bluuebunny/crossref_metadata_2025 huggingface dataset:
Genomic targeting with a positive-selection lox integration vector allows highly reproducible gene expression in mammalian cells.
Authors: Fukushige S , Sauer B
Published: May 2006

Stable transformants of mammalian cells from gene transfer often show extreme variability in expression of the introduced transgene. This occurs from the highly variable number of copies integrated into the genome and from position effects on gene expression due to random integration. We have eliminated both of these constraints on predictable gene expression by use of a lox recombination vector. The positive selection vector system is designed to directly select Cre-mediated DNA integration at a lox target previously placed into the genome of cultured mammalian cells. Proper targeting activates expression of a defective lox-neomycin phosphotransferase (neo) fusion gene target. With CHO cell lines containing this target, almost all of the selected transformants (54 of 56 independent G418-resistant colonies) were simple single-copy integrants of the targeting DNA. To monitor gene expression at a single chromosomal site, we used a beta-actin promoter-lacZ reporter construct. Independent G418-resistant colonies from site-specific integration of the reporter gene all showed nearly identical levels of beta-galactosidase activity when the reporter construct integrated at a particular chromosomal position. The same construct integrated at a second chromosomal position exhibited a slightly different level of activity, characteristic of that second position. These results show that Cre-mediated site-specific integration can facilitate the construction of isogenic cell lines and thereby permit reproducible gene expression in stably transformed cell lines.
A major binding protein for leukemia inhibitory factor in normal mouse serum: identification as a soluble form of the cellular receptor.
Authors: Layton M J , Cross B A , Metcalf D , Ward L D , Simpson R J , Nicola N A
Published: May 2006

A protein that specifically binds leukemia inhibitory factor (LIF) has been isolated from normal mouse serum by using four successive fractionation steps: chromatography on a LIF affinity matrix, anion-exchange chromatography, size-exclusion chromatography, and preparative native gel electrophoresis. The purified LIF-binding protein (LBP) is a glycoprotein with an apparent molecular mass of 90 kDa that specifically binds 125I-labeled murine LIF with an affinity comparable to that of the low-affinity cellular LIF receptor (Kd = 600 pM). N-terminal sequencing has identified this protein as a soluble truncated form of the alpha chain of the cellular LIF receptor. LBP is present in normal mouse serum at high levels (1 microgram/ml) and these levels are elevated in pregnant mice and reduced in neonatal mice. Since normal serum concentrations of LBP can block the biological actions of LIF in culture, LBP may serve as an inhibitor of the systemic effects of locally produced LIF.
An anergic, islet-infiltrating T-cell clone that suppresses murine diabetes secretes a factor that blocks interleukin 2/interleukin 4-dependent proliferation.
Authors: Díaz-Gallo C , Moscovitch-Lopatin M , Strom T B , Kelley V R
Published: May 2006

The mechanism of peripheral immunological tolerance has not been fully established. While anergic T cells have been noted in tolerant hosts, the mechanism by which they contribute to the induction and maintenance of tolerance has not been defined. As we previously reported, an accelerated form of diabetogenic autoimmunity in nonobese diabetic mice can be blocked by passive transfer of a CD3+, CD8+, beta-chain variable region 11-positive islet-infiltrating T-cell clone (IS-2.15). In this report we examine the properties of this T-cell clone. We have established that this clone is unresponsive to mitogenic concentrations of anti-T-cell receptor or anti-CD3 monoclonal antibodies and is only weakly responsive to syngeneic islet and spleen cells. Moreover, these T cells secrete an inhibitory factor(s) that irreversibly inhibits interleukin (IL) 2/IL-4-driven proliferation of IL-2/IL-4 indicator T-cell lines. This noncytotoxic factor, which possesses an apparent size of 10-30 kDa, does not interfere with low-affinity IL-2 receptor expression. These data indicate that at least some anergic T cells can play an active role in peripheral tolerance by secreting suppressor factor(s) that regulate IL-2/IL-4-dependent proliferation.
Developmentally regulated Drosophila gene family encoding the fork head domain.
Authors: Häcker U , Grossniklaus U , Gehring W J , Jäckle H
Published: May 2006

We have isolated seven Drosophila genes by means of low-stringency hybridization to a DNA probe containing the coding sequence for the protein domain shared by the rodent hepatocyte-enriched nuclear transcription factor HNF3A (alpha) and the product of the Drosophila region-specific homeotic gene fork head (fkh). The previously unreported genes encode a 110-amino acid conserved sequence, which we call the fork head (fkh) domain. Two of these fkh-domain-encoding genes ("FD genes") map to the sloppy paired locus (slp), which exerts segmentation gene function. The expression patterns of the other FD genes suggest that their protein products are likely to be involved in gut formation, mesoderm specification, and some specific aspects of neural development. The FD gene products presumably represent a family of transcription factors that, like the previously identified DNA-binding proteins, contribute to early developmental decisions in cell fates during embryogenesis.
Hair shaft elongation, follicle growth, and spontaneous regression in long-term, gelatin sponge-supported histoculture of human scalp skin.
Authors: Li L , Margolis L B , Paus R , Hoffman R M
Published: May 2006

In order to better understand the molecular mechanisms of human hair growth control and to test hair growth-modulatory drugs, appropriate in vitro models are required. Here, we report the long-term growth, shaft elongation, and spontaneous regression of human hair follicles in histoculture of intact scalp skin. Human scalp skin with abundant hair follicles in various stages of the hair growth cycle was grown for up to 40 days in a gelatin sponge-supported histoculture system at the air/liquid interface. Isolated follicles placed in the gelatin-sponge matrix also supported hair shaft elongation, with the hair follicle cells remaining proliferative and viable for very long periods. Hair shaft elongation occurred mainly during the first 10 days of histoculture of both intact skin and isolated follicles. However, hair follicles were viable and follicle keratinocytes continued to incorporate [3H]thymidine for up to several weeks after shaft elongation had ceased as shown by fluorescent-dye double staining, measured by confocal laser scanning microscopy, and by histological autoradiography of [3H]thymidine incorporation, respectively. Hair follicles could continue their cycle in histoculture; for example, apparent spontaneous catagen induction was observed both histologically and by the actual regression of the hair follicle. In addition, vellus follicles were shown to be viable at day 40 after initiation of culture. In the histocultured human scalp we demonstrated the association of mast cells with anagen follicles and macrophages with catagen follicles, suggesting a role of these cells in the hair cycle. This histoculture technique should serve as a powerful tool for future hair research in the human system as well as a screening assay for compounds that can perturb the hair cycle.
Differential expression of arachidonate 5-lipoxygenase transcripts in human brain tumors: evidence for the expression of a multitranscript family.
Authors: Boado R J , Pardridge W M , Vinters H V , Black K L
Published: May 2006

In addition to the important role of leukotrienes as mediators in allergy and inflammation, these compounds are also linked to pathophysiological events in the brain including cerebral ischemia, brain edema, and increased permeability of the blood-brain barrier in brain tumors. Although brain tumors have been shown to secrete leukotrienes, no studies to date have provided evidence for the tumor expression of genes encoding enzymes involved in leukotriene production. Therefore, the present study determined the abundance of the mRNA for arachidonate 5-lipoxygenase (5-LO; arachidonate:oxygen 5-oxidoreductase, EC 1.13.11.34), which is the rate-limiting enzyme in leukotriene synthesis, in a series of human brain tumors. Macrophage/monocyte infiltration of the tumor was estimated by measuring the abundance of the transcript for the 91-kDa glycoprotein phagocyte-specific oxidase (gp91-phox), which is the phagocyte-specific cytochrome b heavy chain. The present study shows that (i) the 5-LO transcript is expressed in normal bovine brain and in human brain tumors; (ii) the 5-LO gene in human brain tumors and in the dimethyl sulfoxide-induced promyelocytic human leukemic HL-60 cells is expressed as a multitranscript family (2.7, 3.1, 4.8, 6.4, 8.6 kilobases); and (iii) the abundance of 5-LO transcripts, the expression of the larger transcripts, and the 5-LO/gp91-phox ratio correlate with the tumor malignancy. Overall, the present study supports the hypothesis that the 5-LO gene product may play a role in human tumor-induced brain edemas and provides evidence for tumor-associated expression of high molecular weight 5-LO transcripts in human brain tumors.
Perforin and granzyme A expression identifying cytolytic lymphocytes in rheumatoid arthritis.
Authors: Griffiths G M , Alpert S , Lambert E , McGuire J , Weissman I L
Published: May 2006

Lymphocytes from the synovial fluid of patients with rheumatoid arthritis were examined for the expression of granzyme A and perforin. Previous studies have demonstrated that the expression of these proteins, which are implicated as mediators of cytotoxicity, can be used to identify putative cytolytic lymphocytes in vivo. Twenty-two synovial fluid samples were analyzed by in situ hybridization and immunohistochemistry. In six patients receiving low doses of immunosuppressant, a population of granzyme A- and perforin-expressing lymphocytes could be identified. In contrast, lymphocytes from patients who were receiving high doses of immunosuppressant did not contain any granzyme A- or perforin-expressing lymphocytes. Synovial fluid lymphocytes from patients with osteoarthritis did not express either marker. The expression of these markers demonstrates the presence of potentially functional cytolytic lymphocytes, expressing proteins required to mediate killing, in the synovial fluid of patients with rheumatoid arthritis. This suggests that cytolytic lymphocytes may be involved in the pathogenesis of rheumatoid arthritis.
Discrimination between related DNA sites by a single amino acid residue of Myc-related basic-helix-loop-helix proteins.
Authors: Dang C V , Dolde C , Gillison M L , Kato G J
Published: May 2006

A yeast genetic system was developed to study how the basic regions of basic-helix-loop-helix (bHLH) proteins distinguish between related consensus bHLH binding sites, with nucleotide sequence CANNTG. The yeast bHLH protein CBF1 binds to the sequence CAC(A/G)TG found in the yeast centromere element CDE1 and in promoter regions of several yeast genes involved in methionine biosynthesis. Using a functional assay to rescue a mutant cbf1 yeast strain from methionine auxotrophy, we determined that the basic region of CBF1 could be replaced by the homologous region of either the vertebrate USF transcription factor or c-Myc, both of which bind CACGTG. The homologous region of the AP4 transcription factor, which recognizes the sequence CAGCTG, could not functionally replace the CBF1 basic region. However, only a single substitution, Met----Arg, in the AP4 basic region of the inactive chimera CBF-AP4 was sufficient to restore CBF1 function. In randomization experiments, only arginine or lysine provided functional substitutions at the AP4 methionine position. The results suggest that this conserved arginine residue in the basic regions of Myc-related bHLH proteins discriminates between CAC(A/G)TG and related sites.
Regulation of the yeast DNA replication genes through the Mlu I cell cycle box is dependent on SWI6.
Authors: Verma R , Smiley J , Andrews B , Campbell J L
Published: May 2006

In Saccharomyces cerevisiae, at least 17 DNA replication genes are coordinately expressed at the G1/S boundary during the cell cycle. All of these genes have the DNA sequence element ACGCGT in their 5' upstream regulatory regions. This sequence has been shown to be essential for periodic expression of the POL1, CDC9, and TMP1 genes. The cyclin (CLN1 and CLN2) and HO genes are another subset of genes that are expressed with the same timing as the DNA replication genes. Their periodic expression requires the participation of two well-characterized transcriptional activators: the SWI4 and SWI6 gene products. In this study, we present evidence that SWI6 contributes to the regulation of DNA replication genes as well. Surprisingly, a preferential requirement for SWI6 over SWI4 is observed in our studies of ACGCGT-dependent reporter gene expression in vivo. This selectivity has not been observed for the other G1/S genes. Correlating with the in vivo results, protein-DNA complexes formed in vitro on multimeric ACGCGT elements are either abolished or reduced in swi6 delta deletion mutants.
Structure of the host-specific toxins produced by the fungal pathogen Periconia circinata.
Authors: Macko V , Stimmel M B , Wolpert T J , Dunkle L D , Acklin W , Bänteli R , Jaun B , Arigoni D
Published: May 2006

Four metabolites named peritoxins A and B and periconins A and B have been isolated together with the known metabolite circinatin from culture filtrates of the fungal pathogen Periconia circinata. Peritoxins A and B, which correspond to the P. circinata toxins Ia and IIa partially characterized in previous work, are selectively toxic to genotypes of Sorghum bicolor susceptible to the pathogen, whereas periconins A and B are biologically inactive. Combination of instrumental analysis and chemical degradation has led to structural assignments for each of the four compounds; only the configuration at some of the chiral centers remains undefined. Structural comparison suggests a precursor role for circinatin in the formation of the peritoxins and the periconins.
Low frequency of p53 mutations observed in a diverse collection of primary hepatocellular carcinomas.
Authors: Buetow K H , Sheffield V C , Zhu M , Zhou T , Shen F M , Hino O , Smith M , McMahon B J , Lanier A P , London W T
Published: May 2006

Recent studies of the p53 tumor suppressor locus (designated TP53) in primary hepatocellular carcinoma (PHC) have identified a high frequency of codon 249 mutations. Due to the geographic location from which the samples were obtained and the substitution observed, the mutation was suggested to be attributable to aflatoxin B1 (AFB1) exposure. To determine the generality of this phenomenon, we have examined PHC tissues from 107 geographically and ethnically diverse sources. The frequency of p53 gene mutations was evaluated by using PCR/restriction-digest methods, GC-clamp (G+C-rich sequence) denaturing gradient gel electrophoresis, and DNA sequencing. The mutation rate observed in tumors from high-AFB1-exposure regions (25%) was more than double the rate observed in low-exposure regions (12%) but lower than the 50% frequency previously reported. Codon 249 mutations occurred at a much lower frequency than previously reported (2 of 107 samples examined). These results suggest that changes in DNA encoding p53 may not represent primary oncogenic effects but instead represent genetic changes related to tumor progression. High AFB1 levels may facilitate the generation of these progressional changes, but not by inducing a specific p53 gene mutation at codon 249 as previously reported.
Recombinant antibody-metallothionein: design and evaluation for radioimmunoimaging.
Authors: Das C , Kulkarni P V , Constantinescu A , Antich P , Blattner F R , Tucker P W
Published: May 2006

We have produced a chimeric antibody (Ab) in which metallothionein, a well-characterized biological chelator of metals, was genetically fused to the F(ab') domain of the S107 Ab heavy chain. Coexpression with the Ab light chain that conveys specificity for the synthetic antigen phosphocholine was achieved in plasmacytoma cells. Metal- and antigen-binding domains of the Ab-metallothionein hybrid function with normal avidity and specificity. Ab-metallothionein can be efficiently loaded with 99mTc and used to specifically bind phosphocholine-haptenated cells in vitro or to localize plasma-cell ascites tumors in mice. The approach offers potential advantages for producing radiolabeled Ab for targeted radiotherapy and diagnostic imaging.
Extended x-ray absorption fine structure studies of a retrovirus: equine infectious anemia virus cysteine arrays are coordinated to zinc.
Authors: Chance M R , Sagi I , Wirt M D , Frisbie S M , Scheuring E , Chen E , Bess J W , Henderson L E , Arthur L O , South T L
Published: May 2006

Zinc finger arrays have been established as a critical structural feature of proteins involved in DNA recognition. Retroviral nucleocapsid proteins, which are involved in the binding of viral RNA, contain conserved cysteine-rich arrays that have been suggested to coordinate zinc. We provide metalloprotein structural data from an intact virus preparation that validate this hypothesis. Extended x-ray absorption fine structure (EXAFS) spectroscopy of well-characterized and active preparations of equine infectious anemia virus, compared with a peptide with known coordination and in combination with available biochemical and genetic data, defines a Cys3His1 coordination environment for zinc. The average of the Zn-S distances is 2.30(1) A and that of the Zn-N distance (to histidine) is 2.01(3) A.
Cholinergic agonists and interleukin 1 regulate processing and secretion of the Alzheimer beta/A4 amyloid protein precursor.
Authors: Buxbaum J D , Oishi M , Chen H I , Pinkas-Kramarski R , Jaffe E A , Gandy S E , Greengard P
Published: May 2006

Activation of protein kinase C by phorbol esters is known to accelerate the processing and secretion of the beta/A4 amyloid protein precursor. We have now examined various first messengers that increase protein kinase C activity of target cells for their ability to affect beta/A4 amyloid protein precursor metabolism. Acetylcholine and interleukin 1, which are altered in Alzheimer disease, were shown to increase processing of the beta/A4 amyloid protein precursor via the secretory cleavage pathway. Cholinergic agonists stimulated secretion in human glioma and neuroblastoma cells as well as in PC12 cells transfected with the M1 receptor, while interleukin 1 stimulated secretion in human endothelial and glioma cells.
Immobilization of DNA for scanning probe microscopy.
Authors: Allison D P , Bottomley L A , Thundat T , Brown G M , Woychik R P , Schrick J J , Jacobson K B , Warmack R J
Published: May 2006

Reproducible scanning tunneling microscope and atomic force microscope images of entire molecules of uncoated plasmid DNA chemically bound to surfaces are presented. The chemically mediated immobilization of DNA to surfaces and subsequent scanning tunneling microscope imaging of DNA molecules demonstrate that the problem of molecular instability to forces exerted by the probe tip, inherent with scanning probe microscopes, can be prevented.
Obliterative bronchiolitis after lung transplantation: a fibroproliferative disorder associated with platelet-derived growth factor.
Authors: Hertz M I , Henke C A , Nakhleh R E , Harmon K R , Marinelli W A , Fox J M , Kubo S H , Shumway S J , Bolman R M , Bitterman P B
Published: May 2006

Fibroproliferative disorders are characterized by accumulations of mesenchymal cells and connective tissue in critical locations, leading to organ dysfunction. We examined the role of platelet-derived growth factor (PDGF) in the pathogenesis of obliterative bronchiolitis, a fibroproliferative process that occurs after lung transplantation and results in small airway occlusion. Bronchoalveolar lavage fluid from obliterative bronchiolitis patients significantly stimulated fibroblast migration, whereas fluid from patient controls did not. Quantitation by radioligand binding assay demonstrated increased concentrations of PDGF in lavage fluid from obliterative bronchiolitis patients (patients, 104 +/- 26.9 pM; controls, 8.4 +/- 6.9 pM; P < 0.01). Heparin affinity, gel filtration, and Western blot analysis confirmed the presence of PDGF in lavage fluid. Immunohistochemical and in situ hybridization studies of histologic sections and bronchoalveolar lavage cells suggest that alveolar macrophages are one cellular source. Prospective evaluation of sequential bronchoalveolar lavage samples from a patient who developed obliterative bronchiolitis demonstrated markedly increased PDGF concentrations before the onset of irreversible airflow obstruction. These findings are consistent with a role for PDGF in the fibroproliferative changes observed in obliterative bronchiolitis.
Construction of chimeric vaccinia viruses by molecular cloning and packaging.
Authors: Scheiflinger F , Dorner F , Falkner F G
Published: May 2006

Foreign DNA was inserted into unique restriction endonuclease cleavage sites (Sma I or Not I) of the 200,000-base-pair vaccinia virus genome by direct molecular cloning. The modified vaccinia virus DNA was packaged in fowlpox virus-infected avian cells, and chimeric vaccinia virus was isolated from mammalian cells not supporting the growth of the fowlpox helper virus. In contrast to the classical "in vivo" recombination technique, chimeric viruses with inserts in both possible orientations and families of chimeras with multiple inserts were obtained. The different genomic configurations of chimeric viruses provide a broader basis for screening of optimal viruses. In addition to packaging in avian cells, a second packaging procedure for vaccinia DNA, based on the abortive infection of mammalian cells with the fowlpox helper virus, was developed. This procedure permits simultaneous packaging and host-range selection for the packaged virus. The cloning/packaging procedure allows the direct insertion of foreign DNA without the need for plasmids having flanking regions homologous to viral nonessential regions and is independent of inefficient in vivo recombination events. By direct cloning and packaging, about 5-10% of the total vaccinia virus yield consisted of chimeras. The procedure is, therefore, a useful tool in molecular virology.
On the London equations.
Authors: Sternberg S
Published: May 2006

The classical London equations for superconductors are written in differential form notation and recast in relativistic form, where they yield the Proca equation. In particular, the field itself acts as its own charge carrier.
Cloning and surface expression of Pseudomonas aeruginosa O antigen in Escherichia coli.
Authors: Goldberg J B , Hatano K , Meluleni G S , Pier G B
Published: May 2006

As a step toward developing recombinant oral vaccines, we have explored the feasibility of expression of O polysaccharide antigens from Pseudomonas aeruginosa by Escherichia coli. We cloned in E. coli HB101 a 26.2-kilobase DNA fragment from P. aeruginosa strain PA103 that specifies the production of the O polysaccharide of Fisher immunotype 2 (IT-2) strains. The recombinant organism incorporated the P. aeruginosa IT-2 O polysaccharide onto the core of the E. coli lipopolysaccharide (LPS). Transfer of the recombinant plasmid to three LPS-rough strains of P. aeruginosa resulted in synthesis of IT-2 O antigen, and two of these transconjugant strains also synthesized a second O polysaccharide, presumably representing expression of a repressed, or an incomplete, set of genes for an endogenous O polysaccharide. Rabbits injected with the purified recombinant LPS made antibody specific for P. aeruginosa IT-2 O side chains, as did mice fed the recombinant E. coli strain. Expression of P. aeruginosa O antigens by enteric bacteria makes it possible to study these recombinant strains as oral vaccines to prevent P. aeruginosa infections.
A host-inducible cytochrome P-450 from a host-specific caterpillar: molecular cloning and evolution.
Authors: Cohen M B , Schuler M A , Berenbaum M R
Published: May 2006

Cytochrome P-450 monooxygenases (P-450s) play a critical role in the detoxification of natural and synthetic toxins in a wide range of organisms. We have isolated and sequenced cDNA clones encoding a P-450, CYP6B1, from larvae of Papilio polyxenes (Lepidoptera: Papilionidae), the black swallowtail butterfly. This P-450, cloned from a herbivorous insect, is highly inducible by xanthotoxin, a secondary metabolite abundant in the host plants of this specialized herbivore. On Northern blots, mRNAs crossreactive with CYP6B1 were detected in three Papilio species that, like the black swallowtail, have high levels of xanthotoxin-metabolic P-450 activity and encounter xanthotoxin or related compounds in their host plants; in contrast, no crossreactive mRNAs were detectable in three papilinid species that never encounter xanthotoxin in their host plants and lack detectable xanthotoxin-metabolic activity. These results provide evidence that new P-450s can arise as herbivores colonize different host plants and support the hypothesis that interactions between herbivores and their toxin-producing host plants have contributed to the diversification of the P-450 superfamily.
Induction of aP2 gene expression by nonmetabolized long-chain fatty acids.
Authors: Grimaldi P A , Knobel S M , Whitesell R R , Abumrad N A
Published: May 2006

Long-chain fatty acids (FA) have been shown to regulate expression of the gene for the adipocyte FA-binding protein aP2. We examined whether this effect was exerted by FA themselves or by a FA metabolite. The alpha-bromo derivative of palmitate, an inhibitor of FA oxidation, was synthesized in the radioactive form, and its metabolism was investigated and correlated with its ability to induce aP2 in Ob1771 preadipocytes. alpha-Bromopalmitate was not utilized by preadipocytes. It was not cleared from the medium over a 24-hr period and was not incorporated into cellular lipids. Short incubations indicated that alpha-bromopalmitate exchanged across the preadipocyte membrane but remained in the free form inside the cell. In line with this, preadipocyte homogenates did not activate alpha-bromopalmitate to the acyl form. However, although it was not metabolized, bromopalmitate was much more potent than native FA in inducing aP2 gene expression. Induction exhibited the characteristics previously described for native FA, indicating that a similar if not identical mechanism was involved. The data indicated that induction of aP2 was exerted by unprocessed FA. Finally, in contrast to preadipocytes, adipocytes metabolized bromopalmitate. This reflected increased activity with cell differentiation of a palmitoyl-CoA synthase that could activate palmitate and bromopalmitate at about one-fifth the rate for palmitate. In preadipocytes, the predominant fatty-acyl-CoA synthase, arachidonyl-CoA synthase, had very low affinity for both FA. Increased activity of the palmitoyl-CoA synthase, which has a wider substrate range, is likely to be important for initiation of lipid deposition.
The 5' splice site consensus RNA oligonucleotide induces assembly of U2/U4/U5/U6 small nuclear ribonucleoprotein complexes.
Authors: Hall K B , Konarska M M
Published: May 2006

A short RNA oligonucleotide comprising the 5' splice site consensus sequence (5'SS RNA oligo) efficiently inhibits splicing of mRNA precursors in HeLa cell nuclear extracts. Addition of 5'SS RNA oligo inhibits early, but not late, steps in the splicing reaction, affecting the process of spliceosome assembly. In the presence of 5'SS RNA oligo a majority of U4/U5/U6 triple small nuclear ribonucleoprotein (snRNP) complex present in HeLa nuclear extracts associates with U2 snRNP to form a multi-snRNP complex, which could account for the observed inhibition of splicing by the oligo. This same set of snRNPs has been shown to assemble on pre-mRNAs during in vitro splicing to form splicing complex B. Removal of the 5' end of U1 snRNA, which is complementary to the 5' splice site, does not prevent association of snRNPs into U2/U4/U5/U6 complex in the presence of 5'SS RNA oligo. This suggests that interactions other than U1 snRNA.5'SS RNA oligo base pairing are used in recognition of the oligo sequence. 5'SS RNA oligo-induced assembly of the multi-snRNP complex may thus serve as a model to study the mechanism of 5' splice site recognition during splicing.
A conserved double-stranded RNA-binding domain.
Authors: St Johnston D , Brown N H , Gall J G , Jantsch M
Published: May 2006

We have identified a double-stranded (ds)RNA-binding domain in each of two proteins: the product of the Drosophila gene staufen, which is required for the localization of maternal mRNAs, and a protein of unknown function, Xlrbpa, from Xenopus. The amino acid sequences of the binding domains are similar to each other and to additional domains in each protein. Database searches identified similar domains in several other proteins known or thought to bind dsRNA, including human dsRNA-activated inhibitor (DAI), human trans-activating region (TAR)-binding protein, and Escherichia coli RNase III. By analyzing in detail one domain in staufen and one in Xlrbpa, we delimited the minimal region that binds dsRNA. On the basis of the binding studies and computer analysis, we have derived a consensus sequence that defines a 65- to 68-amino acid dsRNA-binding domain.
Identification of a pathogenic epitope involved in initiation of Heymann nephritis.
Authors: Kerjaschki D , Ullrich R , Diem K , Pietromonaco S , Orlando R A , Farquhar M G
Published: May 2006

Heymann nephritis is an experimental autoimmune disease model for human membranous nephropathy. We have recently identified a pathogenic epitope, clone 14 (C14), responsible for formation and deposition of glomerular immune complexes that is contained within the small subunit of the Heymann nephritis antigenic complex (HNAC). HNAC is a heterodimer composed of a large subunit designated gp330 and a smaller (44 kDa) subunit, which is immunologically identical to the receptor-associated protein. In this study, we prepared antibodies to fusion proteins with C-terminal deletions in the C14 sequence and assessed their ability to promote formation of immune deposits (IDs). When IgG specific for the shortest truncated fusion protein (C14/delta 3; 86 amino acids) was injected into rats, small IDs developed. In contrast, when IgG raised against the full-length C14 sequence was depleted of its reactivity toward the C14/delta 3 fusion protein (C14/delta 3-fp), no IDs could be detected. These data indicate that at least one pathogenic epitope is contained within the N-terminal 86 amino acids of C14. Since the IDs induced with the C14/delta 3-fp-specific IgG are smaller than those induced with the poly-epitope-specific anti-gp330 antibodies, it is likely that other epitopes in addition to those expressed by the C14/delta 3-fp are required for formation and growth of immune complexes.
Drosophila homolog of the human S6 ribosomal protein is required for tumor suppression in the hematopoietic system.
Authors: Watson K L , Konrad K D , Woods D F , Bryant P J
Published: May 2006

The tumor suppressor gene lethal(1)aberrant immune response 8 (air8) of Drosophila melanogaster encodes a homolog of the human S6 ribosomal protein. P element insertions that prevent expression of this gene cause overgrowth of the lymph glands (the hematopoietic organs), abnormal blood cell differentiation, and melanotic tumor formation. They also cause delayed development, inhibit growth of most of the larval organs, and lead to larval lethality. Mitotic recombination experiments indicate that the normal S6 gene is required for clone survival in the germ line and imaginal discs. The S6 gene produces a 1.1-kilobase transcript that is abundant throughout development in wild-type animals and in revertants derived from the insertional mutants but is barely detectable in the mutant larvae. cDNAs corresponding to this transcript show a 248-amino acid open reading frame with 75.4% identity and 94.8% similarity to both human and rat S6 ribosomal protein sequences. The results reveal a regulatory function of this ribosomal protein in the hematopoietic system of Drosophila that may be related to its developmentally regulated phosphorylation.
Morphological changes in liposomes caused by polymerization of encapsulated actin and spontaneous formation of actin bundles.
Authors: Miyata H , Hotani H
Published: May 2006

Spherical giant liposomes that had encapsulated skeletal-muscle G-actin were made by swelling a dried lipid mixture of dimyristoyl phosphatidylcholine/cardiolipin, 1:1 (wt/wt), in a solution of G-actin/CaCl2 at 0 degree C. Polymerization of the encapsulated G-actin into actin filaments was achieved by raising the temperature to 30 degrees C. We observed the subsequent shape changes of the liposomes by dark-field and differential interference-contrast light microscopy. After approximately 40 min, which was required for completion of actin polymerization, two shapes of liposome were evident: dumbbell and disk. Elongation of the dumbbell-shaped liposomes was concomitant with actin polymerization. Polarization microscopy showed that actin filaments formed thick bundles in the liposomes and that these filaments lay contiguous to the periphery of the liposome. Localization of actin filaments in the liposomes was confirmed by observation of rhodamine phalloidin-conjugated actin filaments by fluorescence microscopy. Both dumbbell- and disk-shaped liposomes were rigid and kept their shapes as far as actin filaments were stabilized. In contrast, liposomes containing bovine serum albumin were fragile, and their shapes continually fluctuated from Brownian motion, indicating that the actin bundles served as mechanical support for the liposome shapes.
Purification of human leukotriene C4 synthase.
Authors: Penrose J F , Gagnon L , Goppelt-Struebe M , Myers P , Lam B K , Jack R M , Austen K F , Soberman R J
Published: May 2006

Leukotriene (LT) C4 synthase, the enzyme that catalyzes the conjugation of LTA4 with reduced glutathione to form LTC4, was purified to homogeneity from the KG-1 myeloid cell line after solubilization of the microsomes utilizing a combination of 0.4% sodium deoxycholate and 0.4% Triton X-102. The solubilized enzyme was then applied to an S-hexyl-glutathione-agarose column that was eluted by the use of 7.5 mM probenecid. After removal of the probenecid by sequential concentration and dilution in an Amicon concentrator, the enzyme was additionally purified and concentrated by binding to and elution from approximately 75 mg of S-hexyl-glutathione-agarose. The enzyme was further resolved by electrophoresis with a nondenaturing Tris-glycine gel, and the LTC4 synthase activity was localized to slices 3 and 4. When the remainder of the eluate from the nondenaturing gel was precipitated by acetone and analyzed by 14% SDS/PAGE with silver staining, a single protein band of 18 kDa was associated with LTC4 synthase activity and was not present in the eluates of slices lacking activity. The overall recovery was 12.5%. In a separate preliminary purification, in which the yield was only approximately 1%, the eluates of the nondenaturing gel had also revealed a single protein of 18 kDa by SDS/PAGE, which was present only in the eluates with LTC4 synthase activity. These data identify LTC4 synthase as a protein of 18 kDa, a size consistent with its membership in the microsomal glutathione S-transferase family.
Characterization of the tumor suppressor protein p53 as a protein kinase C substrate and a S100b-binding protein.
Authors: Baudier J , Delphin C , Grunwald D , Khochbin S , Lawrence J J
Published: May 2006

We report here that the negative cell cycle regulator protein p53 is an in vivo and in vitro substrate for protein kinase C, a cellular receptor for the tumor-promoter phorbol esters. We also demonstrate that p53 interacts in a calcium-dependent manner with S100b, a member of the S100 protein family involved in cell cycle progression and cell differentiation, and that such an interaction inhibits in vitro p53 phosphorylation by protein kinase C. The interaction between p53 and S100b was utilized for the purification of cellular and recombinant murine p53 by affinity chromatography with S100b-Sepharose. Furthermore, and of particular interest, we have shown that purified p53 undergoes temperature-dependent oligomerization and that the interaction between S100b and p53 not only induces total inhibition of p53 oligomerization but also promotes disassembly of the p53 oligomers. We suggest that these effects result from the binding of S100b to the multifunctional basic C-terminal domain of p53 and propose that p53 may be a cellular target for the S100 protein family members involved in the control of the cell cycle at the G0-G1/S boundary.
1H NMR studies on the catalytic subunit of aspartate transcarbamoylase.
Authors: Cohen R E , Takama M , Schachman H K
Published: May 2006

The 1H NMR spectrum of the catalytic subunit of Escherichia coli aspartate transcarbamoylase (EC 2.1.3.2) was simplified by using strains auxotrophic for the aromatic amino acids and a growth medium containing fully deuterated Trp, Phe, and His and partially deuterated Tyr. 1H resonances for Tyr in the catalytic trimer (M(r) = 10(5)) were partially resolved into five peaks at 27 degrees C, which above 50 degrees C were further resolved to give a distinct resonance for each of the eight Tyr residues in the polypeptide chain. Experiments on chemically modified catalytic subunits and on a mutant form in which Tyr-165 was converted to Ser-165 led to the assignment of resonances for Tyr-165, Tyr-240, and Tyr-185. Binding of the substrate, carbamoyl phosphate, caused shifts of two of the unassigned resonances, and the subsequent binding of the aspartate analog succinate perturbed the resonances corresponding to Tyr-165 and Tyr-240. The bisubstrate analog N-(phosphonacetyl)-L-aspartate produced a spectrum differing considerably from that caused by the combination of carbamoyl phosphate and succinate. The NMR spectrum for the Tyr-165-->Ser mutant trimer showed clearly that the single amino acid substitution caused conformational changes affecting the environment of residues remote from the position of the replacement. In contrast, the inactive mutant subunit in which Gly-128 was replaced by Asp exhibited a spectrum virtually identical to that of the wild-type protein. However, addition of the substrate carbamoyl phosphate caused a marked change in the spectrum of the mutant enzyme, whereas that of the wild-type trimer was altered only slightly, showing that the effect of the amino acid substitution was manifested in the NMR spectrum only with the liganded enzyme.
DNA strand-specific repair of (+-)-3 alpha,4 beta-dihydroxy-1 alpha,2 alpha-epoxy-1,2,3,4-tetrahydrobenzo[c]phenanthrene adducts in the hamster dihydrofolate reductase gene.
Authors: Carothers A M , Zhen W , Mucha J , Zhang Y J , Santella R M , Grunberger D , Bohr V A
Published: May 2006

We evaluated the formation and removal of (+-)-3 alpha,4 beta-dihydroxy-1 alpha,2 alpha-epoxy-1,2,3,4- tetrahydrobenzo[c]phenanthrene (BcPHDE)-DNA adducts in two Chinese hamster ovary (CHO) cell lines. One line of repair-proficient cells (MK42) carries a stable 150-fold amplification of the dihydrofolate reductase (DHFR) locus. The other line of repair-deficient cells (UV-5) is diploid for this gene and is defective in excision of bulky DNA lesions. Two methods were used to quantitate adduct levels in treated cells: Escherichia coli UvrABC excision nuclease cleavage and 32P-postlabeling. DNA repair was examined in the actively transcribed DHFR gene, in an inactive region located 25 kilobases downstream, and in the overall genome. Between 8 and 24 hr after BcPHDE exposure, preferential repair of the DHFR gene compared to the noncoding region was apparent in MK42 cells. This gene-specific repair was associated with adduct removal from the DHFR transcribed strand. However, UV-5 cells showed no lesion reduction from this strand of the gene. By both quantitation methods, regions accessible to repair in MK42 cells showed a 2-fold reduction in DNA adduct levels by 24 hr. That the decline in adducts reflects genomic repair was demonstrated by the constant damage level remaining in UV-5 cells. Since BcPHDE-induced mutations in DHFR apparently arise from adducted purines on the nontranscribed strand, results from the present study support the idea that a consequence of strand-specific repair is strand-biased mutations.
Costs of deception: cheaters are punished in rhesus monkeys (Macaca mulatta).
Authors: Hauser M D
Published: May 2006

From a functional perspective, deception can evolve in animal populations but should be constrained by the costs associated with detection. It then follows that withholding information should be more prevalent as a form of deception than active falsification of information because of the relative difficulties associated with detecting cheaters. Empirical studies of deception have focused on the benefits of cheating but have provided no data on the costs associated with being detected as a cheater. I present results from field experiments on rhesus monkeys (Macaca mulatta) which show that individuals discovering food announce their discoveries by calling on 45% of all trials. Discoverers who failed to call, but were detected with food by other group members, received significantly more aggression than vocal discoverers. Moreover, silent female discoverers ate significantly less food than vocal females. This demonstrates that there are significant costs to withholding information. Such costs may constrain the frequency with which deception occurs in this and other populations.
A new approach to evaluating carcinogenic risk.
Authors: Pariza M W
Published: May 2006

Carcinogenic risk assessments are based on extrapolating from high-dose chronic rodent-feeding studies to human-exposure levels. A serious problem is that about half of all substances tested at their respective maximum tolerated dose (MTD) are found to induce cancer. The MTD as currently defined has been criticized because it may stimulate cell proliferation in susceptible tissues. Such chemically induced mitogenesis is postulated to increase the probability that neoplasia will develop at the affected site. It is proposed that, in the development of an MTD for a given substance, chemically induced mitogenesis be considered an undesirable toxic manifestation. Hence, mitogenesis should not be induced by a substance fed at its true MTD. Since MTDs determined in this fashion are likely to be lower than those developed using current criteria, an added level of protection is introduced by employing a safety factor similar to that used now in determining acceptable daily intakes for noncarcinogenic food additives. In calculating acceptable daily intakes, the usual safety factor is 100; i.e., the acceptable daily intake is set at 1% of the no-observed-effect level. Hence it is proposed that the acceptable daily level of exposure to a substance that does not induce cancer at its MTD as defined herein be set at 1% of that MTD. On the other hand, a chemical that induces cancer at its MTD as defined herein would continue to be regulated as is customary now.
Adaptive evolution of degrees and kinds of neoplastic transformation in cell culture.
Authors: Rubin H
Published: May 2006

Human cancers undergo protracted complex development from benign to malignant states, as most thoroughly documented in the mole-to-melanoma sequence. The early stages of the sequence tend to redifferentiate into normal tissues; the later stages progress to ever increasing multiplication and malignancy. When placed under the growth constraint of either crowding or low serum concentrations, the NIH 3T3 line of mouse cells readily undergoes transformation, expressed in the development of foci of cells that continue to multiply at confluence when the rest of the population has stopped. If the nontransformed cells are maintained for 3 months by frequent low-density passages in high concentrations of calf serum, they gradually lose the capacity to undergo transformation when the constraints are applied. The same conditions of passage have been used to reverse the transformation, both processes resembling in principle the reversal of the early stages of the mole-to-melanoma sequence. When the frequent low-density passages are made in high concentrations of fetal bovine serum, which supports a slightly lower growth rate than an equal concentration of calf serum, the degree of transformation is gradually increased, so that the foci become more numerous, broader, and thicker, reaching a maximum in successive assays at about 3 months of passaging. A diversity of focal morphologies is sporadically generated in the calf serum passage by exposing the cells to various concentrations of calf serum for 14 days of growth and confluency before assaying them. The dependence of the number, density, and morphology of foci on the environment in which the cells had been grown before assay reinforces the evidence that the transformation is an epigenetic process. The fact that these effects develop in culture gradually over an extended period of time suggests parallels to the characteristically long-term early regression and later progression, as well as the diversity of the mole-to-melanoma sequence, and may also be representative of other cancers.
Histones H2A/H2B inhibit the interaction of transcription factor IIIA with the Xenopus borealis somatic 5S RNA gene in a nucleosome.
Authors: Hayes J J , Wolffe A P
Published: May 2006

A Xenopus borealis somatic 5S RNA gene was assembled with either the complete octamer of histones, (H2A/H2B/H3/H4)2, or the (H3/H4)2 tetramer of histones that comprises the central protein kernel of the nucleosome. Gel-mobility shifts, DNase I protection, and immunoblotting assays demonstrate that the class III transcription factor IIIA (TFIIIA) readily interacts with 5S DNA associated with the tetramer but that little or no binding is detected when 5S DNA is associated with the full octamer of histones. Thus, the presence of histones H2A and H2B in the 5S nucleosome significantly inhibits the interaction of TFIIIA with its cognate binding site within the 5S RNA gene. We propose that either the depletion of histones H2A and H2B from preexisting nucleosomes or the staged assembly of chromatin after replication in which a tetramer of histones H3/H4 associates with DNA before histones H2A/H2B will facilitate the binding of transcription factors to their cognate DNA sequences.