bluuebunny/crossref_metadata_2025 huggingface dataset:
Detection of an altered DNA conformation at specific sites in chromatin and supercoiled DNA.
Authors: Kohwi-Shigematsu T , Gelinas R , Weintraub H
Published: May 2006

We show that bromoacetaldehyde, which reacts selectively at the N-1 and N-6 positions of unpaired adenine and at the N-3 and N-4 positions of unpaired cytosine residues reacts with chromosomal DNA in intact cells at probable regulatory sequences near active genes. A region of about 200 base pairs 5' to the chicken beta A-globin gene, which contains sites sensitive to nuclease S1, to several restriction endonucleases, and to very low levels of DNase I, also contains DNA structures that are preferentially sensitive to bromoacetaldehyde. These altered DNA structures are found at reproducible positions relative to the beta A-globin gene regardless of whether the bromoacetaldehyde is presented to intact erythrocytes, erythrocyte nuclei, or the beta A-globin gene itself carried in pBR322 as purified supercoiled DNA. The unpaired DNA 5' to the adult beta A-globin gene in adult erythrocytes is not detectable in embryonic erythrocytes that express embryonic beta-globin in contrast to adult beta A-globin. Our results suggest that well-defined regions of DNA with effectively unpaired bases occur in intact nuclei and that these structures may be important for specific recognition because they are tissue specific and are found at putative regulatory regions.
Role for the 3' end of the genome in determining disease specificity of Friend and Moloney murine leukemia viruses.
Authors: Chatis P A , Holland C A , Hartley J W , Rowe W P , Hopkins N
Published: May 2006

To probe the genetic basis of disease specificity of nondefective murine type C viruses, we are constructing recombinants in vitro between molecular clones of Friend murine leukemia virus (Fr-MuLV) and Moloney murine leukemia virus (Mo-MuLV). Fr-MuLV induces erythroleukemias when injected into newborn NFS mice, whereas Mo-MuLV almost invariably induces T-cell lymphomas. We find that a recombinant whose genome is derived primarily from Fr-MuLV but which has 621 nucleotides of Mo-MuLV information at its 3' end induces almost exclusively thymic lymphomas. The sequences derived from Mo-MuLV include 99 nucleotides encoding the carboxyl terminus of Prp15E, the origin of DNA +-strand synthesis, all of the U3 region, and 36 nucleotides of the R portion of the long terminal repeat. When the segment of Mo-MuLV was removed and replaced with the comparable segment from Fr-MuLV, the virus was again erythroblastosis-inducing. These results, in conjunction with studies from other laboratories [Laimins, L. A., Khoury, G., Gorman, C., Howard, B. & Gruss, P. (1982) Proc. Natl. Acad. Sci. USA 79, 6453-6457], suggest that transcriptional signals in U3 may determine tissue tropism and hence influence disease specificity ("targeting") of murine leukemia viruses.
Targeted mutation at cytosine-containing pyrimidine dimers: studies of Escherichia coli B/r with acetophenone and 313-nm light.
Authors: Fix D , Bockrath R
Published: May 2006

We have tested the two-event model for UV mutagenesis producing class 2 suppressor mutations at glutamine tRNA genes in Escherichia coli. In the model used, the induction/indexing lesion is any type of pyrimidine dimer and the premutational photoproduct at the target site is a cytosine-containing dimer. Specific mutation-frequency responses were analyzed under conditions in which the ratio of thymine-thymine dimers to cytosine-containing dimers was modified by using 313-nm light and 0.0%, 0.1%, or 0.2% acetophenone. Changes observed in the production of class 2 suppressor mutations were consistent with the model and suggested that the G X C leads to A X T transitions responsible for class 2 suppressor mutations are targeted by cytosine-containing pyrimidine dimers at the mutational sites.
(Dichloromethylene)diphosphonate-induced impairment of T-lymphocyte function.
Authors: Milhaud G , Labat M L , Moricard Y
Published: May 2006

The daily subcutaneous administration of (dichloromethylene)diphosphonate (clodronate) to 3-day-old normal inbred Wistar-Furth rats for 30 days produces osteopetrotic bone lesions resembling those of the osteopetrotic mutants. Furthermore, tooth eruption is prevented, growth slows down, and signs of runt disease appear. The weight of the thymus is decreased, and T cells from the thymus and spleen respond weakly to mitogens. These thymic disorders associated with defective bone resorption are very similar to those previously reported in the osteopetrotic mutant op rat and support the hypothesis of a link between the thymus and normal bone modeling and remodeling.
Dielectric constant of water at very high temperature and pressure
Authors: Pitzer Kenneth S.
Published: May 2006

Pertinent statistical mechanical theory is combined with the available measurements of the dielectric constant of water at high temperature and pressure to predict that property at still higher temperature. The dielectric constant is needed in connection with studies of electrolytes such as NaCl/H
2
O at very high temperature.
Importance of secondary structure in the signal sequence for protein secretion.
Authors: Emr S D , Silhavy T J
Published: May 2006

Mutant Escherichia coli strains in which export of the LamB protein (coded for by the lamB gene) to the outer membrane of the cell is prevented have been described previously. One of these mutant strains contains a small (12-base pair) deletion mutation within the region of the lamB gene that codes for the NH2-terminal signal sequence. In this mutant strain, export but not synthesis of the LamB protein is blocked. We have isolated pseudorevertants that restore export of functional LamB protein to the outer membrane. DNA sequence analysis showed that two of the revertants contain a point mutation in addition to the original deletion. These point mutations lead to amino acid substitutions within the signal sequence. Our results indicate that these secondary mutations efficiently suppress the export defect caused by the deletion mutation. Analysis of the secondary structure of the wild-type, mutant, and pseudorevertant LamB signal sequences suggests that the secondary mutations restore export by allowing the formation of a stable alpha-helical conformation in the central, hydrophobic region of the signal sequence.
Methylation status and DNase I sensitivity of immunoglobulin genes: changes associated with rearrangement.
Authors: Mather E L , Perry R P
Published: May 2006

Immunoglobulin V kappa genes are transcriptionally silent in their germline context and become transcriptionally active upon fusion to the J kappa-C kappa region (kappa locus). To elucidate the role of chromosomal structure in this regulatory phenomenon we have investigated the DNase I sensitivity and methylation status of the kappa locus and selected V kappa genes in a variety of alleles exhibiting different rearrangement configurations and different levels of transcriptional activity. Our findings indicate that the kappa locus in either germline or rearranged contexts maintains a distinctive DNase I-sensitive, hypomethylated structure in plasmacytomas and hybridomas, irrespective of its level of transcriptional activity. In contrast, the germline V kappa genes are in less accessible regions of chromatin and more highly methylated regions of DNA. Upon fusion to the kappa locus, V kappa genes become DNase I-sensitive and hypomethylated. This effect extends several kilobases upstream of the transcriptional initiation site but does not extend to the adjacent V kappa gene or to the identical V kappa allele on the other chromosome, indicating that the structural alteration is a localized cis-acting phenomenon.
"Prompt" heat shock proteins: translationally regulated synthesis of new proteins associated with the nuclear matrix-intermediate filaments as an early response to heat shock.
Authors: Reiter T , Penman S
Published: May 2006

The response of mammalian cells, such as HeLa cells, to prolong exposure to increased temperature (termed heat shock) has been well characterized. In these studies new mRNA is synthesized for several proteins whose translation is best seen after a return to 37 degrees C. We show here another response to increased temperature of a distinctively different character. A set of at least 50 newly detectable proteins, exclusively associated with the nuclear matrix-intermediate filaments (NM-IF) fraction, is synthesized immediately upon exposure to high temperature. These are of very low abundance or nonexistent in the unstressed cell and none appear to correspond to the "classic" heat shock proteins produced after new transcription. Prior treatment with actinomycin D has little effect on these "prompt" proteins, and they appear to be made from preexisting mRNAs that are activated at the increased temperature. The protein synthesis in the soluble, cytoskeletal, and chromatin fractions is strongly reduced by the increased temperature, while the labeling of the prompt proteins associated with NM-IF complex rapidly rises severalfold above that in control cells. Additionally these results suggest that the four cell fractions are not arbitrary cell divisions; rather they represent physiologically significant compartments in the cell.
A previously unrecognized influenza B virus glycoprotein from a bicistronic mRNA that also encodes the viral neuraminidase.
Authors: Shaw M W , Choppin P W , Lamb R A
Published: May 2006

RNA segment 6 of the influenza B virus genome codes for a previously unidentified polypeptide designated NB. The reading frame for this polypeptide begins with the first AUG codon on the mRNA and overlaps the reading frame for the viral neuraminidase by 292 nucleotides. The amino acid sequence of polypeptide NB deduced from the nucleotide sequence of the B/Lee/40 strain consists of 100 amino acids with a molecular weight of 11,242. The sequence contains four potential glycosylation sites, and the protein has been found to be glycosylated in infected cells. NB has not been found in virions. Sucrose gradient sedimentation and analysis of the structure of the mRNA by nuclease S1 mapping and sequence analysis by the primer extension method indicated that polypeptide NB and the neuraminidase are translated from a single bicistronic mRNA. A protein analogous to NB has not been found with influenza A virus, and this represents a major difference between the two virus types.
Binding of alpha-bungarotoxin to isolated alpha subunit of the acetylcholine receptor of Torpedo californica: quantitative analysis with protein blots.
Authors: Gershoni J M , Hawrot E , Lentz T L
Published: May 2006

The direct binding of alpha-bungarotoxin to the alpha subunit of the acetylcholine receptor from Torpedo electric organ immobilized onto protein blots was demonstrated. Protein blots were prepared by electrophoretically transferring resolved acetylcholine receptor subunits from 10% polyacrylamide gels onto Zetabind, positively charged nylon membrane filters. Such blots, when incubated with 125I-labeled alpha-bungarotoxin, washed, and autoradiographed, gave rise to a single labeled band corresponding to the alpha subunit of the receptor. The labeling with alpha-bungarotoxin could be inhibited by pretreating the receptor-containing membranes with the affinity ligand 4-(N-maleimido)-alpha-benzyltrimethylammonium iodide. In addition, the association of toxin with the alpha subunit could be inhibited by d-tubocurarine (IC50 = 0.9 mM). Furthermore, removal of high-mannose oligosaccharide chains from the alpha subunit by treatment with endoglycosidase H did not interfere with the observed toxin binding. Thus it is demonstrated that isolated, immobilized alpha subunit of the Torpedo acetylcholine receptor can bind alpha-bungarotoxin. However, the observed binding of alpha-bungarotoxin to immobilized alpha subunit is reduced in affinity to 1/1,000 to 1/10,000 of that obtained with native receptor. The endoglycosidase H-susceptible oligosaccharide side chain(s) is not required for this interaction. Binding of alpha-bungarotoxin is to the physiologically relevant acetylcholine binding site as defined by affinity ligand alkylation.
Genetic analysis of the 15;17 chromosome translocation associated with acute promyelocytic leukemia.
Authors: Sheer D , Hiorns L R , Stanley K F , Goodfellow P N , Swallow D M , Povey S , Heisterkamp N , Groffen J , Stephenson J R , Solomon E
Published: May 2006

Somatic cell hybrids have been constructed between a thymidine kinase-deficient mouse cell line and blood leukocytes from a patient with acute promyelocytic leukemia showing the 15q+;17q- chromosome translocation frequently associated with this disease. One hybrid contains the 15q+ translocation chromosome and very little other human material. We have shown that the c-fes oncogene, which has been mapped to chromosome 15, is not present in this hybrid and, therefore, probably is translocated to the 17q- chromosome. Analysis of the genetic markers present in this hybrid has enabled a more precise localization of the translocation breakpoints on chromosomes 15 and 17. Our experiments also have enabled an ordering and more precise mapping of several genetic markers on chromosomes 15 and 17.
Methanophosphagen: Unique cyclic pyrophosphate isolated from
Methanobacterium thermoautotrophicum
Authors: Kanodia Sushila , Roberts Mary Fedarko
Published: May 2006

A unique cyclic pyrophosphate compound has been detected at 10-12 mM intracellular concentration in
Methanobacterium thermoautotrophicum
by
in vivo
31
P NMR. This compound has been extracted from cells and purified by anion-exchange chromatography. Studies with
1
H,
13
C, and
31
P NMR and fast-atom-bombardment mass spectrometry have identified it as 2,3-cyclopyrophosphoglycerate, an intramolecularly cyclized pyrophosphate of 2,3-diphosphoglycerate. Chemical degradation to 2,3-diphosphoglycerate and synthesis by dicyclohexylcarbodiimide coupling of 2,3-diphosphoglycerate are consistent with this identification. It is suggested that this compound serves as a primary phosphagen in methanogens.
Synthesis and post-translational assembly of intermediate filaments in avian erythroid cells: vimentin assembly limits the rate of synemin assembly.
Authors: Moon R T , Lazarides E
Published: May 2006

The assembly of vimentin intermediate filaments and the high molecular weight filament crosslinking protein, synemin, was studied in erythroid cells from 10-day chicken embryos. Pulse labeling studies show that newly synthesized vimentin is present both in a Triton X-100-insoluble form and in a soluble form. The incorporation of labeled vimentin into the insoluble fraction increases linearly with time, while the soluble pool of labeled vimentin saturates quickly. In contrast, synemin accumulates rapidly in the Triton X-100-soluble fraction and begins to accumulate in the insoluble fraction only after a considerable lag of time. Pulse-chase studies reveal that the detergent-soluble pools of both vimentin and synemin contain precursors for their post-translational assembly into detergent-insoluble filaments and that the half-life of soluble synemin is about twice as long as that of soluble vimentin. Immunoprecipitation of solubilized filaments with synemin antiserum precipitates vimentin with synemin. On the other hand, soluble vimentin does not coimmunoprecipitate with soluble synemin. These results suggest that, in the assembly of vimentin and synemin into intermediate filaments, vimentin filament elongation generates synemin binding sites, and thus the rate of vimentin filament elongation limits the rate of synemin assembly.
Complete primary structures of the E beta chain and gene of the mouse major histocompatibility complex.
Authors: Saito H , Maki R A , Clayton L K , Tonegawa S
Published: May 2006

Using the cross-hybridization with plasmid pDC beta-1, containing the cDNA coding for the DC beta chain of the human major histocompatibility complex class II molecules, we have cloned and subjected to sequence analysis both the cDNA and genomic gene for the E beta chain of the BALB/c (d haplotype) mouse. The nucleotide sequences of the cDNA and genomic DNA clones permitted us to deduce the entire primary structure of the E beta chain and the complete exon-intron structure of the E beta gene. Unlike alpha chain genes that contain five exons, the E beta gene consists of six exons corresponding to the six functional domains--the leader, beta 1 and beta 2 domains, transmembrane peptide, intracytoplasmic peptide, and 3' untranslated region. In addition, two short blocks of sequences common to alpha and beta chain genes were identified in the 5' flanking regions. We propose that these sequences are involved in the coordinate expression of alpha and beta chains.
Normal mode paths for hydrogen exchange in the peptide ferrichrome
Authors: Sheridan Robert P. , Levy Ronald M. , Englander S. W.
Published: May 2006

Possible paths for exposure to solvent and hydrogen exchange of the amide protons of ferrichrome, a cyclic hexapeptide, are examined. The paths are obtained from calculations of the vibrational normal modes of ferrichrome and correspond to low energy atomic displacements away from the local minimum in the multidimensional conformational space of the molecule. Exposure of exchangeable groups along the normal modes was determined by using the solvent accessible surface area algorithm of Lee and Richards. Three of the exchangeable protons (Gly
1,2,3,
) are largely exposed to solvent in the x-ray structure while the remaining three exchangeable protons of the ornithines are totally shielded from solvent. A very small number of normal mode displacements are found to expose the Orn
2
and Orn
3
amide groups while the Orn
1
amide proton remains shielded from solvent for all the paths studied. The effective paths for exposure of Orn
2
and Orn
3
correspond to the lowest frequency (≈18 cm
-1
) motions. The paths are characterized in terms of the magnitude and energy of atomic displacements, correlated changes in dihedral angles, and the resulting changes in exposure and hydrogen bonding of exchangeable groups.
Surface binding and internalization of platelet-derived growth factor in human fibroblasts.
Authors: Nilsson J , Thyberg J , Heldin C H , Westermark B , Wasteson A
Published: May 2006

Surface binding and uptake of platelet-derived growth factor (PDGF) in human fibroblasts cultivated in vitro were studied by quantitative electron microscopic autoradiography using 125I-labeled PDGF and by indirect immunofluorescence using PDGF antibodies. After 120 min at 12 degrees C PDGF was found preferentially in coated regions of the plasma membrane. Warming the cells to 37 degrees C initiated rapid ingestion of the factor via small vesicles, usually lacking a membrane coat. After 10 min PDGF started to appear in lysosomes, and it showed maximal concentration within these organelles after 30 min. There were also signs of passage of PDGF through the Golgi complex, but only after 60 min. Treatment of the cells with chloroquine, a weak base that inhibits intralysosomal degradation, prevented disappearance of tracer from the lysosomes. The observations indicate that PDGF was internalized via coated regions of the plasma membrane and carried to lysosomes for degradation. Subsequent appearance of tracer within the Golgi complex could reflect receptor-ligand complexes that escaped degradation and were recirculated back to the cell surface.
Cell growth on liquid interfaces: Role of surface active compounds
Authors: Keese Charles R. , Giaever Ivar
Published: May 2006

Anchorage-dependent fibroblasts can be cultured by using as a substrate the protein layer that spontaneously forms at the liquid-liquid interface between fluorocarbon fluids and tissue culture medium. For this novel substrate to be effective in supporting confluent cell layers, the protein monolayer must support the stresses exerted by spreading fibroblasts. The composition of the fluorocarbon fluid has a significant effect on the strength of the protein layer and, thus, on the patterns of cell growth. Evidence is presented demonstrating that a protein film, sufficiently strong to support cell growth, does not occur on purified fluorocarbon fluids but requires the presence of trace amounts of polar, surface active compounds. By the addition of small quantities of pentafluorobenzoyl chloride to alumina-treated fluorocarbon fluids, excellent interfacial substrates can be produced. We have applied this understanding to produce a liquid microcarrier system capable of general use with a variety of cells, including human fibroblasts. A microcarrier in which the fluorocarbon is replaced with polydimethyldiphenyl siloxane is also described.
The transforming function of bovine papillomavirus DNA.
Authors: Nakabayashi Y , Chattopadhyay S K , Lowy D R
Published: May 2006

When bovine papillomavirus (BPV) or its 7.9-kilobase full viral DNA genome induces focal transformation of mouse cells, the viral DNA is maintained in the transformed cells as multiple episomal copies. This transforming capacity and maintenance of the episomal state previously has been localized to a 69% subgenomic fragment of the viral DNA genome. We now have characterized further the BPV DNA sequences that can encode the transforming function. We first created a series of BPV DNA deletion mutants and correlated the location of the deletions with the capacity of the deleted viral DNAs to induce transformation of mouse cells. The results indicated that two discontinuous segments of the viral DNA were required for transformation. One segment, near the 5' end of the 69% transforming fragment, probably represents a control element of the viral DNA. The second segment, which lies within the 3' end of the 69% fragment, encodes transforming sequences of the viral DNA; ligation of a retroviral control element (the long terminal repeat DNA of Harvey murine sarcoma virus) to the 2.3-kilobase segment at the 3' end of the 69% fragment induces transformation of mouse cells. In contrast to mouse cells transformed by the full-length BPV DNA genome, the cells transformed by the deleted BPV DNA genomes contained few viral DNA copies; at least some copies appeared to be integrated. We conclude that different viral functions mediate cellular transformation and maintain the viral DNA in its episomal state.
Type I and type II keratins have evolved from lower eukaryotes to form the epidermal intermediate filaments in mammalian skin.
Authors: Fuchs E , Marchuk D
Published: May 2006

We have traced the evolutionary origins of keratin-like sequences to the genomes of lower eukaryotes. The proteins encoded by these genes have evolved to form the intermediate filaments that comprise the backbone of vertebrate skin cells. Two related but distinct types of keratins encoded by two separate multigene subfamilies are expressed in the epidermal keratinocytes of vertebrate species from fish to human. Both at the level of protein and at the level of DNA, these two classes of keratins are coordinately conserved throughout vertebrate evolution, indicating the central role that both types of keratins must play in the assembly and structure of the 8-nm filament.
Chlamydomonas
agglutinin is a hydroxyproline-rich glycoprotein
Authors: Cooper James B. , Adair W. Steven , Mecham Robert P. , Heuser John E.
Published: May 2006

The
mt
+
sexual agglutinin from
Chlamydomonas reinhardi
is shown to contain ≈12% hydroxyproline, and two inhibitors of hydroxyproline formation, α,α′-dipyridyl and 3,4-dehydroproline, are shown to block the production of agglutinin activity in an
in vivo
bioassay system. These results indicate that the agglutinin glycoprotein may be related to a class of hydroxyproline-rich glycoproteins found in the extracellular matrix of higher plants, several of which have been shown to have lectin activity.
Construction and characterization of Moloney murine leukemia virus mutants unable to synthesize glycosylated gag polyprotein.
Authors: Fan H , Chute H , Chao E , Feuerman M
Published: May 2006

Murine leukemia virus (MuLV) encodes two independent pathways for expression of the gag gene. One pathway results in processing and cleavage of the precursor Pr65gag to yield the internal capsid proteins of the virion and is analogous to gag polyprotein precursors for all classes of retroviruses. The other pathway, which is not encoded by several other classes of retroviruses, begins with a glycosylated polyprotein gPr80gag . gPr80gag is synthesized independently of Pr65gag; it contains Pr65gag peptides and additional amino-terminal protein. It is modified by further addition of carbohydrate, exported to the cell surface, and released from the cell but does not appear in virus particles. To investigate the role of glycosylated gag in MuLV infection, two mutants of Moloney MuLV (M-MuLV) deficient for synthesis of gPr80gag but able to synthesize Pr65gag were constructed. The mutants were obtained by substitution into a molecular clone of M-MuLV DNA by DNA from two acutely transforming viruses, Ableson MuLV (Ab-MuLV) and Moloney murine sarcoma virus (M-MSV). Both Ab-MuLV and M-MSV are derived from M-MuLV and they express M-MuLV gag sequences, but some strains do not synthesize glycosylated gag protein. For Ab-MuLV, a 177-base-pair Pst I fragment from the P90 strain containing the initiation codon for Pr65gag was substituted for the equivalent fragment in M-MuLV DNA. For M-MSV, 1.5 kilobases at the 5' end of the genome was substituted. Transfection of the recombined DNAs onto NIH-3T3 cells produced infectious M-MuLV, although the infected cells did not produce gPr80gag. Therefore glycosylated gag is not absolutely required for MuLV replication. Deletion of the glycosylated gag pathway did not significantly reduce the level of virus production, although a minor difference in XC plaque morphology was observed.
Restoration of ovulatory cycles by young ovarian grafts in aging mice: potentiation by long-term ovariectomy decreases with age.
Authors: Felicio L S , Nelson J F , Gosden R G , Finch C E
Published: May 2006

The relative contributions of ovarian and hypothalamic-pituitary factors to the anovulatory status of aging mice were evaluated by measuring the capacity of mice to resume ovulatory cyclicity after receiving young ovaries under the renal capsule. Young grafts partially restored cyclicity if old hosts were acutely ovariectomized but almost fully restored cyclic ovulatory function if the old hosts had been ovariectomized early in adulthood. With advancing age, however, the efficacy of the grafts declined progressively in both acute and long-term ovariectomized groups. These data show that both ovarian and hypothalamic-pituitary aging contribute to the etiology of anovulation. Although chronic withdrawal from ovarian secretions retards the age of onset of hypothalamic-pituitary aging, the duration of this ameliorative effect is limited by progressive ovary-independent neuroendocrine dysfunction.
Inhibition of thromboxane A2 synthesis in human platelets by coagulation factor Xa.
Authors: Sinha A K , Rao A K , Willis J , Colman R W
Published: May 2006

Factor Xa binds to platelets provided that factor Va is present on the platelet surface, an interaction that results in a striking acceleration of the conversion of prothrombin to thrombin. Thrombin then initiates fibrin formation, induces platelet aggregation, and stimulates the intraplatelet synthesis of thromboxane A2 (TXA2). Addition of thrombin (2.4-14.4 nM) to platelet-rich plasma increased the basal level of TXA2, measured as thromboxane B2, from less than 0.5 pmol per 10(8) platelets to (mean +/- SEM) 100 +/- 22 and 250 +/- 10 pmol per 10(8) platelets, respectively. Treatment of platelet-rich plasma with increasing concentrations of factor Xa (1-12 nM) prior to the addition of thrombin progressively inhibited the production of TXA2. Thrombin (9.6 nM), which produced 93% of the maximal formation of TXA2, was inhibited 70% by factor Xa (10 nM). To identify which of these steps in thromboxane synthesis was inhibited by factor Xa, platelets labeled with [14C]arachidonic acid were exposed to thrombin and products of prostaglandin synthesis were separated by thin-layer chromatography. In contrast to the inhibition of TXA2 synthesis, prostaglandin E2 and prostaglandin F2 alpha synthesis were not inhibited suggesting that neither phospholipase(s) nor cycloxygenase was involved. The inhibition of TXA2 formation by factor Xa could be reversed by increasing the molar ratio of thrombin to factor Xa to 5.5. Incubation of platelets with an IgG fraction of a human monoclonal antifactor V antibody, previously shown to inhibit factor Xa binding, was found to block factor Xa inhibition of TXA2 synthesis. The inhibition of TXA2 synthesis requires the presence of the active site serine of factor Xa and is not specific for TXA2 formation induced by thrombin because it is also demonstrable when the agonist is ADP. Further, factor Xa does not require additional plasma components for its action because its inhibitory effects are detected in gel-filtered platelets. The effect of factor Xa was evident at physiological (1.3 mM) calcium concentrations. These results indicate that factor Xa binding to platelets through factor Va not only stimulates thrombin formation but also has a countervailing effect by inhibiting TXA2 formation.
Efficient expression of influenza virus NS1 nonstructural proteins in Escherichia coli.
Authors: Young J F , Desselberger U , Palese P , Ferguson B , Shatzman A R , Rosenberg M
Published: May 2006

RNA segment 8 of the influenza A virus genome codes for two nonstructural proteins, NS1 and NS2, for which the functions are unknown. Cloned cDNA copies of this gene from three different influenza A virus strains were inserted into an Escherichia coli plasmid expression vector, pAS1, carrying the strong regulatable lambda phage promoter, PL. After induction, the NS1 proteins were overproduced to levels of 20-25% of total cellular protein. This was surprising in that the codon composition for these eukaryotic genes is similar to that for weakly expressed proteins in E. coli. Thus, under the appropriate conditions, it appears that high level expression of genes containing a relatively large proportion of minor codons can be obtained. The NS1 protein produced in bacteria from a cloned cDNA copy of the A/PR/8/34 virus NS gene was purified to apparent homogeneity and used to generate a high-titer monospecific rabbit antiserum. Immunoprecipitation studies showed this antibody to be crossreactive against the NS1 proteins produced by several different influenza A virus strains. Immunofluorescence experiments in Madin-Darby canine kidney cells showed the NS1 proteins to be located in the nucleoplasm early in infection for all strains examined. With some of the strains, NS1-specific immunofluorescence was observed predominantly in the nucleoli later in infection. This technology can be used to obtain other viral proteins in pure form for structural, functional, and immunological studies.
Characterization of a calmodulin-binding protein that is deficient in trifluoperazine-resistant variants of the macrophage-like cell line J774.
Authors: Speaker M G , Orlow S J , Sturgill T W , Rosen O M
Published: May 2006

A calmodulin-binding protein is present in extracts of the macrophage-like mouse cell line J774 and in extracts of thioglycollate-stimulated mouse peritoneal macrophages; it is deficient in variants of J774 resistant to trifluoperazine and in resident peritoneal macrophages. The calmodulin-binding protein [CaMBP (J7)0.5] was purified from J774 and resolved from endogenous cyclic nucleotide phosphodiesterase and protein kinase activities. The protein has an apparent native Mr of 125,000-150,000 and binds calmodulin in a calcium-dependent manner with a Kd of 20 nM. It inhibits the ability of calmodulin to activate phosphodiesterase. Its sedimentation constant in glycerol gradients containing calmodulin was dependent upon the relative concentrations of calmodulin and the calmodulin-binding protein.
Iron-sulfur stoichiometry and structure of iron-sulfur clusters in three-iron proteins: Evidence for [3Fe-4S] clusters
Authors: Beinert Helmut , Emptage Mark H. , Dreyer Jean-Luc , Scott Robert A. , Hahn James E. , Hodgson Keith O. , Thomson Andrew J.
Published: May 2006

Beef heart aconitase contains 3Fe clusters in its inactive and 4Fe clusters in its active form. The fully active form can be restored from the inactive one by insertion of Fe
2+
, whereas S
2-
is not required. Chemical analyses for iron and labile sulfide yield Fe/S
2-
ratios of 0.66-0.74 for the inactive and 0.90-1.03 for the active form. Sulfane sulfur (S
0
) was not detected. We propose on the basis of these data that the inactive form may arise from the active one by loss of one iron only per cluster with the sulfur remaining as S
2-
in a [3Fe-4S] structure. Measurements by extended x-ray absorption fine structure (EXAFS) spectroscopy on the 3Fe form of aconitase yield a Fe··S distance of 2.24 Å and a Fe··Fe distance of 2.71 Å. This Fe··Fe distance is in agreement with that obtained by EXAFS on ferredoxin II of
Desulfovibrio gigas
, another 3Fe protein, but disagrees with Fe··Fe distances observed for the 3Fe cluster of
Azotobacter vinelandii
ferredoxin I by x-ray diffraction—namely, 4.1 Å. We suggest that this difference may be due to the presence of a [3Fe-3S] structure in the
Azotobacter
ferredoxin I crystals vs. a [3Fe-4S] structure in liquid or frozen solutions of aconitase. The [3Fe-3S] cluster has been shown to have a relatively flat twist-boat structure, whereas a [3Fe-4S] cluster could be expected to essentially maintain the compact structure of the [4Fe-4S] cluster. This would explain the differences in Fe··Fe distances. Two possible structural models for a [3Fe-4S] cluster are discussed.
P85

gag-mos

encoded by ts110 Moloney murine sarcoma virus has an associated protein kinase activity
Authors: Kloetzer William S. , Maxwell Steven A. , Arlinghaus Ralph B.
Published: May 2006

A protein identified as P85

gag-mos

was shown to be phosphorylated when immunoprecipitates from ts110 Moloney murine sarcoma virus transformed nonproducer cells (clone 6m2) were incubated with [γ-
32
P]ATP. The
in vitro
-labeled 85,000-dalton phosphoprotein comigrated on NaDodSO
4
/polyacrylamide gels with authentic phosphorylated P85

gag-mos

. Immunoprecipitates obtained with antisera prepared against Rauscher murine leukemia virus core protein p30 were active in the immune complex kinase assay but anti-murine leukemia virus p10 precipitates were not. Previous studies have shown that anti-p30 but not anti-p10 antisera recognize P85

gag-mos

. The 6m2 clone has been shown to express P85

gag-mos

at 33°C but not at 39°C. Anti-p30 immune complexes from 6m2 cells maintained at 39°C failed to phosphorylate the 85,000-dalton protein. Furthermore, the
in vitro
phosphorylated 85,000-dalton protein gave the same pattern of V8 protease-generated cleavage products as
in vivo
32
P-labeled P85

gag-mos

. We conclude from these results that P85

gag-mos

is phosphorylated in anti-p30 immune complex kinase reactions. Phosphoamino acid analyses indicated that the
in vitro
phosphorylated P85

gag-mos

contained phosphoserine and phosphothreonine. Our findings indicate that incubation of anti-p30 immunoprecipitates at 39°C drastically reduced, in a specific way, the kinase activity associated with P85

gag-mos

. This result and other data suggest that the kinase is virus-encoded. Because P85

gag-mos

, but not Pr65

gag

is phosphorylated in anti-p30 immunoprecipitates from MuLV-MuSV ts110 producer cells, the kinase enzyme is associated with P85

gag-mos

and not
gag
gene products. A second major polypeptide of the size of P58

gag

was also phosphorylated in anti-p30 immunoprecipitates from cells maintained at 33°C but not at 39°C. Since 6m2 cells at 39°C contain P58

gag

, this is also consistent with the kinase activity being associated with P85

gag-mos

.
DNA sequence polymorphism in an androgen-regulated gene is associated with alteration in the encoded RNAs.
Authors: Elliott R W , Berger F G
Published: May 2006

We have used plasmid pMK908, whose cDNA insert corresponds to an androgen-inducible RNA from mouse kidney, as a probe to study both the complementary genomic DNA and the encoded RNA sequences in several inbred strains of mice. A polymorphism in the 908 structural gene, revealed by Southern blotting of HindIII-generated DNA fragments, was found to map near the Gpi-1 and Tam-1 loci on chromosome 7. The 908 structural gene has been termed RP2. The 908 RNAs constitute a sequence-related group displaying extensive size heterogeneity. This heterogeneity, reflected in the size distribution of the RNA on electrophoretic blots, is controlled by a genetic site that is tightly linked to RP2. Thus, associated with polymorphism at RP2 is a change in the molecular size pattern of encoded transcripts.
Immunocytochemical identification of epithelium-derived human tumors with antibodies to desmosomal plaque proteins.
Authors: Franke W W , Moll R , Mueller H , Schmid E , Kuhn C , Krepler R , Artlieb U , Denk H
Published: May 2006

Epithelial cells contain desmosomes, special intercellular junctions providing sites of membrane attachment for intermediate-sized filaments of the cytokeratin type (tonofilaments). Such sites of anchorage of tonofilaments appear as dense plaques on the cytoplasmic side of the desmosomal membrane. We have isolated desmosome-enriched fractions from bovine snout epidermis and tongue mucosa and have characterized the major protein associated with the desmosomal plaque. This protein occurs in equimolar amounts of two polypeptides of Mr 250,000 (desmoplakin I) and Mr 215,000 (desmoplakin II) which are chemically and immunologically related. Antibodies raised against desmoplakins allow the identification and localization of this protein in epithelial cells grown in tissues or in vitro and show crossreaction in species as diverse as man, mouse, and chicken. Using immunolocalization at the light and electron microscope levels, we show that these antibodies bind specifically to desmosomal plaques. Antibodies to desmoplakins have been used successfully for detection of desmosomal proteins in a broad variety of epithelium-derived human tumors, including primary carcinomas and their metastases, irrespective of the morphology of the specific tumor. Nonepithelial tumors examined have been negative. We propose to use antibodies to desmoplakins and to cytokeratins in pathological diagnosis as two independent markers for the positive immunocytochemical identification and classification of epithelium derived tumors.
Platelet-derived growth factor stimulates Na+/H+ exchange and induces cytoplasmic alkalinization in NR6 cells.
Authors: Cassel D , Rothenberg P , Zhuang Y X , Deuel T F , Glaser L
Published: May 2006

Stimulation of Na+/H+ exchange by growth factors has been implicated as a mechanism allowing quiescent cells to resume growth because of a predicted elevation of intracellular pH(pHi). We tested this prediction in NR6 cells by using a further development of our technique for pHi measurement, based on introduction of the fluorescent pH indicator 4',5'-dimethylfluorescein (pKa = 6.75) coupled to dextran into the cytoplasm. Addition of the potent mitogens platelet-derived growth factor (PDGF) or serum to NR6 cells stimulates an amiloride-sensitive 22Na+ uptake and causes an elevation of pHi. The PDGF-dependent pHi increase follows a lag period of approximately equal to 2 min, reaches a maximal level within 10 min (delta pHi approximately equal to 0.1 at an external pH of 7.18), and remains at this level for at least 1 hr. Serum addition initially produces a large elevation of pHi, which later declines to a level similar to that obtained with PDGF. The effects of PDGF and serum are partially additive (delta pHi approximately equal to 0.14). The magnitude of pHi elevation by PDGF decreases with increasing extracellular pH. Serum- and PDGF-dependent elevations of pHi are inhibited by amiloride and by eliminating Na+ from the medium. Under conditions in which Na+/H+ exchange is inhibited, PDGF and serum induce an initial cytoplasmic acidification that does not show a lag period. The results show that a single purified growth factor, as well as serum, can promote a sustained elevation of pHi by stimulating Na+/H+ exchange. The extent of pHi elevation may be modulated by the concomitant stimulation by the growth factor of a process generating H+ within the cell.
Intermediate-size filaments in a germ cell: Expression of cytokeratins in oocytes and eggs of the frog Xenopus.
Authors: Franz J K , Gall L , Williams M A , Picheral B , Franke W W
Published: May 2006

Vitellogenic oocytes and eggs of the frog Xenopus laevis contain intermediate-size filaments that are resistant to extractions in high-salt buffers and Triton X-100 and are specifically stained with antibodies to cytokeratins. Gel electrophoresis of cytoskeletal proteins from Xenopus oocytes shows a specific enrichment of three polypeptides designated components 1 [Mr, 56,000; IEP (pI obtained by two-dimensional gel electrophoresis in the presence of 9.5 M urea), ca. 5.9], 2 (Mr, 46,000; IEP, 5.38), and 3 (Mr, 42,000; IEP, ca. 5.3). The same three cytoskeletal polypeptides are found in eggs and early embryos, in intestinal mucosa of adult frogs, and in cultured kidney epithelial cells. They are different from amphibian vimentin and desmin and from the keratins present in the epidermis of adult frogs. Peptide mapping and immunoblotting experiments indicate that Xenopus cytokeratin component 1 is related to cytokeratin A of higher vertebrates but is different from the two smaller cytoskeletal polypeptides 2 and 3. Incorporation of [35 S]methionine shows that all three polypeptides are synthesized in both oocytes and embryos. Our observations show that maternal storage is not only restricted to proteins serving basic cellular functions but also can extend to proteins related to a specific form of cell differentiation (i.e., epithelial formation) in the early embryo. The data suggest that mechanisms of epithelial differentiation in Xenopus embryogenesis are different from those of early mammalian embryos in which no such intermediate-size-filament storage pool has been detected.
Analysis of HLA class I genes with restriction endonuclease fragments: implications for polymorphism of the human major histocompatibility complex.
Authors: Cohen D , Paul P , Font M P , Cohen O , Sayagh B , Marcadet A , Busson M , Mahouy G , Cann H M , Dausset J
Published: May 2006

Cellular DNA from HLA-typed individuals was digested with the restriction endonucleases HindIII, EcoRV, and EcoRI. The separated restriction endonuclease fragments were hybridized with a HLA class I cDNA probe by using the Southern transfer technique. Digestion of cellular DNA with HindIII generated 22 restriction endonuclease fragments, 11 of which showed polymorphism for presence or absence in a population sample. With EcoRV, 13 fragments were identified; 6 showed polymorphism. EcoRI generated 11 fragments, of which 1 was polymorphic. Of these 18 polymorphic fragments generated by the three restriction endonucleases, each of 5 was found to be positively associated with one allele of the HLA-A or -B allelic series (HLA-Aw24, -B8, -B15, -Bw35, and -B40). One fragment was positively associated with two HLA-A series alleles (HLA-A1 and -A11). Another fragment was positively associated with five HLA-B series alleles (HLA-B5, -B7, -B14, -Bw16, and -Bw35) and one fragment was positively associated with alleles at two loci (HLA-B14 and -Cw5). The serologically defined allele HLA-Aw24 was associated with two polymorphic fragments, one association showing a positive correlation and the other a negative correlation. Each informative family studied thus far has shown segregation of the restriction fragment with the associated serologically defined allele. The fragments associated with serologically defined alleles occurred in the population sample studied at low or moderate frequencies. The remaining polymorphic fragments occur at high frequency, suggesting that class I genes not serologically detected show less polymorphism than serologically defined class I genes.
The structure and nucleotide sequence of the 5' end of the human c-myc oncogene.
Authors: Watt R , Nishikura K , Sorrentino J , ar-Rushdi A , Croce C M , Rovera G
Published: May 2006

We have established the structure and nucleotide sequence of the 5' end of the human c-myc oncogene, using a cloned genomic fragment isolated from a fetal liver library (clone lambda MC41) and cloned cDNA from the human leukemic cell line K562. The human c-myc oncogene consists of three exons and two introns. Primer extension of the human c-myc mRNA of three different cell lines and S1 nuclease protection experiments served to establish the position of two transcription initiation sites. The splicing site of the first exon-intron boundary was determined by comparative analysis of the sequences of the genomic and cDNA clones. The first exon contains termination codons in all three reading frames and no translation initiation signals, confirming our previous observation that the c-myc mRNA has a long 5' noncoding sequence. This first exon also was found to be utilized in the formation of c-myc mRNAs in a variety of human cell lines.
Enhancement of specific antitumor immunity in mice fed a diet enriched in vitamin A acetate.
Authors: Malkovský M , Doré C , Hunt R , Palmer L , Chandler P , Medawar P B
Published: May 2006

Age-matched male CBA mice on a conventional or a vitamin A acetate (VAOAc)-rich diet were immunized with irradiated cloned 3-methylcholanthrene- or Harvey sarcoma virus-induced (McSa-1 or HT3-2.1) sarcoma cells and then challenged with viable corresponding or unrelated (non-crossreacting) syngeneic sarcoma cells. The survival of the specifically immunized mice on the VAOAc diet was significantly prolonged in comparison with all control groups of mice as assessed by using logrank tests. Moreover, the specific immunization markedly decreased the incidence of tumors after the McSa-1 (but not HT3-2.1) challenge in a group of mice on the VAOAc diet (5% tumor incidence) compared with the equivalent group on the control diet (50% tumor incidence). Neither the VAOAc diet nor in vivo immunization alone or combined influenced natural killer cell activity. Specific T-cell-mediated cytotoxicity after in vivo priming and in vitro boosting with sarcoma cells was increased in VAOAc-fed mice. However, the marginal increase in cytotoxicity does not in itself explain the strikingly increased resistance to tumor transplants in preimmunized mice on the VAOAc diet in comparison with preimmunized mice on the control diet. The results indicate that a diet enriched in VAOAc can modify the ability of the immune system of a mouse to respond effectively to tumor antigens and can influence whether a tumor grows or regresses.
Solid-phase synthesis of cecropin A and related peptides.
Authors: Andreu D , Merrifield R B , Steiner H , Boman H G
Published: May 2006

Cecropin A, a 37-residue antibacterial peptide amide, was synthesized by the solid-phase method. It was shown to be homogeneous and totally indistinguishable from natural cecropin A by chemical and physical criteria, as well as by its antibacterial activity against several Gram-positive and Gram-negative organisms. The synthetic material was also used to establish unambiguously that the carboxyl-terminal blocking group of natural cecropin A is a primary amide as tentatively proposed earlier. The role of the amino terminus of cecropin A in antibacterial activity was investigated by the synthesis of two analogs.