bluuebunny/crossref_metadata_2025 huggingface dataset:
Translation of bicistronic viral mRNA in transfected cells: regulation at the level of elongation.
Authors: Fajardo J E , Shatkin A J
Published: May 2006

The S1 species of mammalian reovirus mRNA, like a number of other viral but not cellular mRNAs, codes for two dissimilar polypeptides by initiation of translation at two 5'-proximal, out-of-frame AUG codons. To determine if uninfected cells can utilize bicistronic genes, a bovine papilloma virus-based vector system was used to select mouse C127 cell lines containing multiple integrated copies of the reovirus S1 gene. These cell lines produced both reovirus polypeptides from a single mRNA. In addition, studies of COS cells transfected with the S1 gene containing small changes around the first AUG suggest that bicistronic mRNA translation is regulated at the level of elongation. A model is proposed in which ribosomes engaged in translation of one reading frame interfere with movement of ribosomes in the other frame because of differences in codon usage. Expression of bicistronic genes may be similarly regulated in virus-infected cells.
Mutations in the bglY gene increase the frequency of spontaneous deletions in Escherichia coli K-12.
Authors: Lejeune P , Danchin A
Published: May 2006

A strong mutator effect has been observed in Escherichia coli K-12 strains mutated in the bglY gene (27 min). The frequency of point mutations is not modified in bglY mutant strains. In contrast, a strong increase in spontaneous generation of large deletions has been observed in these strains, both for chromosomal markers (10-fold increase of galETK-chlA deletions and 100-fold increase of ptsI-cysK deletions) and for plasmid DNA (100-fold increase of large deletions in the region located upstream of the chloramphenicol-resistance gene in plasmid pGR71). bglY mutations are recessive and can be complemented by a DNA fragment of 900 base pairs assumed to contain the entire bglY wild-type gene. This mutator effect is recA-independent.
Overexpression of fibronectin induced by diabetes or high glucose: phenomenon with a memory.
Authors: Roy S , Sala R , Cagliero E , Lorenzi M
Published: May 2006

To identify events and mechanisms that might contribute to the poor reversibility of diabetic complications, we examined whether diabetes or high glucose induces changes in gene expression and whether such changes outlast the presence of the metabolic abnormalities. The study focused on fibronectin because the increased amounts of this glycoprotein found in diabetic tissues and thickened basement membranes are as yet unexplained. In streptozotocin-induced diabetic rats, fibronectin mRNA levels were increased to 304 +/- 295% of control (mean +/- SD) in the kidney cortex (P less than 0.02), and to 271 +/- 273% of control in the heart (P less than 0.02), while actin mRNA levels remained unchanged. Elevation of fibronectin mRNA persisted for weeks after restoration of near-normoglycemia. In cultured human endothelial cells, high glucose-induced overexpression of fibronectin and collagen IV remained detectable after replating and multiple cell divisions in the absence of high glucose. Cells shifted to normal-glucose medium after prolonged exposure to high glucose also exhibited a proliferative advantage over cells chronically maintained in normal glucose. Thus, diabetes increases fibronectin expression in tissues that are known targets of the complications, and the effect is not readily reversible. The in vitro studies suggest that hyperglycemia may be responsible for these events through induction of self-perpetuating changes in gene expression.
Histidine-94 is the only important histidine residue in the melibiose permease of Escherichia coli.
Authors: Pourcher T , Sarkar H K , Bassilana M , Kaback H R , Leblanc G
Published: May 2006

Oligonucleotide-directed, site-specific mutagenesis has been utilized to modify the melB gene of Escherichia coli such that each of the seven His residues in the melibiose permease has been replaced with Arg. Replacement of His-213, His-442, or His-456 has no significant effect on permease activity, while permease with Arg in place of His-198, His-318, or His-357 retains more than 70% of wild-type activity. In striking contrast, replacement of His-94 with Arg causes a complete loss of sugar binding and transport, although the cells contain a normal complement of permease molecules. Thus, as shown previously with lac permease, only a single His residue is important for activity, but, in the case of mel permease, the critical His residue is present in the 3rd putative transmembrane helix rather than the 10th.
A stable double-stranded DNA-ethidium homodimer complex: application to picogram fluorescence detection of DNA in agarose gels.
Authors: Glazer A N , Peck K , Mathies R A
Published: May 2006

The complex between double-stranded DNA and ethidium homodimer (5,5'-diazadecamethylene)bis(3,8-diamino-6-phenylphenanthridini um) cation, formed at a ratio of 1 homodimer per 4 or 5 base pairs, is stable in agarose gels under the usual conditions for electrophoresis. This unusual stability allows formation of the complex before electrophoresis and then separation and detection in the absence of background stain. Competition experiments between the preformed DNA-ethidium homodimer complex and a 50-fold molar excess of unlabeled DNA show that approximately one-third of the dye is retained within the original complex independent of the duration of the competition. However, dye-extraction experiments show that these are not covalent complexes. After electrophoretic separation, detection of bands containing 25 pg of DNA was readily achieved in 1-mm thick agarose gels with laser excitation at 488 nm and a scanning confocal fluorescence imaging system. The band intensity was linear with the amount of DNA applied from 0.2 to 1.0 ng per lane and with the number of kilobase pairs (kbp) per band within a lane. Analysis of an aliquot of a polymerase-chain-reaction mixture permitted ready detection of 80 pg of a 1.6-kbp amplified fragment. The use of the ethidium homodimer complex together with laser excitation for DNA detection on gels is at least two orders of magnitude more sensitive than conventional fluorescence-based procedures. The homodimer-DNA complex exemplifies a class of fluorescent probes where the intercalation of dye chromophores in DNA forms a stable, highly fluorescent ensemble.
Correction of murine mucopolysaccharidosis VII by a human beta-glucuronidase transgene.
Authors: Kyle J W , Birkenmeier E H , Gwynn B , Vogler C , Hoppe P C , Hoffmann J W , Sly W S
Published: May 2006

We recently described a murine model for mucopolysaccharidosis VII in mice that have an inherited deficiency of beta-glucuronidase (beta-D-glucuronoside glucuronosohydrolase, EC 3.2.1.31). Affected mice, of genotype gusmps/gusmps, present clinical manifestations similar to those of humans with mucopolysaccharidosis VII (Sly syndrome) and are shown here to have secondary elevations of other lysosomal enzymes. The mucopolysaccharidosis VII phenotype in both species includes dwarfism, skeletal deformities, and premature death. Lysosome storage is visualized within enlarge vesicles and correlates biochemically with accumulation of undegraded and partially degraded glycosaminoglycans. In this report we describe the consequences of introducing the human beta-glucuronidase gene, GUSB, into gusmps/gusmps mice that produce virtually no murine beta-glucuronidase. Transgenic mice homozygous for the mucopolysaccharidosis VII mutation expressed high levels of human beta-glucuronidase activity in all tissues examined and were phenotypically normal. Biochemically, both the intralysosomal storage of glycosaminoglycans and the secondary elevation of other acid hydrolases were corrected. These findings demonstrate that the GUSB transgene is expressed in gusmps/gusmps mice and that human beta-glucuronidase corrects the murine mucopolysaccharidosis storage disease.
1,25-Dihydroxy-16-ene-23-yne-vitamin D3 prolongs survival time of leukemic mice.
Authors: Zhou J Y , Norman A W , Chen D L , Sun G W , Uskokovic M , Koeffler H P
Published: May 2006

The 1,25-dihydroxy-16-ene-23-yne-vitamin D3 [1,25(OH)2-16-ene-23-yne-D3] is a vitamin D analog that is very potent in inhibiting proliferation and inducing differentiation of myeloid leukemic cells in vitro. Also, 1,25(OH)2-16-ene-23-yne-D3 is 300 times less active in mediating intestinal calcium absorption and bone calcium mobilization as compared to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], the physiologically active metabolite. Furthermore, 1,25(OH)2-16-ene-23-yne-D3 is 10-25 times less potent than 1,25(OH)2D3 in causing hypercalcemia in BALB/c mice injected intraperitoneally (i.p.) every other day (q.o.d.) for 6 weeks. We explored the therapeutic potential of 1,25(OH)2-16-ene-23-yne-D3 by developing and using the following three leukemia models. (i) Injection of 2.5 x 10(5) myeloid leukemic cells (WEHI 3BD+) into syngeneic BALB/c mice resulted in leukemic death of all diluent-injected mice by day 26. Mice who received the same number of leukemic cells and also received 1,25(OH)2D3 (0.1 micrograms q.o.d., i.p.) had nearly an identical survival curve. Those who received the leukemic cells and 1,25(OH)2-16-ene-23-yne-D3 (1.6 micrograms q.o.d., i.p.) had a significantly (P = 0.003) longer survival, with the last mouse dying of leukemia on day 50. (ii) Injection of 50% fewer leukemic cells (1 x 10(5) cells) into syngeneic BALB/c mice resulted in 86% dead of leukemia at 51 days. Experimental mice who received the same number of leukemic cells and 1,25(OH)2-16-ene-23-yne-D3 (0.8 microgram q.o.d.) had a significantly (P = 0.0006) longer survival than controls; only 53% of the mice were dead by day 100. (iii) After injection of 1.5 x 10(4) leukemic cells, 13% of syngeneic BALB/c mice were free of disease at day 180. In contrast, 43% of mice who received leukemic cells and 1,25(OH)2-16-ene-23-yne-D3 (1.6 micrograms q.o.d.) were still free of disease at day 180. In summary, 1,25(OH)2-16-ene-23-yne-D3 is a vitamin D analog that significantly increased survival of mice who had myeloid leukemia.
High-level production of a functional immunoglobulin heterodimer in a baculovirus expression system.
Authors: Hasemann C A , Capra J D
Published: May 2006

A murine immunoglobulin heterodimer has been expressed in a baculovirus expression system. This was achieved by using both double infection of insect cells with separate heavy- and light-chain-expressing viruses and infection with a double-recombinant virus containing both the immunoglobulin heavy- and light-chain cDNAs. In both cases, the polypeptide chains were correctly processed, glycosylated, and assembled into normal H2L2 (H = heavy, L = light) immunoglobulin monomers. These molecules bound antigen and expressed both polyclonal idiotype and monoclonal idiotopes. Furthermore, the transfer vectors described have been modified to contain the F1 origin of replication for the production of single-stranded DNA, which facilitates site-specific mutations of either the polyhedrin promoter or the inserted foreign gene. Use of this system should significantly advance the analysis of the structural bases for both idiotype expression and antigen binding by immunoglobulin. More importantly, it provides a generic method for the high-level expression of antibodies of diverse interest.
Molecular characterization of germ-line immunoglobulin A transcripts produced during transforming growth factor type beta-induced isotype switching.
Authors: Lebman D A , Nomura D Y , Coffman R L , Lee F D
Published: May 2006

The addition of transforming growth factor type beta to lipopolysaccharide-stimulated murine B-cell cultures enhances isotype switching to IgA and induces the appearance of two sizes of alpha mRNA transcripts. One of these is the same size as mRNA for secreted IgA but the other, which is 300-400 base pairs (bp) shorter, does not correlate in size with any form of productive alpha mRNA. Both sizes of transcript were shown to contain germ-line sequences 5' to the alpha switch region, suggesting that the longer transcripts included both germ-line and productive forms of alpha mRNA, whereas the shorter transcripts were only germ-line alpha mRNA. We isolated cDNA clones corresponding to the shorter, 1.3-kilobase (kb), transcript by using an anchored polymerase chain reaction and a specific primer for the constant region. Analyses of these cDNA clones show that the short transcript consists of a 126-bp exon located approximately 1.5 kb 5' to the alpha switch region spliced to the first exon of the alpha constant region locus. Furthermore, a minor fraction of the longer, approximately 1.7 kb, transcripts also contains this exon. These results demonstrate that transforming growth factor type beta-mediated isotype switching to IgA is preceded by transcriptional activation of the heavy-chain locus.
Peptidyl-prolyl cis-trans-isomerase from Escherichia coli: a periplasmic homolog of cyclophilin that is not inhibited by cyclosporin A.
Authors: Liu J , Walsh C T
Published: May 2006

The prokaryotic peptidyl-prolyl cis-trans-isomerase called "rotamase", a homolog of the human cyclophilin, has been identified in Escherichia coli. The E. coli rotamase, a product of the gene we suggest be called "rot," has been purified to homogeneity after cloning of the gene by the polymerase chain reaction and its overexpression in E. coli. Based on the chymotrypsin-coupled assay using the tetrapeptide substrate succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, the purified protein has rotamase activity identical to human cyclophilin with a catalytic efficiency close to the upper diffusional limit (kcat/Km approximately 1.0 x 10(7) M-1 x S-1 at 10 degrees C). Unlike the human cyclophilins, however, the E. coli rotamase is not significantly inhibited by the immunosuppressant drug cyclosporin A. By spheroplast fractionation of cells harboring the expression vector for the complete rot gene, the rotamase is located in the periplasm, where it could function in refolding of secreted proteins.
Introduction of large linear minichromosomes into Schizosaccharomyces pombe by an improved transformation procedure.
Authors: Allshire R C
Published: May 2006

The efficiency of transformation of Schizosaccharomyces pombe has been increased 10- to 50-fold over previously reported methods. By using 1 microgram of plasmid, 7.0 x 10(5) transformants are regularly obtained. This increased transformation efficiency is mainly due to the inclusion of the cationic liposome-forming reagent Lipofectin in the protocol. Various parameters affecting transformation of Sc. pombe in the presence of Lipofectin have been examined. Lipofectin can also be used to increase transformation efficiency in Saccharomyces cerevisiae. It is also demonstrated that by using this improved transformation procedure, linear minichromosomes of greater than 500 kilobases can be introduced into Sc. pombe with relative ease. These minichromosomes can replicate as stable linear molecules upon reintroduction into Sc. pombe, demonstrating that Sc. pombe telomeres retain function when reintroduced as naked DNA. The ability of Sc. pombe to admit large DNA molecules indicates that it should be feasible to clone large DNA from other organisms in Sc. pombe.
Lesioned corticospinal tract axons regenerate in myelin-free rat spinal cord.
Authors: Savio T , Schwab M E
Published: May 2006

In the adult central nervous system (CNS) of higher vertebrates lesioned axons seemed unable to regenerate and reach their former target regions due to influences of the CNS microenvironment. Evidence from in vitro and biochemical experiments has demonstrated the presence of inhibitory substrate components in CNS tissue, in particular in white matter. These CNS components, which strongly inhibit neurite growth, were identified as minor membrane proteins of defined molecular mass (35 and 250 kDa) in oligodendrocyte membranes and CNS myelin. Oligodendrocyte development and myelin formation can be prevented by x-irradiation of newborn rats. Here we show that in myelin-free spinal cords cortico-spinal tract fibers transected at 2 weeks of age show reelongation of many millimeters within 2-3 weeks after the lesion. In normally myelinated controls, regenerative sprouts grew less than 1.7 mm caudal to the lesion.
Friend spleen focus-forming virus glycoprotein gp55 interacts with the erythropoietin receptor in the endoplasmic reticulum and affects receptor metabolism.
Authors: Yoshimura A , D'Andrea A D , Lodish H F
Published: May 2006

The Friend spleen focus-forming virus envelope glycoprotein, gp55, binds to the murine erythropoietin receptor (EPO-R) and triggers growth activation in the absence of EPO. Interleukin 3-dependent lymphoid cell lines that have been stably transfected with the EPO-R cDNA grow in the presence of EPO or interleukin 3. In these cells, the EPO-R is synthesized as a minor 62-kDa unglycosylated form and a major 64-kDa form carrying one high-mannose N-linked oligosaccharide. A fraction of the 64-kDa form is processed to a 66-kDa species with complex-type sugars. Very little of the EPO-R is expressed on the cell surface and all three forms of EPO-R are degraded rapidly. Cells transfected with both EPO-R and gp55 cDNAs grow in the absence of EPO. Most of the EPO-R associated with gp55 is endoglycosidase H-sensitive, suggesting that the interactions between these proteins occur in the endoplasmic reticulum. Furthermore, the endoglycosidase H-sensitive EPO-R is more stable than in the absence of gp55, a result suggesting that interaction of gp55 with the EPO-R causes it to remain within the rough endoplasmic reticulum. It is possible that gp55 EPO-R complexes within this compartment send a growth-promoting signal to the cell.
Primary structure of a key enzyme in plant tetrapyrrole synthesis: glutamate 1-semialdehyde aminotransferase.
Authors: Grimm B
Published: May 2006

The formation of delta-aminolevulinate from glutamate 1-semialdehyde (GSA) is catalyzed by glutamate 1-semialdehyde aminotransferase (EC 5.4.3.8). The active form of the barley enzyme appears to be a dimer of identical subunits with a molecular mass of 46 kDa. From the purified enzyme, amino acid sequences of the N-terminal ends of the mature protein as well as an internal peptide were determined. DNA primers deduced from these peptide sequences were used to amplify with the polymerase chain reaction a cDNA sequence encoding part of the enzyme. Screening a cDNA library with this DNA fragment identified a full-length clone encoding the 49,540-Da precursor of the GSA aminotransferase. The transit peptide for chloroplast import consists of 34 amino acids. GSA aminotransferase and a precursor form were expressed on a multicopy plasmid in Escherichia coli. Both recombinant gene products reacted with an antibody against the barley GSA aminotransferase. Active barley GSA aminotransferase expressed in E. coli was shown to be active in assays of bacterial cell extracts. As a gene symbol for barley GSA aminotransferase, Gsa is proposed.
Mitochondrial DNA evolution in experimental populations of Drosophila subobscura.
Authors: Fos M , Domínguez M A , Latorre A , Moya A
Published: May 2006

When two mitochondrial DNA (mtDNA) haplotypes of Drosophila subobscura compete in experimental populations with discrete generations, one or the other approaches fixation, depending on the nuclear background with which they are associated. The approach to fixation, however, is strongly dependent on the effective number of females in the population, Nf. Whether or not the ultimate fate of a given mtDNA haplotype is determined by random genetic drift depends on Nf as well as on the relative fitnesses. Our experimental results show that the mtDNA polymorphisms observed in natural populations are affected by interactions among nuclear polymorphisms, random genetic drift, and direct selection on the mtDNA haplotypes.
Doxorubicin selectively inhibits muscle gene expression in cardiac muscle cells in vivo and in vitro.
Authors: Ito H , Miller S C , Billingham M E , Akimoto H , Torti S V , Wade R , Gahlmann R , Lyons G , Kedes L , Torti F M
Published: May 2006

The anthracycline antibiotic doxorubicin produces a characteristic myopathy in cardiac muscle that limits its use in cancer therapy. We have shown in cultured neonatal rat cardiac muscle cells that doxorubicin treatment resulted in a rapid, selective decrease in the expression of muscle-specific genes, which preceded other changes characteristic of doxorubicin cardiomyopathy. Doxorubicin selectively and dramatically decreased the levels of mRNA for the sarcomeric genes, alpha-actin, troponin I, and myosin light chain 2, as well as the muscle-specific, but nonsarcomeric M isoform of creatine kinase. However, doxorubicin did not affect nonmuscle gene transcripts (pyruvate kinase, ferritin heavy chain, and beta-actin). Actinomycin D, an inhibitor of DNA-dependent RNA polymerase, did not show a similar selective decrease of muscle-specific mRNAs but, rather, produced a nonspecific, dose-dependent decrease of muscle and nonmuscle transcripts. The doxorubicin effect on muscle gene expression was limited to cardiac muscle; cultured skeletal myocytes were resistant to the effects of doxorubicin at 100-fold greater doses than those causing changes in mRNA levels in cardiac muscle cells. These effects of doxorubicin were reproduced in vivo; rats injected with doxorubicin showed a dose-dependent decrease in the levels of mRNAs for alpha-actin, troponin I, myosin light chain 2, and M isoform of creatine kinase in cardiac but not skeletal muscle. These selective changes in gene expression in cardiocyte cultures and cardiac muscle precede classical ultrastructural changes and may explain the myofibrillar loss that characterizes doxorubicin cardiac injury.
Neuronal modulation of calcium channel activity in cultured rat astrocytes.
Authors: Corvalan V , Cole R , de Vellis J , Hagiwara S
Published: May 2006

The patch-clamp technique was used to study whether cocultivation of neurons and astrocytes modulates the expression of calcium channel activity in astrocytes. Whole-cell patch-clamp recordings from rat brain astrocytes cocultured with rat embryonic neurons revealed two types of voltage-dependent inward currents carried by Ca2+ and blocked by either Cd2+ or Co2+ that otherwise were not detected in purified astrocytes. This expression of calcium channel activity in astrocytes was neuron dependent and was not observed when astrocytes were cocultured with purified oligodendrocytes.
A murine fibroblast growth factor (FGF) receptor expressed in CHO cells is activated by basic FGF and Kaposi FGF.
Authors: Mansukhani A , Moscatelli D , Talarico D , Levytska V , Basilico C
Published: May 2006

We have cloned a murine cDNA encoding a tyrosine kinase receptor with about 90% similarity to the chicken fibroblast growth factor (FGF) receptor and the human fms-like gene (FLG) tyrosine kinase. This mouse receptor lacks 88 amino acids in the extracellular portion, leaving only two immunoglobulin-like domains compared to three in the chicken FGF receptor. The cDNA was cloned into an expression vector and transfected into receptor-negative CHO cells. We show that cells expressing the receptor can bind both basic FGF and Kaposi FGF. Although the receptor binds basic FGF with a 15- to 20-fold higher affinity, Kaposi FGF is able to induce down-regulation of the receptor to the same extent as basic FGF. The receptor is phosphorylated upon stimulation with both FGFs, DNA synthesis is stimulated, and a proliferative response is produced in cells expressing the receptor, whereas cells expressing the cDNA in the antisense orientation show none of these responses to basic FGF or Kaposi FGF. Thus this receptor can functionally interact with two growth factors of the FGF family.
Early transcription factor subunits are encoded by vaccinia virus late genes.
Authors: Gershon P D , Moss B
Published: May 2006

The vaccinia virus early transcription factor (VETF) was shown to be a virus-encoded heterodimer. The gene for the 82-kDa subunit was identified as open reading frame (ORF) A8L, based on the N-terminal sequence of factor purified by using DNA-affinity magnetic beads. The 70-kDa subunit of VETF was refractory to N-terminal analysis, and so N-terminal sequences were obtained for three internal tryptic peptides. All three peptides matched sequences within ORF D6R. ORFs A8L and D6R are located within the central region of the vaccinia virus genome and are separated by about 13,600 base pairs. Proteins corresponding to the 3' ends of ORFs A8L and D6R were overexpressed in Escherichia coli and used to prepare antisera that bound to the larger and smaller subunits, respectively, of affinity-purified VETF. Immunoblot analysis of proteins from infected cells indicated that both subunits are expressed exclusively in the late phase of infection, just prior to their packaging in virus particles. The two subunits of VETF have no significant local or overall amino acid sequence homology to one another, to other entries in biological sequence data bases including bacterial sigma factors, or to recently determined sequences of some eukaryotic transcription factors. The 70-kDa subunit, however, has motifs in common with a super-family of established and putative DNA and RNA helicases.
Osteopontin--a possible anchor of osteoclasts to bone.
Authors: Reinholt F P , Hultenby K , Oldberg A , Heinegård D
Published: May 2006

A key event in bone resorption is the binding of osteoclasts to the mineral matrix of bone surfaces. A candidate for mediating this binding is osteopontin, a major cell- and hydroxyapatite-binding protein synthesized by osteoblasts. In support of this hypothesis is the fact that the synthesis of osteopontin is stimulated by calcitriol (1,25-dihydroxy-vitamin(D3), a substance that induces bone resorption. The present study demonstrates that osteopontin is highly enriched at regions of the bone surface where osteoclasts are anchored. Furthermore, the vitronectin receptor, which has known specificity for osteopontin, is shown preferentially localized at the corresponding area of the osteoclast plasma membrane. The results thus support the hypothesis that osteoclasts when resorbing bone are anchored by osteopontin bound both to the mineral of bone matrix and to a vitronectin receptor on the osteoclast plasma membrane.
Differential plasmid rescue from transgenic mouse DNAs into Escherichia coli methylation-restriction mutants.
Authors: Grant S G , Jessee J , Bloom F R , Hanahan D
Published: May 2006

Plasmids comprising transgene insertions in four lines of transgenic mice have been retrieved by plasmid rescue into a set of Escherichia coli strains with mutations in different members of the methylation-dependent restriction system (MDRS). Statistical analysis of plasmid rescue frequencies has revealed that the MDRS loci detect differential modifications of the transgene insertions among mouse lines that show distinctive patterns of transgene expression. Plasmids in mice that express hybrid insulin transgenes during development can be readily cloned into E. coli strains carrying mutations in two of the MDRS loci, mcrA and mcrB. In mice in which transgene expression is inappropriately delayed into adulthood, plasmids can only be cloned into E. coli that carry mutations in all known MDRS activities. Differential cloning frequencies in the presence or absence of the various methylation-dependent restriction genes represent a further way to distinguish regions of mammalian chromosomes. These multiply deficient E. coli strains will also facilitate the molecular cloning of modified chromosomal DNA.
Interaction of an NF-kappa B-like factor with a site upstream of the c-myc promoter.
Authors: Duyao M P , Buckler A J , Sonenshein G E
Published: May 2006

The c-myc protooncogene has been implicated in control of growth and differentiation of mammalian cells. For instance, growth arrest is often preceded by reduction in c-myc mRNA and gene transcription. To elucidate the mechanisms of control of c-myc gene transcription, we have begun to characterize the interaction of nuclear factors with the 719-base-pair (bp) c-myc regulatory domain, located 1139-421 bp upstream of the P1 start site of the mouse gene. Nuclear extracts from exponentially growing WEHI 231 murine B-lymphoma cells formed multiple complexes in mobility-shift assays. Changes in complex distribution were observed in growth-arrested WEHI 231 cells, and a major site of this interaction mapped to a 21-bp sequence that is similar to the sequences recognized by the NF-kappa B family of proteins. Binding of NF-kappa B-like factors was demonstrated by oligonucleotide competition. Induction of complex formation upon 70Z/3 pre-B- to B-cell differentiation, enhancement of binding by GTP, and detergent-induced release of inhibitor protein suggested that NF-kappa B itself is one member of the family that can bind. Transfection of thymidine kinase-chloramphenicol acetyltransferase constructs containing the 21-bp c-myc sequence into Jurkat cells demonstrated increased chloramphenicol acetyltransferase activity upon phorbol ester and phytohemagglutinin treatment. These results suggest the involvement of NF-kappa B-like factors in the regulation of c-myc transcription.
All-D amino acid-containing channel-forming antibiotic peptides.
Authors: Wade D , Boman A , Wåhlin B , Drain C M , Andreu D , Boman H G , Merrifield R B
Published: May 2006

The D enantiomers of three naturally occurring antibiotics--cecropin A, magainin 2 amide, and melittin--were synthesized. In addition, the D enantiomers of two synthetic chimeric cecropin-melittin hybrid peptides were prepared. Each D isomer was shown by circular dichroism to be a mirror image of the corresponding L isomer in several solvent mixtures. In 20% hexafluoro-2-propanol the peptides contained 43-75% alpha-helix. The all-D peptides were resistant to enzymatic degradation. The peptides produced single-channel conductances in planar lipid bilayers, and the D and L enantiomers caused equivalent amounts of electrical conductivity. All of the peptides were potent antibacterial agents against representative Gram-negative and Gram-positive species. The D and L enantiomers of each peptide pair were equally active, within experimental error. Sheep erythrocytes were lysed by both D- and L-melittin but not by either isomer of cecropin A, magainin 2 amide, or the hybrids cecropin A-(1-13)-melittin-(1-13)-NH2 or cecropin A-(1-8)-melittin-(1-18)-NH2. The infectivity of the bloodstream form of the malaria parasite Plasmodium falciparum was also inhibited by the D and L hybrids. It is suggested that the mode of action of these peptides on the membranes of bacteria, erythrocytes, plasmodia, and artificial lipid bilayers may be similar and involves the formation of ion-channel pores spanning the membranes, but without specific interaction with chiral receptors or enzymes.
Structural correlates of high antibody affinity: three engineered amino acid substitutions can increase the affinity of an anti-p-azophenylarsonate antibody 200-fold.
Authors: Sharon J
Published: May 2006

The basis for the 200-fold difference in affinity between two hybridoma antibodies specific for the hapten p-azophenylarsonate (Ars) that have diversified by somatic hypermutation was examined. Oligonucleotide-directed mutagenesis was used to sequentially convert the nucleotide sequence of the lower-affinity antibody into that of the higher-affinity one, and the mutant antibodies generated by transfection of hybridoma cells were analyzed for affinity to Ars-tyrosine. The data showed that out of the 19 amino acid differences between the two hybridoma antibodies, the affinity increase could be reproduced by three heavy-chain substitutions that are present in the high-affinity antibody. The combined effect on affinity of amino acid substitutions was generally found to reflect their individual effects. Although the light chain of the high-affinity antibody did not seem to play a major role in the affinity increase, its contribution varied with the kind and number of heavy-chain substitutions. The results hold promise for antibody engineering and are consistent with a stepwise acquisition of somatic hypermutations in which the existing structural context of an antibody most likely influences the affinity-based selection of later substitutions. They further suggest that many substitutions may be tolerated in vivo during the antigen-driven selection process, even though they confer on the antibody no affinity increase.
ATP-induced loss of Alz-50 immunoreactivity with the A68 proteins from Alzheimer brain is mediated by ubiquitin.
Authors: Vincent I J , Davies P
Published: May 2006

The Alz-50 immunoreactive proteins, designated A68, are detected by electrophoretic blot analysis of 100,000 x g pellet fractions of brain tissue from individuals with Alzheimer disease (AD). In exploring the biochemical nature of these proteins, we have found that a preincubation of such fractions with 5 mM ATP results in loss of Alz-50 immunoreactivity on immunoblots. The loss of antigenicity is complete after a 1-hr incubation at 37 degrees C and is stringently dependent on ATP. Hydrolysis of ATP is required, since the inhibition is not supported by the nonhydrolyzable analog adenosine 5'-[gamma-thio]triphosphate (ATP[gamma S]) and is prevented when the ATPase inhibitors o-vanadate and oligomycin are present. Upon further characterization, it was found that certain protease inhibitors, phenylmethylsulfonyl fluoride, antipain, tosylphenylalanine chloromethyl ketone, and aprotonin prevent the loss of the epitope. This suggests that hydrolysis of ATP is coupled with proteolysis of A68, leading to loss of Alz-50 immunoreactivity. Since a variety of proteins are believed to be degraded by an ATP/ubiquitin-dependent pathway, a possible role for ubiquitin (Ub) in this effect was investigated. Two polyclonal antibodies against Ub protected A68 from proteolysis and were also effective in immunoprecipitating A68 after incubation with ATP in the presence of Ub and phenylmethylsulfonyl fluoride. The proteolysis of A68 was also blocked by hemin, an inhibitor of the protease that cleaves Ub-protein conjugates. Taken together, these findings indicate that loss of Alz-50 immunoreactivity with A68 is due to ATP-dependent/Ub-mediated proteolysis. This mechanism may be relevant to the physiological role for A68 in AD or it may simply represent an attempt to abort an aberrant protein.
Islet amyloid polypeptide: pinpointing amino acid residues linked to amyloid fibril formation.
Authors: Westermark P , Engström U , Johnson K H , Westermark G T , Betsholtz C
Published: May 2006

Islet amyloid polypeptide (IAPP), a putative polypeptide hormone, is a product of pancreatic beta-cells and the major constituent of the amyloid deposits seen mainly in islets of type 2 diabetic humans and diabetic cats. The connection between IAPP amyloid formation and diabetes is unknown, but a limited segment of the IAPP molecule, positions 20-29, seems responsible for the aggregation to fibrils. Differences in the amino acid sequence of this region probably determine whether or not islet amyloid can develop in a particular species. Amyloid fibril formation can be mimicked in vitro with the aid of synthetic peptides. With this technique we show that peptides corresponding to IAPP positions 20-29 of human and cat, species that develop IAPP-derived islet amyloid, form amyloid-like fibrils in vitro. The corresponding IAPP segment from three rodent species that do not develop IAPP-derived amyloid did not give rise to fibrils. Substitution of the human IAPP-(20-29) decapeptide with one or two amino acid residues from species without islet amyloid generally reduced the capacity to form fibrils. We conclude that the sequence Ala-Ile-Leu-Ser-Ser, corresponding to positions 25-29 of human IAPP, is strongly amyloidogenic and that a proline-for-serine substitution in position 28, as in several rodents, almost completely inhibits formation of amyloid fibrils.
High-level expression of biologically active human alpha 1-antitrypsin in the milk of transgenic mice.
Authors: Archibald A L , McClenaghan M , Hornsey V , Simons J P , Clark A J
Published: May 2006

Reduced circulating levels of alpha 1-antitrypsin (alpha 1 AT) are associated with certain alpha 1 AT genotypes and increased susceptibility to emphysema. Unfortunately, the amounts of alpha 1 AT that would be required for replacement therapy are beyond the capacity of plasma fractionation and mammalian cell culture systems. Thus, we have examined the potential of transgenic animals as an alternative means of producing human alpha 1 AT. A hybrid gene constructed by using sequences from the ovine milk protein gene beta-lactoglobulin fused to an alpha 1 AT "minigene" was used to generate transgenic mice. Of 13 independent transgenic mice and mouse lines, 5 expressed the hybrid gene in the mammary gland, 5 in the salivary glands, and 2 in both these tissues. Human alpha 1 AT was secreted into the milk of each of the 7 mice and mouse lines that expressed the hybrid gene in the mammary gland. Four of these mammary-expressing transgenic mice and mouse lines produced concentrations of at least 0.5 mg of alpha 1 AT per ml in their milk; one line (AATB 35) produced 7 mg of this protein per ml. alpha 1 AT from transgenic mouse milk was similar in size to human plasma-derived alpha 1 AT and showed a similar capacity to inhibit trypsin. Expression at equivalent levels in transgenic sheep or cattle would yield sufficient alpha 1 AT for therapeutic purposes.
Identification of cytosolic peroxisome proliferator binding protein as a member of the heat shock protein HSP70 family.
Authors: Alvares K , Carrillo A , Yuan P M , Kawano H , Morimoto R I , Reddy J K
Published: May 2006

Clofibrate and many of its structural analogues induce proliferation of peroxisomes in the hepatic parenchymal cells of rodents and certain nonrodent species including primates. This induction is tissue specific, occurring mainly in the liver parenchymal cells and to a lesser extent in the kidney cortical epithelium. The induction of peroxisomes is associated with a predictable pleiotropic response, characterized by hepatomegaly, and increased activities and mRNA levels of certain peroxisomal enzymes. Using affinity chromatography, we had previously isolated a protein that binds to clofibric acid. We now show that this protein is homologous with the heat shock protein HSP70 family by analysis of amino acid sequences of isolated peptides from trypsin-treated clofibric acid binding protein and by cross-reactivity with a monoclonal antibody raised against the conserved region of the 70-kDa heat shock proteins. The clofibric acid-Sepharose column could bind HSP70 proteins isolated from various species, which could then be eluted with either clofibric acid or ATP. Conversely, when a rat liver cytosol containing multiple members of the HSP70 family was passed through an ATP-agarose column, and eluted with clofibric acid, only P72 (HSC70) was eluted. These results suggest that clofibric acid, a peroxisome proliferator, preferentially interacts with P72 at or near the ATP binding site.
Characterization of an autoregulated response element in the mouse retinoic acid receptor type beta gene.
Authors: Sucov H M , Murakami K K , Evans R M
Published: May 2006

A sequence that confers transcriptional responsiveness to retinoic acid was identified in the promoter of the mouse retinoic acid receptor (RAR) beta gene. This response element consists of a direct repeat of the sequence GTTCAC, separated by five nucleotides. Direct binding of the RAR to this sequence was demonstrated by gel retardation and immunoprecipitation assays. This element conferred retinoic acid responsiveness on heterologous promoters via all three subtypes of RAR yet failed to support transcriptional activation by the thyroid hormone, estrogen, glucocorticoid, or vitamin D receptors. Surprisingly, a high level of retinoic acid-dependent activation was seen in the absence of transfected RAR in 10 of 10 vertebrate cell lines, many functionally characterized previously as lacking endogenous receptor. This demonstrates an unusually high sensitivity of the retinoic acid response element to low levels of receptor and suggests expression of RAR in a wide variety of tissue types.
Trophallaxis in a communal halictine bee Lasioglossum (Chilalictus) erythrurum.
Authors: Kukuk P F , Crozier R H
Published: May 2006

Food exchange by means of oral trophallaxis was confirmed for a communal halictine bee Lasioglossum (Chilalictus) erythrurum. These results demonstrate an independent evolution of trophallaxis in bees. The occurrence of trophallaxis in a communal species questions the role of trophallaxis in the evolution of sociality. Neutral arena encounters between one fed and one unfed female indicate that food exchange is not associated with familiarity. Donor females fed nestmates and nonnestmates in the same proportion, even when nonnestmates were from a separate nest aggregation located 7 km away. Such universal acceptance is expected if positive fitness benefits accrue from nearly all interactions with conspecifics in nature.
Isolation of L-3-phenyllactyl-Leu-Arg-Asn-NH2 (Antho-RNamide), a sea anemone neuropeptide containing an unusual amino-terminal blocking group.
Authors: Grimmelikhuijzen C J , Rinehart K L , Jacob E , Graff D , Reinscheid R K , Nothacker H P , Staley A L
Published: May 2006

Using a radioimmunoassay for the carboxyl-terminal sequence Arg-Asn-NH2, we have purified a peptide from acetic acid extracts of the sea anemone Anthopleura elegantissima. By classical amino acid analyses, mass spectrometry, and 1H NMR spectroscopy, the structure of this peptide was determined as 3-phenyllactyl-Leu-Arg-Asn-NH2. By using reversed-phase HPLC and a chiral mobile phase, it was shown that the 3-phenyllactyl group had the L configuration. Immunocytochemical staining with antiserum against Arg-Asn-NH2 showed that L-3-phenyllactyl-Leu-Arg-Asn-NH2 (Antho-RNamide) was localized in neurons of sea anemones. The L-3-phenyllactyl group has not been found earlier in neuropeptides of vertebrates or higher invertebrates. We propose that this residue renders Antho-RNamide resistant to nonspecific aminopeptidases, thereby increasing the stability of the peptide after neuronal release.
Molecular cloning and neurotrophic activities of a protein with structural similarities to nerve growth factor: developmental and topographical expression in the brain.
Authors: Ernfors P , Ibáñez C F , Ebendal T , Olson L , Persson H
Published: May 2006

We have used a pool of degenerate oligonucleotides representing all possible codons in regions of homology between brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) to prime rat hippocampal cDNAs in the polymerase chain reaction. The amplified DNA included a product with significant similarity to NGF and BDNF, which was used to isolate a 1020-nucleotide-long cDNA from a rat hippocampal library. From the nucleotide sequence, a 282-amino-acid-long protein with approximately 45% amino acid similarity to both pig BDNF and rat NGF was deduced. In the adult brain, the mRNA for this protein was predominantly expressed in hippocampus, where it was confined to a subset of pyramidal and granular neurons. The developmental expression in brain showed a clear peak shortly after birth, 1 and 2 weeks earlier than maximal expression of BDNF and NGF, respectively. It was also expressed in several peripheral tissues with the highest level in kidney. The protein, transiently expressed in COS cells, was tested on chicken embryonic neurons and readily stimulated fiber outgrowth from explanted Remak's ganglion and, to a lesser extent, the nodose ganglion. A weak, but consistent, fiber outgrowth response was also seen in the ciliary ganglion and in paravertebral sympathetic ganglia. Moreover, the protein displaced binding of NGF to its receptor, suggesting that it can interact with the NGF receptor. Thus, this factor, although structurally and functionally related to NGF and BDNF, has unique biological activities and represents a member of a family of neurotrophic factors that may cooperate to support the development and maintenance of the vertebrate nervous system.
Site-directed mutagenesis reveals a liver transcription factor essential for the albumin transcriptional enhancer.
Authors: Zaret K S , Liu J K , DiPersio C M
Published: May 2006

A method based on the polymerase chain reaction is described for constructing a clustered set of basepair changes, deletions, or insertions at any site on a DNA fragment. Advantages of the procedure are that virtually every product has the desired sequence alteration and that only a single round of polymerase chain reaction is required. We used this method to demonstrate that the binding of a specific liver nuclear protein, which we call eH-TF, is essential for the function of the enhancer of the mouse albumin gene. The eH-TF binding activity is hepatocyte-specific; it binds to a functional region of the albumin promoter and is distinct from other albumin promoter factors, and part of the eH-TF binding sequence, TGTTTGC, occurs in functional regulatory sites of other liver-specific genes.
Isolation and characterization of sulfhydryl and disulfide peptides of human apolipoprotein B-100.
Authors: Yang C Y , Kim T W , Weng S A , Lee B R , Yang M L , Gotto A M
Published: May 2006

Twenty-three of the 25 cysteine residues in apolipoprotein B-100 have been isolated directly from tryptic or peptic peptide mixtures. Sixteen cysteine residues exist in disulfide forms: Cys-1-Cys-3, Cys-2-Cys-4, Cys-5-Cys-6, Cys-7-Cys-8, Cys-9-Cys-10, Cys-11-Cys-12, Cys-13-Cys-14, and Cys-20-Cys-21. All of these except Cys-20-Cys-21 are recently discovered disulfide linkages. In addition to Cys-22 and Cys-24, which have been described as sulfhydryls on low density lipoprotein, Cys-15 to Cys-18 and Cys-23 are in the reduced form. Cys-19 and Cys-25 are not yet confirmed. Our results revealed that all identified disulfide linkages are located in the trypsin-releasable regions and that all except Cys-1-Cys-3 and Cys-2-Cys-4 are linked to the neighboring cysteine. We propose a linear model of apolipoprotein B-100 in low density lipoprotein that wraps around the low density lipoprotein molecule.
Identification of a ZP3-binding protein on acrosome-intact mouse sperm by photoaffinity crosslinking.
Authors: Bleil J D , Wassarman P M
Published: May 2006

During the process of fertilization in mammals, sperm bind in a relatively species-specific manner to the zona pellucida (ZP) of ovulated eggs. ZP3, a glycoprotein found in the mouse egg zona pellucida, serves as receptor for sperm during gamete adhesion. We report here that a Mr 56,000 protein found on mouse sperm has properties expected for a sperm component that recognizes and binds to ZP3. This sperm protein is radiolabeled preferentially by a photoactivatable heterobifunctional crosslinker ("Denny-Jaffee reagent") covalently linked to purified ZP3, binds very tightly to ZP3-affinity columns, and is localized to heads of acrosome-intact but not acrosome-reacted sperm. These and other findings suggest that this protein may be a "ZP3-binding protein" that, together with the sperm receptor, supports species-specific binding of mouse sperm to unfertilized eggs.
Learning causes synaptogenesis, whereas motor activity causes angiogenesis, in cerebellar cortex of adult rats.
Authors: Black J E , Isaacs K R , Anderson B J , Alcantara A A , Greenough W T
Published: May 2006

The role of the cerebellar cortex in motor learning was investigated by comparing the paramedian lobule of adult rats given difficult acrobatic training to that of rats that had been given extensive physical exercise or had been inactive. The paramedian lobule is activated during limb movements used in both acrobatic training and physical exercise. Acrobatic animals had greater numbers of synapses per Purkinje cell than animals from the exercise or inactive groups. No significant difference in synapse number or size between the exercised and inactive groups was found. This indicates that motor learning required of the acrobatic animals, and not repetitive use of synapses during physical exercise, generates new synapses in cerebellar cortex. In contrast, exercise animals had a greater density of blood vessels in the molecular layer than did either the acrobatic or inactive animals, suggesting that increased synaptic activity elicited compensatory angiogenesis.