sequence. Chimeric constructs were prepared by ligating 858 base pairs or 248 base pairs of 5'-flanking sequence and 22 nucleotides of 5'-untranslated sequence of the 422 gene to the bacterial gene encoding chloramphenicol acetyltransferase (CAT); these constructs (delta 858.CAT and delta 248.CAT) were transfected into 3T3-L1 preadipocytes. When differentiation was initiated by the adipogenic agents methylisobutylxanthine (a cAMP phosphodiesterase inhibitor), dexamethasone, and insulin, expression of both constructs increased, reaching maximal levels within 24 hr. Both constructs were maximally induced 48 hr before appreciable accumulation of the endogenous 422 mRNA. Expression of delta 858.CAT, but not of delta 248.CAT, was induced by dexamethasone, which correlates with deletion of the potential glucocorticoid regulatory element. Expression of both constructs was induced by 8-bromoadenosine 3',5'-cyclic monophosphate, thus implicating the first 248 base pairs of 5'-flanking sequence of the 422 gene in the response to cAMP. Indirect effects by the adipogenic factors on CAT protein or mRNA synthesis and turnover were ruled out, since replacing the 5'-flanking region of the 422 gene constructs with viral promoters abolished the effects of dexamethasone and 8-bromoadenosine 3',5'-cyclic monophosphate on CAT expression. We conclude that the first 858 base pairs of 5'-flanking sequence of the 422 gene contains elements that mediate activation by dexamethasone and cAMP.
Reexpression of poly(sialic acid) units of the neural cell adhesion molecule in Wilms tumor.
Authors: Roth J , Zuber C , Wagner P , Taatjes D J , Weisgerber C , Heitz P U , Goridis C , Bitter-Suermann D
Published: May 2006

A unique structural feature of the neural cell adhesion molecule N-CAM is the presence of homopolymers of alpha (2----8)-linked sialic acid units. We have used two specific probes for the detection of poly(sialic acid) in normal human kidney and Wilms tumor: a monoclonal antibody against meningococci group B capsular polysaccharide (homopolymers of alpha (2----8)-linked sialic acid units), which shows no crossreactivity with polynucleotides and denaturated DNA, and bacteriophage-induced endosialidases specifically hydrolyzing alpha (2----8)-linked poly(sialic acid) units. Additionally, for the detection of N-CAM, antibodies recognizing the polypeptide portion of the molecule and biotinylated antisense RNA transcribed from a cDNA clone for N-CAM were applied. Poly(sialic acid) was regionally detectable in human embryonic kidney but undetectable in normal adult kidney, as already reported for rat kidney. The malignant Wilms tumor, which is characterized by the presence of structural components resembling those found in embryonic kidney, reexpressed poly(sialic acid) units and showed positive immunostaining for the polypeptide portion of N-CAM. Immunoblot analysis of Wilms tumor as well as human embryonic kidney and brain with the monoclonal anti-poly(sialic acid) antibody revealed in each case the same high molecular mass broad band. In situ hybridization demonstrated the presence of mRNA for N-CAM in Wilms tumor. We conclude that poly(sialic acid), most probably present on N-CAM, is an oncodevelopmental antigen in human kidney.
Ubiquitin is a component of the microtubule network.
Authors: Murti K G , Smith H T , Fried V A
Published: May 2006

Immunofluorescence microscopy was used to study the intracellular localization of ubiquitin. Baby hamster kidney cells (BHK cells) and several other cell lines were probed with a well characterized monoclonal antibody to ubiquitin. The antibody stained a complex cellular structure that we identified as the microtubule network. The anti-ubiquitin antibody bound to the microtubule network at all stages of the cell cycle, and we showed that the apparent association of ubiquitin with the microtubule network is not an artifact of crosslinking of free ubiquitin to the cell structure. Immunoblot procedures demonstrated that tubulin itself was not ubiquitinated. We propose that ubiquitin and/or ubiquitin-protein conjugates are associated with those networks as a new class of microtubule-associated protein. The targeting of ubiquitin to specific sites within the cell by its association with the microtubule network may regulate some of the functions of ubiquitin.
A second DNA methyltransferase repair enzyme in Escherichia coli.
Authors: Rebeck G W , Coons S , Carroll P , Samson L
Published: May 2006

The Escherichia coli ada-alkB operon encodes a 39-kDa protein (Ada) that is a DNA-repair methyltransferase and a 27-kDa protein (AlkB) of unknown function. By DNA blot hybridization analysis we show that the alkylation-sensitive E. coli mutant BS23 [Sedgwick, B. & Lindahl, T. (1982) J. Mol. Biol. 154, 169-175] is a deletion mutant lacking the entire ada-alkB operon. Despite the absence of the ada gene and its product, the cells contain detectable levels of a DNA-repair methyltransferase activity. We conclude that the methyltransferase in BS23 cells is the product of a gene other than ada. A similar activity was detected in extracts of an ada-10::Tn10 insertion mutant of E. coli AB1157. This DNA methyltransferase has a molecular mass of about 19 kDa and transfers the methyl groups from O6-methylguanine and O4-methylthymine in DNA, but not those from methyl phosphotriester lesions. This enzyme was not induced by low doses of alkylating agent and is expressed at low levels in ada+ and a number of ada- E. coli strains.
Inheritance of human breast cancer: evidence for autosomal dominant transmission in high-risk families.
Authors: Newman B , Austin M A , Lee M , King M C
Published: May 2006

Segregation analysis of breast cancer in families can provide the logical basis and the specific genetic models for mapping and identifying genes responsible for human breast cancer. Patterns of breast cancer occurrence in families were investigated by complex segregation analysis. In a sample of 1579 nuclear families ascertained through a population-based series of probands, an autosomal dominant model with a highly penetrant susceptibility allele fully explained disease clustering. From the maximum-likelihood Mendelian model, the frequency of the susceptibility allele was 0.0006 in the general population, and lifetime risk of breast cancer was 0.82 among susceptible women and 0.08 among women without the susceptibility allele. Inherited susceptibility affected only 4% of families in the sample: multiple cases of this relatively common disease occurred in other families by chance. The same genetic models, with higher gene frequency, explained disease clustering in an extended kindred at high risk of breast cancer. Evidence for a highly penetrant, autosomal dominant susceptibility allele for breast cancer in a high-risk family and the general population suggests that high-risk families can serve as models for understanding breast cancer in the population as a whole.
Cell-surface glycoproteins of human sarcomas: differential expression in normal and malignant tissues and cultured cells.
Authors: Rettig W J , Garin-Chesa P , Beresford H R , Oettgen H F , Melamed M R , Old L J
Published: May 2006

Normal differentiation and malignant transformation of human cells are characterized by specific changes in surface antigen phenotype. In the present study, we have defined six cell-surface antigens of human sarcomas and normal mesenchymal cells, by using mixed hemadsorption assays and immunochemical methods for the analysis of cultured cells and immunohistochemical staining for the analysis of normal tissues and greater than 200 tumor specimens. Differential patterns of F19 (Mr, 120,000/95,000 glycoprotein), F24 (Mr, 95,000 glycoprotein), G171 (Mr, 75,000 glycoprotein), G253 (Mr, 90,000 glycoprotein), S5 (Mr, 120,000 glycoprotein), and Thy-1 (Mr, 25,000 glycoprotein) antigen expression were found to characterize (i) subsets of cultured sarcoma cell lines, (ii) cultured fibroblasts derived from various organs, (iii) normal resting and activated mesenchymal tissues, and (iv) sarcoma and nonmesenchymal tumor tissues. These results provide a basic surface antigenic map for cultured mesenchymal cells and mesenchymal tissues and permit the classification of human sarcomas according to their antigenic phenotypes.
Chemically induced murine T lymphomas: continued rearrangement within the T-cell receptor beta-chain gene during serial passage.
Authors: Dasgupta U B , Lilly F
Published: May 2006

The first constant region of the Tcrb gene was completely deleted from the DNA of 8/10 mouse cell lines established from 3-methylcholanthrene-induced RF/J thymic lymphomas, but 6/7 primary lymphomas contained the first constant region sequences. DNA from RF/J thymic lymphomas induced by N-methyl-N-nitrosourea was then examined serially as the tumors were passaged in vivo and adapted to growth in culture as uncloned and, in some cases, cloned lines. Patterns of Tcrb-specific restriction fragments from most tumors changed extensively during continued propagation. Analysis of the patterns often suggested that initial DNA rearrangements within the Tcrb complexes of monoclonal tumors had been followed by further rearrangements within the same genes. However, these different patterns may alternatively have represented successive outgrowth of separate lineages from lymphomas that were polyclonal in origin.
Site-directed mutagenesis of the catalytic residues Asp-52 and Glu-35 of chicken egg white lysozyme.
Authors: Malcolm B A , Rosenberg S , Corey M J , Allen J S , de Baetselier A , Kirsch J F
Published: May 2006

The roles of the catalytic active-site residues aspartic acid-52 and glutamic acid-35 of chicken lysozyme (EC 3.2.1.17) have been investigated by separate in vitro mutagenesis of each residue to its corresponding amide (denoted as D52N and E35Q, respectively). The mutant enzyme D52N exhibits approximately 5% of the wild-type lytic activity against Micrococcus luteus cell walls, while there is no measurable activity associated with E35Q (0.1% +/- 0.1%). The measured dissociation constants for the chitotriose-enzyme complexes were 4.1 microM (D52N) and 13.4 microM (E35Q) vs. 8.6 microM for wild type, indicating that the alterations in catalytic properties may be due in part to binding effects as well as to direct catalytic participation of these residues. The mutant lysozymes have been expressed in and secreted from yeast and obtained at a level of approximately 5 mg per liter of culture by high-salt elution from the cell walls.
Cloned avirulence genes from the tomato pathogen
Pseudomonas syringae
pv.
tomato
confer cultivar specificity on soybean
Authors: Kobayashi Donald Y. , Tamaki Stanley J. , Keen Noel T.
Published: May 2006

Three different cosmid clones were isolated from a genomic library of the tomato pathogen
Pseudomonas syringae
pv.
tomato
, which, when introduced into the soybean pathogen
P. syringae
pv.
glycinea
, caused a defensive hypersensitive response (HR) in certain soybean cultivars. Each clone was distinguished by the specific cultivars that reacted hypersensitively and by the intensity of the HR elicited. Unlike wild-type
P. syringae
pv.
tomato
isolates, which elicit the HR on all soybean cultivars, all three clones exhibited cultivar specificities analogous to avirulence genes previously cloned from
P. syringae
pv.
glycinea
. However, the collective phenotypes of the three clones accounted for HRs on all tested soybean cultivars. One of the three
P. syringae
pv.
tomato
clones contained an avirulence gene homologous to
avrA
, which was previously cloned from
P. syringae
pv.
glycinea
race 6. The other two
P. syringae
pv.
tomato
clones expressed unique HR patterns on various soybean cultivars, which were unlike those caused by any known
P. syringae
pv.
glycinea
race or previously cloned
P. syringae
pv.
glycinea avr
gene. Further characterization of the second
P. syringae
pv.
tomato
clone indicated that the avirulence phenotype resided on a 5.6-kilobase
Hin
dIII fragment that, in Southern blot analyses, hybridized to an identical-size fragment in various
P. syringae
pathovars, including all tested
glycinea
races. These results demonstrate that avirulence genes may be distributed among several
P. syringae
pathovars but may be modified so that the HR is not elicited in a particular host plant. Furthermore, the data raise the possibility that avirulence genes may function in host-range determination at levels above race—cultivar specificity.
Functional substitution of mouse ribosomal protein L27' for yeast ribosomal protein L29 in yeast ribosomes.
Authors: Fleming G , Belhumeur P , Skup D , Fried H M
Published: May 2006

A cDNA clone of mouse ribosomal protein L27' was shown previously to be 62% identical in amino acid residues to yeast ribosomal protein L29. The L27' cDNA was expressed in yeast to determine the ability of the mouse protein to substitute for yeast L29 in assembling a functional ribosome. In a yeast strain resistant to cycloheximide by virtue of a recessive mutation in the L29 protein, the murine cDNA did not produce a sensitive phenotype, indicating failure of the mouse L27' protein to assemble into yeast ribosomes. However, when the mouse L27' gene was expressed in cells devoid of L29 and otherwise inviable, the murine protein supported normal growth, demonstrating that mouse ribosomal protein L27' indeed was interchangeable with yeast L29. We conclude that mouse ribosomal protein L27' is assembled into ribosomes in yeast, but yeast L29 is assembled preferentially when both L29 and L27' are present in the same cell.
Positive genetic selection for gene disruption in mammalian cells by homologous recombination.
Authors: Sedivy J M , Sharp P A
Published: May 2006

Efficient modification of genes in mammalian cells by homologous recombination has not been possible because of the high frequency of nonhomologous recombination. An efficient method for targeted gene disruption has been developed. Cells with substitution of exogenous sequences into a chromosomal locus were enriched, by a factor of 100, using a positive genetic selection that specifically selects for homologous recombination at the targeted site. The selection is based on the conditional expression of a dominant selectable marker by virtue of in-frame gene fusion with the target gene. The dominant selectable marker was derived by modification of the Escherichia coli neo gene so that it retains significant activity in mammalian cells after in-frame fusion with heterologous coding sequences. In the example presented here, homologous recombinants were efficiently recovered from a pool in which the targeted gene was disrupted in 1 per 10,000 cells incorporating exogenous DNA.
Detection of serum hepatitis B virus DNA in patients with chronic hepatitis using the polymerase chain reaction assay.
Authors: Kaneko S , Miller R H , Feinstone S M , Unoura M , Kobayashi K , Hattori N , Purcell R H
Published: May 2006

We compared the sensitivity of the polymerase chain-reaction (PCR) assay to that of slot-blot hybridization for detecting hepatitis B virus (HBV) DNA in the serum of 31 patients with chronic hepatitis. Of 14 chronic hepatitis patients positive for both HBV surface and HBV e antigens, 9 were positive for HBV DNA by slot-blot hybridization and all 14 by PCR. Also, of 9 patients positive for HBV surface antigen and antibody against HBV e antigen, 2 were positive for HBV DNA by slot-blot analysis and 8 by PCR. Finally, in 8 patients positive for HBV DNA by slot-blot hybridization, but 4 were positive by PCR. We find that analysis by the PCR technique provides a greater than 10(4)-fold increase in sensitivity over the slot-blot hybridization assay. This result represents an important breakthrough in sensitivity because it is now possible to detect as few as three HBV DNA genomes per sample of serum.
Cloned diphtheria toxin within the periplasm of Escherichia coli causes lethal membrane damage at low pH.
Authors: O'Keefe D , Collier R J
Published: May 2006

Acidic pH within endosomal vesicles of sensitive animal cells triggers a conformational change in diphtheria toxin (DT) that is believed to cause the B chain to insert into the vesicular membrane and the enzymic A chain to be released into the cytosol. In artificial lipid bilayers, DT forms ion-conductive channels under mildly acidic conditions (pH approximately 5). Here we report a related phenomenon in Escherichia coli strains that secrete certain cloned DT-related proteins into their periplasm: the cells are rapidly killed at pH 5 but remain unharmed at pH 7. Expression of full-length DT (an active-site mutant, to comply with the National Institutes of Health recombinant DNA guidelines) causes acid-sensitivity, whereas expression of the A chain alone does not. The killed cells are not lysed, but inner-membrane functions are impaired (membrane potential, active transport, and ion impermeability). We propose that acidification of DT within the periplasm induces its insertion into the inner membrane, lethally damaging the permeability barrier. This discovery provides a potentially important selection procedure for mutations affecting the membrane insertion function of DT. Similar approaches may be useful in studying other proteins that undergo condition-dependent interaction with membranes.
Visual responses of neurons in somatosensory cortex of hamsters with experimentally induced retinal projections to somatosensory thalamus.
Authors: Métin C , Frost D O
Published: May 2006

These experiments investigate the capacity of thalamic and cortical structures in a sensory system to process information of a modality normally associated with another system. Retinal ganglion cells in newborn Syrian hamsters were made to project permanently to the main thalamic somatosensory (ventrobasal) nucleus. When the animals were adults, single unit recordings were made in the somatosensory cortices, the principal targets of the ventrobasal nucleus. The somatosensory neurons responded to visual stimulation of distinct receptive fields, and their response properties resembled, in several characteristic features, those of normal visual cortical neurons. In the visual cortex of normal animals and the somatosensory cortex of operated animals, the same functional categories of neurons occurred in similar proportions, and the neurons' selectivity for the orientation or direction of movement of visual stimuli was comparable. These results suggest that thalamic nuclei or cortical areas at corresponding levels in the visual and somatosensory pathways perform similar transformations on their inputs.
Aldosterone stimulates K secretion across mammalian colon independent of Na absorption.
Authors: Rechkemmer G , Halm D R
Published: May 2006

K transport across guinea pig (Cavia porcellus) distal colon was measured in vitro using isotopically determined unidirectional fluxes. Aldosterone stimulated electrogenic Na absorption, as measured by amiloride-sensitive short-circuit current (Isc), and reduced net K absorption from +2.5 +/- 0.2 microEq/cm2 per hr to +0.8 +/- 0.3 microEq/cm2 per hr (mean +/- SEM). Amiloride addition to the mucosal solution did not enhance net K absorption, as expected if inhibiting active Na absorption would reduce active K secretion as in the distal nephron. The amiloride-insensitive Isc was -1.0 +/- 0.2 microEq/cm2 per hr (mean +/- SEM) and was inhibited by mucosal addition of Ba, a K channel blocker. Addition of bumetanide to the serosal solution also inhibited this negative Isc, and K transport returned to the control level of net absorption. Thus, the amiloride-insensitive, negative Isc is consistent with active K secretion stimulated by aldosterone. This stimulation of an active K secretory pathway by aldosterone occurred without altering the active K absorption pathway that also is present. These results indicate that the aldosterone-stimulated K secretory pathway operates independently of the amiloride-sensitive Na absorption pathway, which also is stimulated by aldosterone.
Heme oxygenase is the major 32-kDa stress protein induced in human skin fibroblasts by UVA radiation, hydrogen peroxide, and sodium arsenite.
Authors: Keyse S M , Tyrrell R M
Published: May 2006

We have shown that UVA (320-380 nm) radiation, hydrogen peroxide, and sodium arsenite induce a stress protein of approximately 32 kDa in human skin fibroblasts. The synthesis and cloning of cDNA from arsenite-induced mRNA populations have now allowed us to unequivocally identify the 32-kDa protein as heme oxygenase. By mRNA analysis we have shown that the heme oxygenase gene is also induced in cultured human skin fibroblasts by UVA radiation, hydrogen peroxide, cadmium chloride, iodoacetamide, and menadione. The known antioxidant properties of heme catabolites taken together with the observation of a high level of induction of the enzyme in cells from an organ not involved in hemoglobin breakdown strongly supports the proposal that the induction of heme oxygenase may be a general response to oxidant stress and constitutes an important cellular defense mechanism against oxidative damage.
Persistence of nonoptimal strategies
Authors: Ceccatto H. A. , Huberman B. A.
Published: May 2006

Metastable configurations in open computational systems with local minima in their optimality functions are shown to be very long lived, which makes them effectively stable. When rare transitions to the global optimum do occur, they happen extremely fast, in analogy to models of punctuated evolution in biology. These results are obtained by introducing a thermodynamic-like formalism that allows for a simple analysis of nonlinear game dynamics in computational ecosystems.
OxyR, a positive regulator of hydrogen peroxide-inducible genes in Escherichia coli and Salmonella typhimurium, is homologous to a family of bacterial regulatory proteins.
Authors: Christman M F , Storz G , Ames B N
Published: May 2006

The oxyR gene is required for the induction of a regulon of hydrogen peroxide-inducible genes in Escherichia coli and Salmonella typhimurium. The E. coli oxyR gene has been cloned and sequenced, revealing an open reading frame (305 amino acids) that encodes a 34.4-kDa protein, which is produced in maxicells carrying the oxyR clone. The OxyR protein shows homology to a family of positive regulatory proteins including LysR in E. coli and NodD in Rhizobium. Like them, oxyR appears to be negatively autoregulated: an oxyR::lacZ gene fusion produced 5-fold higher levels of beta-galactosidase activity in oxyR null mutants compared to oxyR+ controls, and extracts from an OxyR-overproducing strain were able to protect regions (-27 to +21) of the oxyR promoter from DNase I digestion. DNA sequence analysis of the oxyR2 mutation, which causes overexpression of oxyR-regulated proteins in the absence of oxidative stress, showed that the oxyR2 phenotype is due to a missense mutation (C.G to T.A transition) that changes alanine to valine at amino acid position 234 of OxyR.
DNA strand breaks alter histone ADP-ribosylation.
Authors: Boulikas T
Published: May 2006

Histone ADP-ribosylation was studied using two-dimensional gel electrophoresis after cleavage of the nuclear DNA with nucleases. Modified histones carrying different numbers of ADP-ribose groups form a ladder of bands above each variant histone. Cellular lysates containing unfragmented DNA mainly synthesize mono(ADP-ribosylated) histones. Cleavage of the DNA with either DNase I or micrococcal nuclease to fragments of an average size of 10-20 kilobases (kb) dramatically induces the formation of poly(ADP-ribosylated) species of histones in nuclei. As the number of DNA strand breaks produced by either DNase I or micrococcal nuclease increases and a great number of DNA cuts is introduced (fragments of 0.4-0.2 kb), the size of the poly(ADP-ribose) chains on the histones decreases. Finally, in the presence of 10 mM cAMP as an inhibitor of poly(ADP-ribose) glycohydrolase, human lymphoid nuclei synthesize hyper(ADP-ribosylated) histone H2B with at least 40 ADP-ribose groups attached to it. Lateral ladders emanating at precise points of the linear ladder on hypermodified H2B can arise from branching of poly(ADP-ribose) or from multiple monomodifications of glutamic (or aspartic) acid residues. Branching or de novo monomodifications occur after a precise number of ADP-ribose groups have been added to a histone molecule. Poly(ADP-ribosylated) histones thus appear to be intermediates in nuclear processes involving DNA strand breaks.
Purification of replication protein C, a cellular protein involved in the initial stages of simian virus 40 DNA replication in vitro.
Authors: Virshup D M , Kelly T J
Published: May 2006

The replication of simian virus 40 (SV40) DNA is dependent upon a single viral protein [tumor (T) antigen] and multiple cellular proteins. To define the required cellular proteins, we have made use of a cell-free system that supports the replication of plasmid DNA molecules containing the SV40 origin of replication. We report here the purification from HeLa cell extracts of replication protein C (RP-C), a previously undescribed protein that is required to reconstitute efficient DNA replication in vitro. Highly purified preparations of RP-C contain two closely related polypeptides of 32 and 34 kDa. Preincubation of purified RP-C with T antigen and the DNA template largely eliminates the delay normally observed before the onset of rapid DNA synthesis. In addition, RP-C stimulates the unwinding of duplex DNA molecules containing the SV40 replication origin in a reaction that requires T antigen and a single-stranded DNA binding protein. These observations suggest that RP-C is involved in the initial steps of SV40 DNA replication in vitro.
E1A-dependent trans-activation of the human MYC promoter is mediated by the E2F factor.
Authors: Hiebert S W , Lipp M , Nevins J R
Published: May 2006

E2F is a cellular transcription factor that binds to two sites in the adenovirus E2 promoter. Previous experiments have implicated E2F in the E1A-dependent transactivation of the E2 gene since levels of active E2F increase markedly during adenovirus infection in parallel with the increase in E2 transcription, and an E2F binding site can confer E1A inducibility to a heterologous promoter. Here we show that E2F binds to two sequence elements within the P2 promoter of the human MYC gene which are within a region that is critical for promoter activity. The MYC promoter can be trans-activated in an E1A-dependent manner and site-directed mutagenesis demonstrates that these E2F elements are essential for trans-activation. Finally, we also find that adenovirus infection of quiescent cells results in a stimulation of the endogenous MYC gene. We conclude that the activation of the E2F factor, which is likely responsible for the activation of viral E2 transcription, is also responsible for the E1A-dependent induction of MYC transcription.
Influence of abasic and anucleosidic sites on the stability, conformation, and melting behavior of a DNA duplex: correlations of thermodynamic and structural data.
Authors: Vesnaver G , Chang C N , Eisenberg M , Grollman A P , Breslauer K J
Published: May 2006

We report a complete thermodynamic characterization of the impact of abasic and anucleosidic lesions on the stability, conformation, and melting behavior of a DNA duplex. The requisite thermodynamic data were obtained by using a combination of spectroscopic and calorimetric techniques to investigate helix-to-coil transitions in a family of DNA duplexes of the form d(CGCATGAGTACGC).d(GCGTACXCATGCG), where X corresponds to a thymidine residue in the parent Watson-Crick duplex and to an abasic or anucleosidic site in the modified duplexes. The data derived from these studies reveal that incorporation of an abasic site into a DNA duplex dramatically reduces the duplex stability, transition enthalpy, and transition entropy. The magnitudes of these lesion-induced effects are greater than one would expect based on simple nearest-neighbor considerations. Nearly identical thermodynamic data are obtained when the modified duplex contains an anucleosidic site rather than an abasic site. This observation suggests that the thermodynamic impact of these lesions primarily results from removal of the base rather than the sugar ring. Significantly, the melting cooperativities of the abasic and anucleosidic derivatives are identical with each other and with the corresponding unmodified Watson-Crick parent duplex. This result suggests that the phosphodiester backbone, rather than the base-sugar network, serves as the primary propagation path for the communication of cooperative melting effects. We propose molecular interpretations for the thermodynamic data based on the structural picture that has emerged from the NMR studies of Patel and coworkers on the same family of modified and unmodified DNA duplexes [Kalnik, M.W., Chang, C.-N., Grollman, A.P. & Patel, D.J. (1988) Biochemistry 27, 924-931].
Fibrin monomer protects thrombin from inactivation by heparin-antithrombin III: implications for heparin efficacy.
Authors: Hogg P J , Jackson C M
Published: May 2006

Fibrin II monomer has a dramatic inhibitory effect on the rate of heparin-catalyzed inactivation of human alpha-thrombin by antithrombin III. At 6 microM fibrin II monomer, equivalent to the concentration of fibrinogen in plasma, the second-order rate constant was reduced by a factor of 308--from 2.05 x 10(8) M-1.s-1 to 6.65 x 10(5) M-1.s-1. Fibrin II monomer minimally affected the uncatalyzed rate of thrombin inactivation showing a reduction in the second-order rate constant by a factor of only 1.6. Fibrinogen and the product of plasmin degradation of fibrinogen, fragment E, at 6 microM concentrations also decreased the second-order rate constant for heparin-catalyzed thrombin inactivation, but by factors of only 2.7 and 1.9, respectively. On the basis of these observations it is proposed that protection of thrombin from inactivation by heparin-antithrombin III by fibrin II monomer can explain the limited efficacy of heparin in preventing coronary reocclusion in patients treated with tissue plasminogen activator and other fibrinolytic agents.
A glycosylation mutation affects cell fate in chimeras of Dictyostelium discoideum.
Authors: Houle J , Balthazar J , West C M
Published: May 2006

Prestalk and prespore cells form a simple pattern in the pseudoplasmodium of the cellular slime mold Dictyostelium discoideum. Prestalk cells are distinguished from prespore cells by a low level of expression of a glycoantigen on their surfaces and by reduced intercellular cohesion. We examined the possible significance of these differences, using the modB mutation, which eliminates this glycoantigen genetically, leading to reduced intercellular cohesion, modB mutant cells were allowed to develop together with normal cells to form chimeric slugs. Mutant cells labeled by feeding with fluorescent bacteria were highly enriched in the prestalk cell zone at the anterior end of the slug. In contrast, normal cells, if in a minority, were concentrated in the rear part of the prespore cell zone. Immunoblot analysis and cell-by-cell double-label immunofluorescence of these mixtures showed that mutant cells underproduced several prespore cell markers. Mutant cells tended not to form spores in chimeras unless they exceeded a threshold proportion of ca. 30%. However, mutant cells showed no tendency to produce excess prestalk cells when allowed to develop alone. These findings are most simply explained by postulating that reduced glycoantigen expression and intercellular adhesion encourage a more anterior cell localization, which in turn causes differentiation into a prestalk cell. Since normal prestalk cells also show reduced glycoantigen expression and intercellular adhesion, this suggests that a similar mechanism may contribute to pattern formation during normal development.
Inhibition of human tumor growth in nude mice by a conjugate of doxorubicin with monoclonal antibodies to epidermal growth factor receptor.
Authors: Aboud-Pirak E , Hurwitz E , Bellot F , Schlessinger J , Sela M
Published: May 2006

Monoclonal antibodies that recognize the extracellular domain of the epidermal growth factor receptor (mAb108) were conjugated with doxorubicin through a dextran bridge. Several antibody-drug conjugates, containing different amounts of doxorubicin, retained binding capacity to human epidermoid carcinoma (KB) cells overexpressing epidermal growth factor receptors. Slight decrease in drug cytotoxicity was seen in in vitro tests, as determined either by inhibition of thymidine incorporation into cells or by reduction in number and size of KB-cell colonies. Yet, when tested in vivo against KB tumor xenografted into nude mice, the anti-epidermal growth factor-receptor drug conjugates with high drug-substitution levels were significantly more effective than free doxorubicin, antibody alone, mixture of dextran-doxorubicin and antibody, or drug conjugated with irrelevant antibody. When the labile covalent bonds linking antibody to dextran bridge were stabilized by reduction, the therapeutic efficacy of the conjugate was markedly decreased. These results show that antibodies against the extracellular domain of the epidermal growth factor can deliver doxorubicin specifically and efficiently to tumor sites that express high receptor levels exerting a specific antitumor effect.
Expression in transgenic mice of class I histocompatibility antigens controlled by the metallothionein promoter.
Authors: Morahan G , Brennan F E , Bhathal P S , Allison J , Cox K O , Miller J F
Published: May 2006

To study the effects of increased expression of major histocompatibility complex class I molecules on the development of self-tolerance, transgenic mice were produced that expressed the H-2Kb gene under the control of the metallothionein promoter. Administration of zinc enhanced transgene expression in liver, kidney and exocrine pancreas. No evidence suggestive of an autoimmune response was found in transgene-expressing tissues in mice otherwise allogeneic to H-2Kb. Despite this lack of responsiveness in vivo, T cells could be stimulated in vitro to lyse H-2Kb-bearing target cells. No infiltration was detected in transgenic mice after irradiation and reconstitution with bone marrow cells. When spleen cells were used for reconstitution, however, dense lymphocytic infiltration was seen, particularly in the portal tracts of the liver, and this was accompanied by piecemeal necrosis and apoptosis of periportal hepatocytes. This aggressive response progressively diminished with time, and by 12 weeks after reconstitution many of the portal tracts were free of infiltration while the others showed no accompanying necrosis. The picture at this stage was similar to that seen in chronic persistent hepatitis. These results suggest that, in addition to negative selection in the thymus, peripheral mechanisms not involving clonal deletion or permanent clonal anergy can prevent immune responses to self molecules.
Proglucagon gene expression is regulated by a cyclic AMP-dependent pathway in rat intestine.
Authors: Drucker D J , Brubaker P L
Published: May 2006

Expression of the gene encoding preproglucagon gives rise to different glucagon-related peptides in the pancreas and intestine. Glucagon gene expression is regulated by a protein kinase C-dependent pathway in rat islet cell lines, whereas activation of the adenylate cyclase pathway in islet cell lines is without effect. To elucidate the factors important for the control of proglucagon biosynthesis in the intestine, we have studied proglucagon gene expression and proglucagon biosynthesis in rat intestine. Analysis of intestinal cDNA clones encoding preproglucagon indicated that pancreatic and intestinal glucagon mRNA transcripts were identical. The regulation of proglucagon gene expression in rat intestine differed markedly from that previously observed in islet cell lines. Phorbol esters increased the secretion of glucagon-like immunoreactive peptides (GLI) but had no effect on proglucagon mRNA levels in rat intestinal cells. Bombesin also increased the secretion of GLI without affecting proglucagon mRNA levels or biosynthesis. In contrast, dibutyryl cyclic AMP, forskolin, and cholera toxin increased both proglucagon mRNA levels and GLI biosynthesis and secretion, suggesting that proglucagon gene expression in the intestine is regulated by a cyclic AMP-dependent pathway. These observations suggest that tissue-specific differences in both the regulation of proglucagon gene expression and the posttranslational processing of proglucagon contribute to the diversity of glucagon gene expression.
Binding of actinomycin D to DNA: evidence for a nonclassical high-affinity binding mode that does not require GpC sites.
Authors: Snyder J G , Hartman N G , D'Estantoit B L , Kennard O , Remeta D P , Breslauer K J
Published: May 2006

We have employed a combination of temperature-dependent UV absorption spectroscopy, circular dichroism, and batch calorimetry to characterize the binding of actinomycin D to a series of oligomeric DNA duplexes. We find the duplex [d(CGTCGACG)]2 to be unique in its ability to bind actinomycin D strongly despite the absence of a classic GpC site. We present evidence that this non-GpC-containing duplex binds two actinomycin D molecules in an apparently cooperative manner to form a complex that exhibits aberrant spectroscopic and calorimetric behavior. We propose that these observations are consistent with actinomycin D exhibiting a high-affinity, sequence-dependent DNA-binding mode distinct from its classic binding to isolated GpC sites.
Location of the Bombyx mori specificity domain on a Bacillus thuringiensis delta-endotoxin protein.
Authors: Ge A Z , Shivarova N I , Dean D H
Published: May 2006

Bacillus thuringiensis produces different types of insecticidal crystal proteins (ICPs) or delta-endotoxins. In an effort to identify the insect specificity of ICP toxins, two icp genes were cloned into the Escherichia coli expression vector pKK223-3, and bioassays were performed with purified crystals. The type A protein [from an icpA1, or 4.5-kilobase (kb) gene, from B. thuringiensis var. kurstaki HD-1] was found to be 400 times more active against Bombyx mori than type C protein (from an icpC73, or 6.6-kb gene, from B. thuringiensis var. kurstaki HD-244). The type C protein was 9 times more active against Trichoplusia ni than the type A protein, while both have similar activity against Manduca sexta. To locate the specificity domain of the type A protein for B. mori, site-directed mutagenesis was used to introduce or remove restriction enzyme sites, facilitating the exchange of regions of the two genes. The hybrid genes were overexpressed, and purified ICP was used in bioassays. The B. mori specificity domain for the ICP A toxin is located in the amino-terminal portion of the hypervariable region between amino acids 332 and 450.
Survey of amino-terminal proteolytic cleavage sites in mitochondrial precursor proteins: leader peptides cleaved by two matrix proteases share a three-amino acid motif.
Authors: Hendrick J P , Hodges P E , Rosenberg L E
Published: May 2006

We have compiled sequences of precursor proteins for 50 mitochondrial proteins for which the mature amino terminus has been determined by amino acid sequence analysis. Included in this set are 8 precursors that have leader peptides that are cleaved in two places by mitochondrial matrix proteases. When these eight leader peptides are aligned and compared, a highly conserved three-amino acid motif is identified as being common to this class of leader peptides. This motif includes an arginine at position -10, a hydrophobic residue at position -8, and serine, threonine, or glycine at position -5 relative to the mature amino terminus. The initial cleavage of these peptides by matrix processing protease occurs within the motif, between residues at -9 and -8, such that arginine at position -10 is at position -2 relative to the cleaved bond. The rest of the motif is within the octapeptide removed by subsequent cleavage catalyzed by intermediate-specific protease. An additional 14 leader peptides in this collection (all of those that contain an arginine at -10) conform to this motif. Assuming that these 14 precursors are matured in two steps, we compared the internal cleavage sites at position -8 with the ends of the other 30 leader peptides in the collection. We find that 74% of matrix processing protease cleavage sites follow an arginine at position -2 relative to cleavage.
Effect of manganese ions on the incorporation of dideoxynucleotides by bacteriophage T7 DNA polymerase and Escherichia coli DNA polymerase I.
Authors: Tabor S , Richardson C C
Published: May 2006

Incorporation of dideoxynucleotides by T7 DNA polymerase and Escherichia coli DNA polymerase I is more efficient when Mn2+ rather than Mg2+ is used for catalysis. Substituting Mn2+ for Mg2+ reduces the discrimination against dideoxynucleotides approximately 100-fold for DNA polymerase I and 4-fold for T7 DNA polymerase. With T7 DNA polymerase and Mn2+, dideoxynucleotides and deoxynucleotides are incorporated at virtually the same rate. Mn2+ also reduces the discrimination against other analogs with modifications in the furanose moiety, the base, and the phosphate linkage. A metal buffer, isocitrate, expands the MnCl2 concentration range effective in catalyzing DNA synthesis. The lack of discrimination against dideoxynucleoside triphosphates using T7 DNA polymerase and Mn2+ results in uniform terminations of DNA sequencing reactions, with the intensity of adjacent bands on polyacrylamide gels varying in most instances by less than 10%.
Mutational analysis of a yeast transcriptional terminator.
Authors: Osborne B I , Guarente L
Published: May 2006

We have isolated and mutagenized a DNA fragment from Saccharomyces cerevisiae that specifies mRNA 3' end formation for the convergently transcribed CYC1 and UTR1 genes. An in vivo plasmid supercoiling assay previously showed that this fragment is a transcriptional terminator, and "run-on" assays shown here are consistent with this interpretation. The poly(A) sites in the mRNAs formed by the fragment are the same whether the fragment resides at the native location or at a heterologous location. No single linker substitution abolishes the fragment's activity, whereas certain large, nonoverlapping deletions have strong, deleterious effects. Therefore, the yeast terminator behaves more like rho-dependent bacterial terminators than terminators of higher eukaryotes. That a number of deletions or substitutions have different effects in the two orientations suggests that the fragment contains the sequences of two, unidirectional terminator elements.