bluuebunny/crossref_metadata_2025 huggingface dataset:
Factors involved in specific transcription by mammalian RNA polymerase II: identification of general transcription factor TFIIG.
Authors: Sumimoto H , Ohkuma Y , Yamamoto T , Horikoshi M , Roeder R G
Published: May 2006

We have identified and partially characterized another human general transcription factor, TFIIG. Using a reconstituted in vitro system comprised of purified RNA polymerase II, TFIIB, TFIID, TFIIE, and TFIIF, we found that TFIIG was essential for specific initiation from all class II genes tested. In this system TFIIA could partially replace TFIIG; however, even at saturating concentrations of TFIIA, addition of TFIIG further stimulated transcription. Since the chromatographic properties of TFIIG differed significantly from those of TFIIA, we concluded that TFIIA and TFIIG are distinct but functionally related transcription factors. Heparin challenge assays showed that TFIIG is required for the assembly of a functional preinitiation complex. However, it must act after template commitment by TFIID, since this step did not require, and was unaffected by, either TFIIG or TFIIA.
Molecular characterization of inherited medium-chain acyl-CoA dehydrogenase deficiency.
Authors: Kelly D P , Whelan A J , Ogden M L , Alpers R , Zhang Z F , Bellus G , Gregersen N , Dorland L , Strauss A W
Published: May 2006

Deficiency of medium-chain acyl-CoA dehydrogenase (MCAD) is a common inherited defect in energy metabolism. Characterization of the mRNA encoding MCAD in a Dutch MCAD-deficient patient revealed an A----G change at nucleotide position 985 of the MCAD mRNA coding region. This point mutation results in the substitution of a glutamic acid for a lysine at amino acid position 304 of the mature protein. The single base change was not found in any wild-type MCAD mRNAs. A mutant allele-specific oligonucleotide probe was used in a hybridization analysis of amplified genomic DNA of MCAD-deficient family members, a carrier, and normal individuals. The hybridization analysis specifically identified individuals who were heterozygotes or homozygotes. In addition to the point mutation, a significant proportion of the index patient's MCAD mRNA contained a variety of deletions and insertions as a result of exon skipping and intron retention. The missplicing occurred in multiple regions throughout the MCAD mRNA. Analysis of the patient's MCAD gene in the regions where the missplicing occurred most frequently did not reveal a mutation in the splicing acceptor or donor sites. Therefore, the molecular characterization of this family revealed a crucial point mutation in the MCAD gene and an unusual abnormality in MCAD pre-mRNA splicing.
Evidence for a biological role in photosynthesis for cytochrome b-559--a component of photosystem II reaction center.
Authors: Canaani O , Havaux M
Published: May 2006

Absorbance changes in untreated intact leaf discs, produced upon excitation with high-intensity red light, were shown to be due to the photooxidation of cytochrome b-559. At low intensities (<100 W/m2), photooxidation was almost undetectable. Photooxidation occurred with a half-time of 4.3 sec and an extent of 0.64 mol of cytochrome per 320 mol of chlorophyll. Upon transition to darkness, an additional oxidation occurred that exhibited faster kinetics (t/12 < 100 msec) and 0.32 mol of cytochrome was oxidized per 320 mol of chlorophyll. Photooxidation was inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea and was specifically induced by red light since far-red light did not cause any absorbance decrease. These results suggest that the redox changes of cytochrome b-559 are driven by photosystem II. Photooxidation was increased by 67% and its initial rate was doubled upon incubation of the leaf in carbonylcyanide p-trifluoromethoxy-phenylhydrazone. Exposure of the leaf to mild water stress or mild heat stress resulted in an increase in the extent of photooxidation and in a 6-fold decrease in the rate constant. Mild heat stress also induced a large increase of the rate constant for the dark reduction of the cytochrome. The dependence of photooxidation on high-intensity red light, its inhibition criteria, the fast transient dark oxidation, and enhancement of both photooxidation and dark transient oxidation by treatments that affect Z, the primary donor to P680, indicate that cytochrome b-559 in vivo is involved in cyclic electron flow around photosystem II. Its primary role in photosynthesis is to divert excess photons from a linear to a cyclic electron flow at high light intensities for protection of the D1 and D2 proteins against photodamage. Dark oxidation of the cytochrome is suggested to reflect a second role, that of deactivation of the powerful oxidant Z+ in the dark.
Repetitive increases in cytosolic Ca2+ of guard cells by abscisic acid activation of nonselective Ca2+ permeable channels.
Authors: Schroeder J I , Hagiwara S
Published: May 2006

Many signal-transduction processes in higher plant cells have been suggested to be triggered by signal-induced opening of Ca2+ channels in the plasma membrane. However, direct evidence for activation of plasma-membrane Ca2+ channels by physiological signals in higher plants has not yet been obtained. In this context, several lines of evidence suggest that Ca2+ flux into the cytosol of guard cells is a major factor in the induction of stomatal closing by abscisic acid (ABA). ABA closes stomatal pores, thereby reducing transpirational loss of water by plants under drought conditions. To directly investigate initial events in ABA-induced signal transduction in guard cells, we devised an experimental approach that allows simultaneous photometric measurements of cytosolic Ca2+ and patch-clamp recordings of ion currents across the plasma membrane of single Vicia faba guard cells. Using this approach, we found that the resting cytosolic Ca2+ concentration was 0.19 +/- 0.09 microM (n = 19). In responsive guard cells, external exposure to ABA produced transient repetitive increases in the cytosolic free Ca2+ concentration. These Ca2+ transients were accompanied by concomitantly occurring increases in an inward-directed ion current. Depolarization of the membrane terminated both repetitive elevations in cytosolic Ca2+ and inward-directed ion currents, suggesting that ABA-mediated Ca2+ transients were produced by passive influx of Ca2+ from the extracellular space through Ca2(+)-permeable channels. Detailed voltage-clamp measurements revealed that ABA-activated ion currents could be reversed by depolarizations more positive than -10 mV. Interestingly, reversal potentials of ABA-induced currents show that these currents are not highly Ca2(+)-selective, thereby permitting permeation of both Ca2+ and K+. These results provide direct evidence for ABA activation of Ca2(+)-permeable ion channels in the plasma membrane of guard cells. ABA-activated ion channels allow repetitive elevations in the cytosolic Ca2+ concentration, which, in turn, can modulate cellular responses promoting stomatal closure.
Evolution of the insulin superfamily: cloning of a hybrid insulin/insulin-like growth factor cDNA from amphioxus.
Authors: Chan S J , Cao Q P , Steiner D F
Published: May 2006

Although insulin and the insulin-like growth factors (IGFs) share marked similarities in amino acid sequence and biological activity, their evolutionary origins have not been resolved. To investigate this issue, we recently cloned a cDNA encoding an insulin-like peptide (ILP) from a primitive chordate species, amphioxus (Branchiostoma californiensis). The deduced sequence of amphioxus preproILP indicates that it is a hybrid molecule containing features characteristic of both insulin and IGF. Like proinsulin, amphioxus proILP contains a C-peptide, which is flanked by paired basic residues and is probably removed by proteolysis. However, proILP also contains an extended carboxyl-terminal peptide region that can be divided into D and E domains similar to those of proIGF. Sequence comparisons show that the amphioxus ILP A and B domains are equally homologous to those of human insulin and IGF-I and -II. Based on these results and the exon-intron organization of the amphioxus ILP gene, we propose that IGF emerged at a very early stage in vertebrate evolution from an ancestral insulin-type gene.
Tumor necrosis factor-induced degranulation in adherent human neutrophils is dependent on CD11b/CD18-integrin-triggered oscillations of cytosolic free Ca2+.
Authors: Richter J , Ng-Sikorski J , Olsson I , Andersson T
Published: May 2006

We have recently been able to correlate closely the "spontaneous" oscillatory activity of cytosolic free Ca2+ in adherent human neutrophils with the ability of tumor necrosis factor (TNF) to induce secretion of granule proteins from these cells. In the present work we show with a single-cell technique that preincubation of human neutrophils with antibodies to CD18, the common beta chain of leukocyte adhesion proteins, inhibits TNF-induced secretion of lactoferrin in a time- and concentration-dependent manner. Similar effects of CD18 antibodies were found on chemotactic factor (fMet-Leu-Phe)- but not on phorbol 12-myristate 13-acetate-induced secretion, suggesting that cell-surface-receptor-mediated secretion is dependent on integrin-associated signals. Similarly, antibodies to CD11b (alpha chain of macrophage 1) also inhibited TNF- and fMet-Leu-Phe- but not phorbol 12-myristate 13-acetate-stimulated release of lactoferrin. Antibodies to CD11a (alpha chain of lymphocyte function-associated antigen 1) or CD11c (alpha chain of p150,95) had only a minimal effect on agonist-induced secretion. Data obtained in several laboratories, including our own, made us suspect that integrin interaction with the surface is responsible for the oscillatory activity of cytosolic free Ca2+ in adherent cells. Indeed, preincubation with antibodies to either CD18 or CD11b, but not to CD11c, inhibited the oscillations of cytosolic free Ca2+ in adherent neutrophils. This inhibitory effect was evident both as a reduction of the number of responding cells and as a reduction of the oscillatory activity in the cells. In conclusion, the oscillatory activity of cytosolic free Ca2+ in adherent neutrophils is mediated through the CD18/CD11b integrins. The generation of this Ca2+ signal may explain how adherence, by way of the integrins, changes the functional properties of the cell and enables TNF to induce secretion.
Vaccination against tumor cells expressing breast cancer epithelial tumor antigen.
Authors: Hareuveni M , Gautier C , Kieny M P , Wreschner D , Chambon P , Lathe R
Published: May 2006

Ninety-one percent of breast tumors aberrantly express an epithelial tumor antigen (ETA) identified by monoclonal antibody H23. Vaccinia virus recombinants expressing tumor antigens have considerable promise in the active immunotherapy of cancer, and we have evaluated the potential of vaccinia recombinants expressing the secreted (S) and cell-associated (transmembrane, T) forms of H23 ETA to elicit immunity to tumor cells expressing ETA. Tumorigenic ras-transformed Fischer rat fibroblast lines FR-S and FR-T, expressing the S or T form of H23 ETA, respectively, were constructed for use in challenge experiments. Expression of H23 ETA in these lines was confirmed by Western blotting and immunofluorescence. When challenged by subcutaneous seeding of tumor cells, 97% (FR-S) and 91% (FR-T) of syngeneic Fischer rats rapidly developed tumors that failed to regress. Vaccination with recombinant vaccinia virus expressing ETA-T prior to challenge prevented tumor development in 82% of animals seeded with FR-T cells but in only 61% of animals seeded with FR-S. The vaccinia recombinant expressing the S form was a less effective immunogen, and vaccination protected only 29-30% of animals from developing tumors upon challenge with either FR-S or -T cells. The increased immunogenicity of the recombinant expressing ETA-T was reflected in elevated levels of ETA-reactive antibody in vaccinated animals, confirming that secreted antigens expressed from vaccinia virus are less effective immunogens than their membrane-associated counterparts.
Transgene-induced mutation of the murine steel locus.
Authors: Keller S A , Liptay S , Hajra A , Meisler M H
Published: May 2006

The product of the steel locus is essential for normal development of three distinct populations of stem cells--the neural crest-derived melanoblasts, germ cells, and blood cell precursors. Many mutant alleles at steel are lethal in homozygotes and produce coat color dilution in heterozygotes. We have identified a transgenic mouse with diluted pigmentation that closely resembles that of steel heterozygotes. We have demonstrated that the site of transgene insertion is genetically linked to the phenylalanine hydroxylase locus on mouse chromosome 10. In addition, the chromosome carrying the transgene fails to complement the recessive lethality of the Sl allele of steel and the pigmentation defect of the Slpan allele. The data indicate that the inserted transgene has disrupted the steel locus. The resulting allele, designated Sltg, provides a molecular tag for isolation of the steel gene, as well as a new allele for characterization of this developmentally important locus.
A bacterial peptide acting as a plant nuclear targeting signal: the amino-terminal portion of Agrobacterium VirD2 protein directs a beta-galactosidase fusion protein into tobacco nuclei.
Authors: Herrera-Estrella A , Van Montagu M , Wang K
Published: May 2006

Agrobacterium tumefaciens is a soil bacterium capable of transferring DNA to the genome of higher plants. Of the virulence region-encoded proteins of the tumor-inducing (Ti) plasmid of A. tumefaciens, the VirD1 and VirD2 proteins are essential for T-DNA transfer to plant cells. These two proteins have been shown to be directly responsible for the formation of T-strands. VirD2 was also shown to be firmly attached to the 5' termini of T-strands; these facts have led to its postulation as a pilot protein in the T-DNA transfer process and as a nucleus-targeting signal in plants. We have constructed a chimeric gene by fusing the virD2 gene and the Escherichia coli lacZ gene. Cell fractionation and electron microscopy studies with transgenic tobacco plants containing the VirD2-LacZ fusion protein indicate that the first 292 amino acids of VirD2 are able to direct the cytoplasmic protein beta-galactosidase to the plant nucleus. This provides an example of cross-kingdom nuclear localization between two free-living organisms: a bacterial peptide is capable of acting as a eukaryotic (plant) nuclear targeting signal.
Evolution of Drosophila mitochondrial DNA and the history of the melanogaster subgroup.
Authors: Satta Y , Takahata N
Published: May 2006

The nucleotide sequences of a common region of 15 mitochondrial DNAs (mtDNAs) sampled from the Drosophila melanogaster subgroup were determined. The region is 2527 base pairs long, including most of the NADH dehydrogenase subunit 2 and cytochrome oxidase subunit 1 genes punctuated by three tRNA genes. The comparative study revealed (i) the extremely low saturation level of transitional differences, (ii) recombination or variable substitution rates even within species, (iii) long persistence times of distinct types of mtDNA in Drosophila simulans and Drosophila mauritiana, and (iv) an apparent lack of within-type variations in island species. Also found was a high correlation among the transitional rate, the saturation level, and the G + C content (or codon usage). It appears that D. simulans and D. mauritiana have maintained highly structured populations for more than 1 million years. Such structures are consistent with the origination of Drosophila sechellia from D. simulans. Yet geographic isolation is so weak as to show no evidence for further speciation. Moreover, one type of mtDNA shared by D. simulans and D. mauritiana suggests either recent divergence or ongoing introgression.
Stability and activity of human immunodeficiency virus protease: comparison of the natural dimer with a homologous, single-chain tethered dimer.
Authors: Cheng Y S , Yin F H , Foundling S , Blomstrom D , Kettner C A
Published: May 2006

A single-chain tethered dimer of human immunodeficiency virus protease (HIV-PR) was produced by expression of a synthetic gene in Escherichia coli. The tethered dimer, which consists of two 99-amino acid HIV-PR subunits linked together by a pentapeptide, was isolated from inclusion bodies and refolded as an active protease with enzymatic properties very similar to those of the natural dimer at pH 5.5. In addition to demonstrating that the tethered dimer is active, we have shown that the tethered dimer is more stable than the natural HIV-PR dimer at pH 7.0. This is attributed to dissociation of the natural HIV-PR dimer, for which a surprisingly high dissociation constant, 5 X 10(-8) M was measured. Furthermore, the tethered dimer offers an opportunity to produce asymmetric dimer mutants and thereby determine the effect of changes in one of the two subunits on protease activity. In one such mutant, a single active-site aspartic residue was changed to a glycine residue. This protein was inactive, consistent with a requirement for an aspartic residue from each subunit to constitute an active site of HIV-PR.
Expression of the thymus leukemia antigen in mouse intestinal epithelium.
Authors: Hershberg R , Eghtesady P , Sydora B , Brorson K , Cheroutre H , Modlin R , Kronenberg M
Published: May 2006

The Qa and Tla regions of the mouse major histocompatibility complex contain a series of genes encoding proteins with structural similarity to the class I transplantation antigens of the same complex. In contrast to the genes encoding the transplantation antigens, the Qa and Tla genes show very little polymorphism. Function(s) of the proteins encoded by the Qa and Tla loci remain an enigma. Recently, the protein products of the Qa and Tla loci, often referred to as class Ib major histocompatibility complex molecules, have been proposed to present antigen to gamma delta T cells. In mice, gamma delta T cells have been found concentrated in several epithelial barriers and in the skin; yet, expression of serologically detectable Tla antigens is believed restricted to thymocytes, activated T lymphocytes, and some T-cell leukemias. Here we report that luminal epithelial cells of the mouse small intestine express the thymus leukemia antigen (TLA). We also find that, unlike T cells in Peyer's patches, a significant fraction of intestinal epithelial lymphocytes also express TLA. RNA prepared from intestinal cells contains transcripts of the T18d gene, which encodes TLA. These data extend the known expression profile of TLA molecules to mature lymphocytes and to nonhematopoietic cells. These data also demonstrate the specific expression of TLA on antigen-presenting cells in a site enriched for T cells that express gamma delta T-cell antigen receptor.
Structure of miniature swine class II DRB genes: conservation of hypervariable amino acid residues between distantly related mammalian species.
Authors: Gustafsson K , Germana S , Hirsch F , Pratt K , LeGuern C , Sachs D H
Published: May 2006

As part of our studies of the class II genes of miniature swine, we have isolated and characterized cDNA clones corresponding to DRB genes from two major histocompatibility complex homozygous strains. Comparison of the sequences of these clones to those of human DRB genes revealed a striking amino acid homology between the hypervariable residues of SLA-DRBc and the human DRB1-0101 allele. The percentage of differences in these residues between the pig DRBc allele and the human DRB1-0101 allele was significantly lower (29%) than that between the DRB1-0101 allele and all other human alleles (average, 66.2%). This similarity was not seen in a comparison of the number of silent substitutions, by which the swine DRBc and the human DRB-0101 differed. Since phenotypic selection operates at the level of protein products rather than nucleotide sequences, these data suggest the existence of selective mechanisms that have resulted in similar hypervariable regions in certain alleles even in these widely disparate species. Consistent with this hypothesis, an examination of available murine and bovine class II sequences revealed a homology in hypervariable residues between the human DRB1-1401 allele and the mouse E beta s allele as well as a cow DRB allele. Consideration of these data along with intraspecies allelic sequence comparisons suggests that at least some of the interspecies similarities have emerged as the result of convergent evolution, possibly as the result of a need to react to common pathogens.
A myo-inositol pool utilized for phosphatidylinositol synthesis is depleted in sciatic nerve from rats with streptozotocin-induced diabetes.
Authors: Zhu X , Eichberg J
Published: May 2006

Peripheral nerve from experimentally diabetic rats exhibits lowered levels of myo-inositol (MI) and decreased incorporation of [3H]MI into phosphatidylinositol (PI). There are indications that diminished PI turnover may be causally related to reduced Na+,K(+)-ATPase activity in diabetic nerve. We have investigated whether a metabolic compartment of MI that is essential for PI synthesis is decreased in this tissue. Sciatic nerve segments from streptozotocin-induced diabetic and age-matched normal rats were incubated in vitro with either 32Pi or [3H]cytidine in the presence of propranolol. This cationic amphiphilic agent redirected nerve phospholipid metabolism to produce enhanced 32P incorporation into PI and decreased labeling of phosphatidylcholine and phosphatidyl-ethanolamine. The accumulation of phosphatidyl CMP (CMP-PA) was also demonstrated by chromatographic and enzymatic means. The incorporation of [3H]cytidine into CMP-PA in normal nerve increased up to 15-fold when 0.6 mM propranolol was present. In diabetic nerve, the liponucleotide incorporated 2- to 3-fold more isotope and was more readily labeled at lower drug concentrations as compared to normal nerve. The buildup of [3H]CMP-PA was reduced in a dose-dependent manner in the presence of MI in the incubation medium at concentrations up to 3 mM. However, if MI was added after liponucleotide accumulation, preformed CMP-PA could not be utilized for PI synthesis. The difference in liponucleotide labeling between normal and diabetic nerve was nearly abolished at 0.3 mM medium MI, a concentration much less than the level of cyclitol in the tissue. These results strongly suggest the presence in nerve of a pool of MI that is not in equilibrium with the bulk of nerve MI and that is preferentially used for PI synthesis. This metabolic compartment is depleted in diabetic nerve but can be readily replenished by exogenous MI and may correspond to the MI pool that has been proposed to be required for the turnover of a portion of tissue PI involved in maintenance of normal Na+,K(+)-ATPase activity.
Molecular epitope identification by limited proteolysis of an immobilized antigen-antibody complex and mass spectrometric peptide mapping.
Authors: Suckau D , Köhl J , Karwath G , Schneider K , Casaretto M , Bitter-Suermann D , Przybylski M
Published: May 2006

Sequences of antigenic determinants were identified by limited proteolysis of peptide antigens bound to an immobilized monoclonal antibody and direct molecular weight determination of the monoclonal antibody-bound peptide fragments by 252Cf plasma desorption mass spectrometry. The epitope peptides to the monoclonal antibody h453 [Burger, R., Zilow, G., Bader, A., Friedlein, A. & Naser, W. (1988) J. Immunol. 141, 553-558] were isolated from immobilized antigen-antibody complexes by partial trypsin digestion. A synthetic eicosapeptide comprised of the C-terminal sequence of the human complement component polypeptide des-Arg77-C3a as well as guinea pig des-Arg78-C3a was used as an antigen. Conditions were developed under which trypsin specifically degraded the antigens without inactivation of the immobilized antibody. After proteolysis, epitope peptides were dissociated from the antibody with 4 M MgCl2. The antigenic peptides were purified by HPLC and identified by 252Cf plasma desorption mass spectrometry. The epitope recognized by h453 resides on the C-terminal tryptic peptides of human (residues 70-76) and guinea pig (residues 70-77) C3a. As an estimation of accuracy this method is able to provide, trypsin digestion of immune complexes caused cleavage of the antigen within a distance of two amino acid residues upstream from the epitope.
Cytogenetic damage induced by folate deficiency in mice is enhanced by caffeine.
Authors: MacGregor J T , Schlegel R , Wehr C M , Alperin P , Ames B N
Published: May 2006

Folate deficiency in Swiss mice increased the incidence of micronuclei in peripheral blood erythrocytes, indicating increased chromosomal damage in nucleated erythrocyte precursors. Caffeine enhanced the incidence of micronuclei in blood and bone marrow by up to 5-fold in folate-deficient mice at doses that did not significantly alter the micronucleus frequency in the presence of adequate dietary folate. The lower dose of caffeine used in this study (75 mg/kg) approaches doses received by humans who consume large amounts of caffeinated beverages. Since folate deficiency and caffeine consumption are highly prevalent in the human population, the potential for a similar interaction in man should be evaluated.
Identification of a DNA sequence responsible for binding of the 1,25-dihydroxyvitamin D3 receptor and 1,25-dihydroxyvitamin D3 enhancement of mouse secreted phosphoprotein 1 (SPP-1 or osteopontin) gene expression.
Authors: Noda M , Vogel R L , Craig A M , Prahl J , DeLuca H F , Denhardt D T
Published: May 2006

Secreted phosphoprotein 1 (Spp-1; osteopontin) is one of the abundant noncollagenous proteins in bone matrix and is produced by osteoblasts. We examined the promoter region of the mouse Spp-1 gene and identified a sequence responsible for 1,25-dihydroxyvitamin D3 enhancement of the Spp-1 gene expression. This 24-base-pair (bp) sequence (vitamin D response element) is located 761 bp upstream of the transcription start site and consists of two direct repeats of a unique 9-bp motif, AGGTTCACG. The vitamin D response element confers responsiveness of a heterologous promoter to 1,25-dihydroxyvitamin D3 in a position- and orientation-independent and copy-number-dependent manner. The basal level of expression of the reporter constructs containing this sequence and its response to 1,25-dihydroxyvitamin D3 were not affected by cotreatment with transforming growth factor beta or the tumor promoter phorbol 12-myristate 13-acetate or by cotransfection with a JUN expression vector. The vitamin D response element forms DNA-protein complexes, as indicated by gel-retardation assays. The addition of a monoclonal antibody raised against the vitamin D receptor further retarded the mobility of the DNA-protein complex. Another antibody that recognizes the DNA binding region of the vitamin D receptor attenuated its binding to the sequence. These results indicate that this 24-bp sequence containing two 9-bp motifs binds to the vitamin D receptor and mediates the vitamin D3 enhancement of murine Spp-1 gene expression.
Erythrophagocytosis induces heat shock protein synthesis by human monocytes-macrophages.
Authors: Clerget M , Polla B S
Published: May 2006

Exposure of cells to elevated temperatures and other environmental stresses results in the expression of specific genes encoding the so-called heat shock proteins (HSPs). Since exogenous H2O2 induces in human monocytes the synthesis of HSPs, and previous induction of HSPs protects these cells from oxidative injury, we investigated whether HSP synthesis was also induced during generation of reactive oxygen species by the phagocyte itself during phagocytosis. As a model system, we analyzed the effects of erythrophagocytosis on protein synthesis by the human premonocytic line U937, in which phagocytosis is induced during differentiation with 1,25-dihydroxyvitamin D3. Exposure to whole erythrocytes, but not to erythrocyte ghosts, induced in the phagocytic cells only the synthesis of the 70- and 83- to 90-kDa HSPs and a 32-kDa oxidation-related stress protein identical by partial peptide mapping to heme oxygenase. The radioprotective aminothiol N-(2'-mercaptoethyl)-1,3-propanediamine (WR-1065), which can substitute for glutathione as hydrogen donor, prevented this induction. These results suggest that oxygen free radicals generated in the presence of hemoglobin-derived iron and consecutive glutathione depletion are involved in induction of stress protein synthesis during erythrophagocytosis. HSPs synthesized during phagocytosis may play a role in the phagocyte's defense mechanisms and in protective immunity.
Cloning of cDNA coding for the beta chain of human complement component C4b-binding protein: sequence homology with the alpha chain.
Authors: Hillarp A , Dahlbäck B
Published: May 2006

The major form of complement component C4b-binding protein, a regulator of the complement system, is composed of seven identical 70-kDa alpha chains, each containing a binding site for the complement protein C4b. We recently showed that C4b-binding protein also contains a unique 45-kDa beta chain. It is disulfide-linked to the central core and contains a binding site for the vitamin K-dependent protein S. We have now isolated and characterized full-length cDNA clones for the beta chain. In addition, 57% of the structure was determined by protein sequencing of tryptic and chymotryptic peptides. Two clones, A8 and C1, isolated from different libraries were sequenced. Except for a deleted triplet encoding Ala-3 in clone A8, the two clones were identical and coded for a leader sequence of 17 amino acids and a mature protein of 235 amino acids (including Ala-3). By N-terminal amino acid sequencing, the Ala-3 heterogeneity was confirmed and a third beta-chain species starting at Glu-4 was identified. The beta chain contains five potential N-linked glycosylation sites, and endoglycosidase digestion suggested that the beta chain contained multiple complex carbohydrate side chains. Northern blot analysis of human liver mRNA, using the beta-chain cDNA as the probe, demonstrated a major mRNA species of approximately 1.0 kilobase. From the N terminus, the beta chain contains three tandem repeat units (60 amino acids long) that are homologous to those present in the alpha chain. The C-terminal region, which was unrelated to the tandem repeats, demonstrated sequence similarity with the corresponding region of the alpha chain. In both alpha and beta chains these regions contain two cysteine residues that probably form the interchain disulfide bridges.
Systematic screening of yeast artificial-chromosome libraries by use of the polymerase chain reaction.
Authors: Green E D , Olson M V
Published: May 2006

We have developed an approach for screening ordered arrays of yeast artificial-chromosome (YAC) clones containing human DNA that is based on the polymerase chain reaction (PCR). This approach is designed to determine the locations of positive clones within a YAC library that is stored as individual clones in 96-well microtiter plates. The high sensitivity and specificity of the PCR allow the detection of target sequences in DNA prepared from pools of 1920 or more YAC clones. The PCR-based screening protocol is performed in two successive stages, which effectively limit the location of a positive clone to four microtiter plates (384 clones). Final localization of each positive clone is accomplished by conventional DNA.DNA hybridization using a single filter containing the YAC clones from the appropriate four microtiter plates. This PCR-based screening strategy has proven highly efficient, allowing the identification and isolation of numerous YAC clones containing specific human genes. The prospects of developing a strategy for screening YAC libraries based completely on PCR assays are discussed, as are the potential applications of this approach to the systematic analysis of the human genome.
Identification of the HeLa tumor-associated antigen, p75/150, as intestinal alkaline phosphatase and evidence for its transcriptional regulation.
Authors: Latham K M , Stanbridge E J
Published: May 2006

Prior studies identified a cell-surface antigen, p75/150, that exclusively associated with the tumorigenic phenotype of the HeLa parent and the tumorigenic phenotype of the HeLa parent and the tumorigenic segregants of suppressed, nontumorigenic HeLa x human fibroblast cell hybrids. Candidate p75/150 cDNA clones were isolated from a D98/AH.2 (HeLa) cDNA library using oligonucleotide probes derived from p75/150 partial peptide sequence data. A data base search revealed close similarity of p75/150 with intestinal alkaline phosphatase (IAP) [Berger, J., Garantini, E., Hua, J. C. & Udenfriend, S. (1987) Proc. Natl. Acad. Sci. USA 84, 695-698]. We demonstrate that p75/150 is identical to HeLa IAP by the following criteria: (i) 47/49 amino acid identity of p75 peptide sequence with IAP, (ii) restriction maps for the p75/150 candidate cDNA clone and IAP are identical, (iii) partial DNA sequence analysis of p75/150 candidate cDNA clones revealed complete nucleotide identity with IAP, except for a single nucleotide substitution in the 5' untranslated region, (iv) transfection of a p75/150 cDNA expression vector into the nontumorigenic hybrid, CGL1, yielded p75/150 antibody-positive transfectants that also expressed partially heat-resistant alkaline phosphatase activity. Northern blot analysis demonstrated that high levels of HeLa IAP mRNA were expressed in D98/AH.2 and the tumorigenic segregant CGL4; however, no mRNA was detected in CGL1. Nuclear run-on analyses indicate that HeLa IAP mRNA expression in the HeLa x fibroblast hybrids is regulated at the level of transcription initiation. Furthermore, evidence is discussed supporting the involvement of a chromosome 11 tumor suppressor locus in the regulation of HeLa IAP gene expression.
RNA processing and ribonucleoprotein assembly studied in vivo by RNA transfection.
Authors: Kleinschmidt A M , Pederson T
Published: May 2006

We present a method for studying RNA processing and ribonucleoprotein assembly in vivo, by using RNA synthesized in vitro. SP6-transcribed 32P-labeled U2 small nuclear RNA precursor molecules were introduced into cultured human 293 cells by calcium phosphate-mediated uptake, as in standard DNA transfection experiments. RNase protection mapping demonstrated that the introduced pre-U2 RNA underwent accurate 3' end processing. The introduced U2 RNA was assembled into ribonucleoprotein particles that reacted with an antibody specific for proteins known to be associated with the U2 small nuclear ribonucleoprotein particle. The 3' end-processed, ribonucleoprotein-assembled U2 RNA accumulated in the nuclear fraction. When pre-U2 RNA with a 7-methylguanosine group at the 5' end was introduced into cells, it underwent conversion to a 2,2,7-trimethylguanosine cap structure, a characteristic feature of the U-small nuclear RNAs. Pre-U2 RNA introduced with an adenosine cap (Ap-ppG) also underwent processing, small nuclear ribonucleoprotein assembly, and nuclear accumulation, establishing that a methylated guanosine cap structure is not required for these steps in U2 small nuclear ribonucleoprotein biosynthesis. Beyond its demonstrated usefulness in the study of small nuclear ribonucleoprotein biosynthesis, RNA transfection may be of general applicability to the investigation of eukaryotic RNA processing in vivo and may also offer opportunities for introducing therapeutically targeted RNAs (ribozymes or antisense RNA) into cells.
Activation of phosphatidylinositol 3-kinase by insulin.
Authors: Ruderman N B , Kapeller R , White M F , Cantley L C
Published: May 2006

Insulin action appears to require the protein-tyrosine kinase domain of the beta subunit of the insulin receptor. Despite this, the identities and biochemical functions of the cellular targets of this tyrosine kinase are unknown. A phosphatidylinositol 3-kinase (PI 3-kinase) that phosphorylates the D-3 position of the inositol ring associates with several protein-tyrosine kinases. Here we report that PI 3-kinase activity is immunoprecipitated from insulin-stimulated CHO cells by antiphosphotyrosine and anti-insulin receptor antibodies. Insulin as low as 0.3 nM increased immunoprecipitable PI 3-kinase activity within 1 min. Increases in activity were much greater in CHO cells expressing the human insulin receptor (100,000 receptors per cell) than in control CHO cells (2000 receptors per cell). During insulin stimulation, various lipid products of the PI 3-kinase either appeared or increased in quantity in intact cells, suggesting that the appearance of immunoprecipitable PI 3-kinase reflects an increase in its activity in vivo. These results indicate that insulin at physiological concentrations regulates the PI 3-kinase and suggest that this regulation involves a physical association between the insulin receptor and the PI 3-kinase and tyrosyl phosphorylation.
Focal accumulation of an apolipoprotein B-based synthetic oligopeptide in the healing rabbit arterial wall.
Authors: Shih I L , Lees R S , Chang M Y , Lees A M
Published: May 2006

The functions of surface-accessible domains of apolipoprotein (apo) B, the protein moiety of low density lipoprotein (LDL), are unknown, aside from the LDL receptor-binding domain, which lies toward the carboxyl-terminal end of apoB. Since LDL accumulation in arterial lesions does not depend on recognition of LDLs by a cell-surface receptor, we synthesized an oligopeptide with the sequence of the trypsin-accessible domain of apoB that lies closest to the amino-terminal end of the protein and compared its biological activity to that of another synthetic oligopeptide with the sequence of the heparin- and apoB/apoE receptor-binding domains of apoE. (Tyrosine was added at the amino-terminal end of each peptide to facilitate radiolabeling.) The 18-amino acid apoB-based peptide included residues 1000-1016 of apoB, for which no function has been previously described. In radioautographs, the 125I-labeled peptide accumulated focally at the healing edges of regenerating endothelial islands in the balloon-catheter deendothelialized rabbit aorta. In contrast, the 21-residue apoE-based peptide, which included residues 129-148 of apoE, accumulated diffusely and uniformly throughout the deendothelialized areas of the aorta. The data show that focal binding of the apoB-based peptide can delineate arterial lesions and suggest that this arterial wall-binding domain of apoB mediates accumulation of LDLs in arterial lesions.
Induction and therapy of autoimmune diabetes in the non-obese diabetic (NOD/Lt) mouse by a 65-kDa heat shock protein.
Authors: Elias D , Markovits D , Reshef T , van der Zee R , Cohen I R
Published: May 2006

Insulin-dependent diabetes mellitus is caused by autoimmune destruction of the insulin-producing beta cells of the pancreas. The results described here indicate that a beta-cell target antigen in non-obese diabetic (NOD/Lt) mice is a molecule cross-reactive with the 65-kDa heat shock protein (hsp65) of Mycobacterium tuberculosis. The onset of beta-cell destruction is associated with the spontaneous development of anti-hsp65 T lymphocytes. Subsequently hsp65 cross-reactive antigen becomes detectable in the sera of the prediabetic mice and some weeks later anti-hsp65 antibodies, anti-insulin antibodies, and anti-idiotypic antibodies to insulin antibodies become detectable. The hsp65-cross-reactive antigen, the autoantibodies, and the T-cell reactivity then decline with the development of overt insulin-dependent diabetes. The importance of hsp65 in the pathogenesis of insulin-dependent diabetes was confirmed by the ability of clones of anti-hsp65 T cells to cause insulitis and hyperglycemia in young NOD/Lt mice. Moreover, hsp65 antigen could be used either to induce diabetes or to vaccinate against diabetes, depending on the form of its administration to prediabetic NOD/Lt mice. Other antigens such as the 70-kDa heat shock protein (hsp70) had no effect on the development of diabetes.
Soluble antigen abrogates the appearance of anti-protein IgG1-forming cell precursors during primary immunization.
Authors: Nossal G J , Karvelas M
Published: May 2006

The anti-human serum albumin (HSA) B-cell repertoire of C57BL/6 mice was examined by culturing splenocytes at limiting dilution following polyclonal stimulation with Escherichia coli lipopolysaccharide and a lymphokine mixture. The frequency of anti-HSA precursors was determined before and after immunization with alum-precipitated HSA and 10(9) killed Bordetella pertussis organisms, by submitting clonal supernatants to an ELISA. Anti-HSA IgG1-forming precursors were rare in unimmunized spleens, representing approximately equal to 1 in 500,000 splenocytes or only approximately equal to 100 cells per spleen. Between day 5 and day 7 after immunization, this figure increased to approximately equal to 20,000 cells per spleen. Over the following 3 weeks, there was a progressive increase in the mean optical density generated in the clonal ELISA, presumably due to affinity maturation of the B-cell population. When freshly deaggregated HSA was injected before or even up to 4 days after challenge immunization, the appearance of anti-HSA IgG1-forming cell precursors was largely prevented. The effect was most marked with 5 mg or 1 mg of soluble HSA, but impressive partial effects could be seen with as little as 10 micrograms of HSA if administered before challenge immunization. Most of the few clones seen after the higher doses of the toleragen appeared to make antibody of low affinity. The capacity to influence the B-cell pool by soluble antigen administered just 1-2 days before the sudden appearance of IgG1 precursors argues against the totality of the effect being due to T-cell-mediated suppression and in favor of a direct effect on B cells.
Dextrorphan binds to opioid receptors in guinea-pig brain membranes and is an antagonist at opioid receptors in myenteric plexus.
Authors: Goldstein A , Naidu A
Published: May 2006

Dextrorphan (+)-tartrate, purified by repeated crystallization to remove all traces of the enantiomer levorphanol, binds to mu, delta, and kappa sites on guinea-pig brain membranes with lower affinities (by a factor of 400-3200) than levorphanol. In the guinea-pig ileum myenteric plexus longitudinal muscle preparation (GPI), dextrorphan, at 100-200 microM, inhibits the electrically stimulated twitch, but this action is not blocked or reversed by naloxone; both (+)- and (-)-naloxone produce similar non-opioid twitch inhibition at comparable concentrations. At 10-20 microM, dextrorphan blocks and reverses the twitch inhibition due to mu and kappa agonists, but the blockade can be overcome only partially by increasing the agonist concentration. We conclude that dextrorphan is an opioid ligand with low affinity and with antagonist effect on opioid receptors in the GPI.
Grazing in a turbulent environment: energy dissipation, encounter rates, and efficacy of feeding currents in Centropages hamatus.
Authors: Marrasé C , Costello J H , Granata T , Strickler J R
Published: May 2006

The creation of feeding currents by calanoid copepods increases encounter rates of copepods with their food and provides and advantage in dilute nutritional environments. Small-scale turbulence has also been hypothesized to increase the encounter rate between planktonic predators and their food. Centropages hamatus was exposed to turbulent and nonturbulent environments at two prey concentrations to quantify the influence of turbulence on feeding current efficacy. Turbulent energy dissipation rates used in the experiment were in the range of 0.05-0.15 cm2. sec-3. In the nonturbulent environments, feeding currents increased the encounter rates of C. hamatus 3-5 times that of control encounter areas. In turbulent environments, encounter rates were not increased by feeding currents, yet C. hamatus continued to create feeding currents. Energetic calculations indicate a tradeoff in the value of turbulence to a copepod feeding on phytoplankton. While turbulence is probably beneficial at low food concentrations, it may be deleterious at high food concentrations.
Chromosome ends in Drosophila without telomeric DNA sequences.
Authors: Biessmann H , Carter S B , Mason J M
Published: May 2006

We have recovered terminal chromosome deletions of the X chromosome of Drosophila [Df(1)RT; RT = receding tips] that break in various positions of the yellow gene (y) region and delete all distal DNA sequences. Terminal DNA fragments are heterogeneous in length. Molecular cloning and sequencing of the terminal DNA fragments revealed that the broken ends of the deleted chromosomes do not carry any telomeric DNA sequences, yet the broken chromatids do not fuse to one another. Moreover, we confirmed by sequence analysis of 49 independently cloned terminal DNA fragments from two RT lines collected at different times that they lose DNA sequences from their distal ends at a rate of 70-75 base pairs per fly generation. We calculate that the rate of loss from these ends is consistent with the removal of an octanucleotide RNA primer at each round of DNA replication in the germ line.
Large sequence divergence among mitochondrial DNA genotypes within populations of eastern African black-backed jackals.
Authors: Wayne R K , Meyer A , Lehman N , Van Valkenburgh B , Kat P W , Fuller T K , Girman D , O'Brien S J
Published: May 2006

In discussions about the relative rate of molecular evolution, intraspecific variability in rate is rarely considered. An underlying assumption is that intraspecific sequence differences are small, and thus variations in rate would be difficult to detect or would not affect comparisons among distantly related taxa. However, several studies on mammalian mitochondrial DNA (mtDNA) have revealed considerable intraspecific sequence divergence. In this report, we test for differences in the rate of intraspecific evolution by comparing mtDNA sequences, as inferred from restriction site polymorphisms and direct sequencing, between mtDNA genotypes of the eastern African black-backed jackal, Canis mesomelas elongae, and those of two other sympatric jackal species. Our results are unusual for several reasons. First, mtDNA sequence divergence within several contiguous black-backed jackal populations is large (8.0%). Previous intraspecific studies of terrestrial mammals have generally found values of less than 5% within a single population, with larger divergence values most often occurring among mtDNA genotypes from geographically distant or isolated localities. Second, only 4 mtDNA genotypes were present in our sample of 64 jackals. The large sequence divergence observed among these mtDNA genotypes suggests there should be many more genotypes of intermediate sequence divergence if they had evolved in sympatry. Finally, estimates of the rate of mtDNA sequence evolution differ by approximately 2- to 4-fold among black-backed jackal mtDNA genotypes, thus indicating a substantial heterogeneity in the rate of sequence evolution. The results are difficult to reconcile with ideas of a constant molecular clock based on random fixation of selectively neutral or nearly neutral mtDNA sequence mutations.