bluuebunny/crossref_metadata_2025 huggingface dataset:
Allogeneic stimulation of cytotoxic T cells by supported planar membranes.
Authors: Brian A A , McConnell H M
Published: May 2006

Phospholipid vesicles containing the transmembrane protein H-2Kk spontaneously fuse to form planar membranes when incubated on treated glass surfaces. Pattern photobleaching of fluorescent lipid probes indicates that these planar membranes are continuous and that the lipids are as mobile as they are in conventional fluid bilayers or monolayers. H-2Kk molecules in these planar membranes are immobile. These membranes stimulate cytotoxic T lymphocytes when cultured with immune spleen cells. The response to H-2Kk in planar membranes is greatly enhanced by the addition of supernatant from concanavalin A-stimulated spleen cells, indicating that relatively little antigen processing or presentation by accessory cells occurs. Cytotoxic T cells induced by purified alloantigen are found to be as susceptible to antibody blockade as are effectors from conventional mixed lymphocyte culture, where the antibody is directed against a T-cell surface antigen reputed to strengthen target cell adhesion through an interaction independent of major histocompatibility antigens.
Monoclonal antibodies against the voltage-sensitive Na+ channel from mammalian skeletal muscle.
Authors: Casadei J M , Gordon R D , Lampson L A , Schotland D L , Barchi R L
Published: May 2006

A panel of 13 monoclonal antibodies against the voltage-sensitive Na+ channel of rat skeletal muscle has been characterized. Each of these antibodies reacted with the purified Na+ channel protein in a solid-phase radioimmunoassay. Nine antibodies specifically immunoprecipitated the Na+ channel in a form that retained its characteristic high affinity for saxitoxin, and 11 recognized the channel in a crude mixture of solubilized membrane proteins separated on a Sepharose CL-6B column. Six antibodies specifically labeled skeletal muscle in immunofluorescence techniques. In each case, antibody was localized only to the surface membrane of the muscle fibers. Eleven antibodies produced detectable reaction on immunoblot transfers of sarcolemmal membrane proteins; each of these bound to a diffuse 160- to 200-kDa band that comigrated with the large glycoprotein subunit of the purified Na+ channel. Further studies were carried out with one of these antibodies, L/D3. In immunoblots of a glycoprotein fraction prepared from muscle that had been homogenized rapidly in a solution containing detergent, EGTA, and protease inhibitors, L/D3 recognized only a single 260-kDa band. Incubation of solubilized muscle proteins at 4 degrees C for 24 hr without EGTA prior to isolation of the glycoprotein fraction resulted in partial conversion of this 260-kDa component to a smaller component between 160 and 200 kDa that comigrated with the principal immunoreactive component of sarcolemma. Based on its immunoreactivity with monoclonal antibodies, the large subunit of the rat skeletal muscle Na+ channel appears to be approximately equal to 260 kDa in its native state but may be sensitive to proteolysis during the isolation of sarcolemmal membranes.
Nuclear matrix DNA from chicken erythrocytes contains beta-globin gene sequences.
Authors: Hentzen P C , Rho J H , Bekhor I
Published: May 2006

Nuclear matrices containing residual DNA were isolated from chicken erythrocytes after extraction of purified nuclei with buffered 2 M NaCl. After further purification of this residual DNA, it was found to contain high concentrations of beta-globin gene sequences as assayed by dot hybridization with 32P-labeled nick-translated pHB1001. Electron microscopy of a random sample of this residual DNA fraction shows the DNA to be intimately associated with protein at various intervals. A hypothesis for enrichment of active genes in residual DNA from purified chromatin or in nuclear matrix DNA is also discussed.
Cloning of a cDNA encoding rat intestinal fatty acid binding protein.
Authors: Alpers D H , Strauss A W , Ockner R K , Bass N M , Gordon J I
Published: May 2006

Intestinal fatty acid binding protein mRNA is one of the most abundant mRNA species in the rat small intestinal epithelium. RNA transfer blot analyses disclosed that the mRNA encoding intestinal fatty acid binding protein is approximately equal to 900 nucleotides long and not represented in liver RNA. We have identified 564 nucleotides of this mRNA, including 12 nucleotides of the 5' nontranslated region, the coding portion, and 155 nucleotides of the 3' nontranslated domain. The primary translation product encoded by this mRNA contains 132 amino acids and has a Mr of 15,062. The derived protein sequence was verified by automated sequential Edman degradation of the intact polypeptide isolated from a wheat germ cell-free system. The in vitro product is NH2-terminally acetylated, a finding that is consistent with its ultimate cytoplasmic destination. Comparison of the amino acid sequence of this protein with liver fatty acid binding protein, a polypeptide specified by the most abundant small intestinal epithelial mRNA, revealed significant homology and similarity in the predicted secondary structures of their NH2-terminal domains.
Purification of bovine bone morphogenetic protein by hydroxyapatite chromatography.
Authors: Urist M R , Huo Y K , Brownell A G , Hohl W M , Buyske J , Lietze A , Tempst P , Hunkapiller M , DeLange R J
Published: May 2006

Bovine bone morphogenetic protein (bBMP) induces differentiation of mesenchymal-type cells into cartilage and bone. bBMP has an apparent Mr of 18,500 +/- 500 and represents less than 0.001% of the wet weight of bone tissue. A Mr 34,000 protein resembling osteonectin is separated by extraction with Triton X-100. A Mr 24,000 protein and about half of a Mr 22,000 protein are disassociated from bBMP by precipitation in 1.5 M guanidine hydrochloride. Aggregates of bBMP and a Mr 14,000 protein are insoluble in aqueous media; the bBMP becomes soluble when the Mr 14,000 protein is disassociated in 6 M urea and removed from the solution by ultrafiltration. Three separate molecular species with apparent Mrs 18,500, 17,500, and 17,000 are eluted at 0.10, 0.15, and 0.20 M phosphate ion concentrations, respectively, from a hydroxy-apatite column. The Mr 18,500 protein has the amino acid composition of acidic polylpeptide and includes four half-cystine residues; the pI is 4.9-5.1. The Mr 22,000 component is a chromoprotein resembling ferritin. The NH2-terminal amino acid sequence of the Mr 17,500 protein simulates histone H2B. The Mr 17,000 protein may possess calmodulin activity. Aggregates of the Mr 18,500 and other proteins induce formation of large deposits of bone; the Mr 18,500 protein alone is rapidly absorbed and induces formation of small deposits. None of the other proteins induces bone formation.
Single-chain structure of human ceruloplasmin: the complete amino acid sequence of the whole molecule.
Authors: Takahashi N , Ortel T L , Putnam F W
Published: May 2006

We have determined the amino acid sequence of the amino-terminal 67,000-dalton (67-kDa) fragment of human ceruloplasmin and have established overlapping sequences between the 67-kDa and 50-kDa fragments and between the 50-kDa and 19-kDa fragments. The 67-kDa fragment contains 480 amino acid residues and three glucosamine oligosaccharides. These results together with our previous sequence data for the 50-kDa and 19-kDa fragments complete the amino acid sequence of human ceruloplasmin. The polypeptide chain has a total of 1,046 amino acid residues (Mr 120,085) and has attachment sites for four glucosamine oligosaccharides; together these account for the total molecular mass of human ceruloplasmin (132 kDa). The sequence analysis of the peptides overlapping the fragments showed that one additional amino acid, arginine, is present between the 67-kDa and 50-kDa fragments, and another, lysine, is between the 50-kDa and 19-kDa fragments. Only two apparent sites of amino acid interchange have been identified in the polypeptide chain. Both involve a single-point interchange of glycine and lysine that would result in a difference in charge. The results of the complete sequence analysis verified that human ceruloplasmin is composed of a single polypeptide chain and that the subunit-like fragments are produced by proteolytic cleavage during purification (and possibly also in vivo).
Von Willebrand protein binds to extracellular matrices independently of collagen.
Authors: Wagner D D , Urban-Pickering M , Marder V J
Published: May 2006

Von Willebrand protein is present in the extracellular matrix of endothelial cells where it codistributes with fibronectin and types IV and V collagen. Bacterial collagenase digestion of endothelial cells removed fibrillar collagen, but the pattern of fibronectin and of von Willebrand protein remained undisturbed. Exogenous von Willebrand protein bound to matrices of different cells, whether rich or poor in collagen. von Willebrand protein also decorated the matrix of cells grown in the presence of alpha, alpha'-dipyridyl, a collagen-secretion inhibitor. These results indicate that the presence of von Willebrand protein in the extracellular matrix of endothelial cells and the binding of von Willebrand protein to foreign matrices do not depend on the matrix collagen.
Localization of the structural gene for human apolipoprotein A-I on the long arm of human chromosome 11.
Authors: Cheung P , Kao F T , Law M L , Jones C , Puck T T , Chan L
Published: May 2006

Apolipoprotein A-I (apo A-I), the major apolipoprotein in human high density lipoproteins, is involved in the disease atherosclerosis. Cloned apo A-I cDNA (pA1-3) was used as a probe in chromosome mapping studies to detect the human apo A-I structural gene sequence in human-Chinese hamster cell hybrids. Southern blot analysis of 13 hybrids localized the gene to human chromosome 11. Confirmation of the chromosomal assignment was obtained by analysis of a hybrid (J1) containing a single human chromosome, no. 11. Regional mapping was achieved by using deletion subclones of J1 that localized the human apo A-I structural gene to the region 11q13 leads to qter. Since the human apolipoprotein C-III (apo C-III) structural gene is closely linked to apo A-I, it can be assigned to the same region on the long arm of chromosome 11. By extension of methods previously described, it now appears possible to carry out fine-structure analysis of this and related gene regions on chromosome 11 and to study the biochemical concomitants of these genes and of genes on other chromosomes for analysis of their role in atherosclerosis.
Hepatitis B surface antigen and polymerized albumin binding activity in sheep serum.
Authors: Franklin S G , Millman I , Blumberg B S
Published: May 2006

Sera from sheep and other domestic animals contain a substance that gives a strongly positive test for antibody to hepatitis B virus surface antigen by the accepted radioimmunoassay procedure. We have purified this substance from sheep serum to near homogeneity by ion-exchange, affinity, and molecular exclusion chromatography and have identified it to be an IgM. We present evidence that this sheep IgM is an antibody to polymerized sheep albumin. This antibody may arise due to infection by hepatitis B virus, hepatitis B virus-like viruses, or other pathological agents and may react with hepatitis B virus surface antigen by combining with polymerized albumin bound to the hepatitis B virus receptor for this polymer.
Immunoprecipitation of cell surface structures of cloned cytotoxic T lymphocytes by clone-specific antisera.
Authors: Kranz D M , Sherman D H , Sitkovsky M V , Pasternack M S , Eisen H N
Published: May 2006

Clones of cytotoxic T lymphocytes (CTL) differ in their specific reactivity with diverse target cell antigens. To learn about the uniqueness of individual CTL clones we injected rats and mice with cloned CTL in an effort to prepare clone-specific antisera and to analyze the CTL surface molecules that were immunoprecipitated by these antisera. Three clones were studied. They were all derived from BALB.B mice and were specific for antigens encoded by the major histocompatibility complex of the H-2d haplotype. Antisera raised in rats against individual clones contained antibodies to lymphocyte function-associated antigen type 1 (LFA-1) and inhibited the cytotoxic activity of all of the clones. In contrast, BALB/c and BALB.K mice injected with individual clones consistently yielded alloantisera that were clone specific in their ability to inhibit CTL-mediated lysis of target cells (P815). In addition, these alloantisera immunoprecipitated from extracts of 125I-radiolabeled CTL a disulfide-bonded dimer consisting of approximately equal to 45-kilodalton subunits. This dimer resembles the putative T-cell antigen-recognition receptor recently identified in several laboratories. The alloantisera also immunoprecipitated CTL surface molecules that were associated with beta 2-microglobulin and that differed in apparent molecular mass (37-38 kilodaltons) in different clones.
Multiple-step relayed correlation spectroscopy: sequential resonance assignments in oligosaccharides.
Authors: Homans S W , Dwek R A , Fernandes D L , Rademacher T W
Published: May 2006

A general property of the high-resolution proton NMR spectra of oligosaccharides is the appearance of low-field well-resolved resonances corresponding to the anomeric (H1) and H2 protons. The remaining skeletal protons resonate in the region 3-4 ppm, giving rise to an envelope of poorly resolved resonances. Assignments can be made from the H1 and H2 protons to their J-coupled neighbors (H2 and H3) within this main envelope by using 1H-1H correlated spectroscopy. However, the tight coupling (J congruent to delta) between further protons results in poor spectral dispersion with consequent assignment ambiguities. We describe here three-step two-dimensional relayed correlation spectroscopy and show how it can be used to correlate the resolved anomeric (H1) and H2 protons with remote (H4, H5) protons directly through a linear network of couplings using sequential magnetization transfer around the oligosaccharide rings. Resonance assignments are then obtained by inspection of cross-peaks that appear in well-resolved regions of the two-dimensional spectrum. This offers a general solution to the assignment problem in oligosaccharides and, importantly, these assignments will subsequently allow for the three-dimensional solution conformation to be determined by using one-dimensional and two-dimensional nuclear Overhauser experiments.
Synthesis in Escherichia coli of human adenovirus type 12 transforming proteins encoded by early region 1A 13S mRNA and 12S mRNA.
Authors: Kimelman D , Lucher L A , Brackmann K H , Symington J S , Ptashne M , Green M
Published: May 2006

Human adenovirus (Ad)-encoded early region 1A (E1A) tumor (T) antigens have been implicated in the positive regulation of viral early genes, the positive and negative regulation of some cellular genes, and cell immortalization and transformation. To further study the Ad E1A T antigens and to facilitate their purification, we have cloned cDNA copies of the Ad12 E1A 13S mRNA and 12S mRNA downstream of a hybrid Escherichia coli trp-lac (tac) promoter. Up to 8% of the protein synthesized in E. coli cells transformed by each of the two different Ad12 E1A cDNA constructs were immunoprecipitated as a Mr 47,000 protein by antibody to a synthetic peptide encoded in the Ad12 E1A DNA sequence. Both proteins produced in E. coli appear to be authentic and complete Ad12 E1A T antigens because they possess (i) the Ad12 E1A NH2-terminal amino acid sequence predicted from the DNA sequence; (ii) the Ad12 E1A COOH-terminal sequence, as shown by immunoprecipitation with anti-peptide antibody; and (iii) a molecular weight and an acidic isoelectric point similar to that of the E1A T antigens synthesized in Ad12-infected and transformed mammalian cells. The T antigens were purified to near homogeneity in yields of 100-200 micrograms per g wet weight of transformed E. coli cells.
Human alcohol dehydrogenase: structural differences between the beta and gamma subunits suggest parallel duplications in isoenzyme evolution and predominant expression of separate gene descendants in livers of different mammals.
Authors: Bühler R , Hempel J , Kaiser R , von Wartburg J P , Vallee B L , Jörnvall H
Published: May 2006

Human alcohol dehydrogenase (ADH; alcohol:NAD+ oxidoreductase, EC 1.1.1.1) occurs in multiple forms, which exhibit distinct electrophoretic mobilities and enzymatic properties. The homogeneous isoenzymes beta 1 beta 1 and gamma 1 gamma 1 were isolated from livers of Caucasians with "typical" ADH phenotype by double ternary complex affinity chromatography and ion exchange chromatography. The differences between the beta 1 and gamma 1 subunits were determined by structural analysis of all tryptic peptides from the carboxymethylated proteins. The human beta 1 and gamma 1 chains differ at 21 of the 373 positions (5.6%). Ten tryptic peptides account for the differences. All residue substitutions are compatible with one-base mutations and result in largely unaltered properties, but five lead to charge differences. Sixteen substitutions are at positions corresponding to the catalytic domain of the well-known horse enzyme; five correspond to the coenzyme-binding domain. Substitutions adjacent to important regions may correlate with differences in coenzyme binding, substrate specificities, and active-site relationships. The residue replacements between the beta 1 and gamma 1 subunits of human ADH are not identical to the known substitutions between ethanol-active (E) and steroid-active (S) subunits of horse ADH. Thus, the duplication leading to human beta 1 and gamma 1 subunits is separate and different from that leading to equine E and S subunits. Both duplications are likely to have occurred after the ancestral separation of human and equine ADH. Of the 21 residues that are different between beta 1/gamma 1, 13 in gamma 1 but only 6 in beta 1 are identical to those of the horse E chain. This suggests a closer relationship between gamma 1 and E, although beta 1 in man and E in the horse are the subunits recovered in highest yield from liver ADH preparations. Consequently, in these two mammalian species, relative activities of genes for an isoenzyme family appear to be different.
Quantitative assay for albumin-producing liver cells after simian virus 40 transformation of rat hepatocytes maintained in chemically defined medium.
Authors: Isom H C , Georgoff I
Published: May 2006

Transformation of rat hepatocytes by simian virus 40 in chemically defined medium was examined. When hepatocytes plated on collagen-coated plates were infected with simian virus 40, colonies of replicating cells appeared as early as 40 days after infection, whereas no colonies appeared in control cultures. Medium from 85% of the transformed cultures contained albumin. When collagen was eliminated and hepatocytes were plated on Primaria plastic cell culture dishes, transformation occurred; medium from 86% of the transformed cultures contained albumin but the maximum albumin level secreted per culture was only 62% of that produced by cultures on collagen-coated plates. Quantitative assays for transformation were established. Transformation was linear after infection with 2-50 plaque-forming units of virus per hepatocyte, and the transformation frequency was the same on the two plating surfaces. An immuno-overlay technique made it possible to identify, purify, and determine the morphology of the albumin-producing cells. When ornithine was substituted for arginine in the medium, the transformation frequency decreased markedly while the percentage of colonies producing albumin increased from 30% to 100%. We conclude that we have defined an assay for quantifying transformation of a normal hepatocyte population and for identifying and enumerating epithelial liver cell transformants that produce albumin.
Amino acid sequence and post-translational modification of human interleukin 2.
Authors: Robb R J , Kutny R M , Panico M , Morris H R , Chowdhry V
Published: May 2006

Human interleukin 2 was separated into multiple molecular forms by selective immunoaffinity chromatography and chromatofocusing. For the most part, this heterogeneity was attributed to variations in glycosylation of the threonine residue in position 3 of the polypeptide chain. The various molecular forms of interleukin 2 had nearly identical specific activities in the in vitro proliferation assay, indicating that the glycosylation had no significant effect on this response. The entire primary sequence of interleukin 2, including the location of the intramolecular disulfide bridge, was determined by a combination of peptide mapping and protein sequencing. This information should aid in the determination of the active site(s) of the molecule.
Cross-reacting material in Gaucher disease fibroblasts.
Authors: Beutler E , Kuhl W , Sorge J
Published: May 2006

Glucocerebrosidase is the enzyme that is deficient in Gaucher diseases. Four monoclonal antibodies reacting with at least two different epitopes of this enzyme have been produced. The amounts of glucocerebrosidase in fibroblasts of patients with all three types of Gaucher disease were investigated by radioiodinating two of the antibodies and measuring their binding to fibroblast extracts immobilized on nitrocellulose filters. The amount of glucocerebrosidase antigen was decreased in all cases of Gaucher disease, particularly in the fibroblasts of patients with the more severe neuronopathic forms of the disorder, types II and III. The catalytic activity was reduced to a greater extent than the amount of antigen in all cases, so that the specific activity of the residual enzyme was found to be diminished. Although measurements in individual cases were quite reproducible and the amount of antigen detected by monoclonal antibodies reacting with different epitopes was quite similar, there was considerable variation between patients. This finding is consistent with the apparent within-type genetic heterogeneity of Gaucher disease, even within the Ashkenazi Jewish population in which it is most prevalent.
Theory of the intermediate rippled phase of phospholipid bilayers.
Authors: Marder M , Frisch H L , Langer J S , McConnell H M
Published: May 2006

A mathematical model of the periodic, rippled P beta' phase of phosphatidylcholine is described. In this model the P beta' phase shows a periodic variation in membrane properties corresponding to a lateral periodic variation between fluid-like and crystal-like states. The model is based on a curvature-dependent Landau-de Gennes free-energy functional. Our proposed form of the curvature-dependent free energy remedies a subtle mathematical flaw in an earlier model having similar physical content [Falkovitz, M. F., Seul, M., Frisch, H. L. & McConnell, H. M. (1982) Proc. Natl. Acad. Sci. USA 79, 3918-3921].
F-actin is intermolecularly crosslinked by N,N'-p-phenylenedimaleimide through lysine-191 and cysteine-374.
Authors: Elzinga M , Phelan J J
Published: May 2006

The bifunctional reagent N,N'-p-phenylenedimaleimide (PDM) is being used in an attempt to measure distances between specific side chains in adjacent monomers within F-actin. [14C]PDM was synthesized and was used to crosslink F-actin. Uncrosslinked actin was removed by gel filtration, and, from an arginine-specific tryptic digest of the covalently crosslinked dimers and higher oligomers, one radioactive crosslinked peptide was obtained in high yield. Amino acid composition and sequence analysis indicated that it comprises residues 184-196 of one monomer and 373-375 of an adjacent actin molecule, bridged by PDM through Cys-374 and Lys-191. Thus, these groups are shown to be 1.2-1.4 nm apart in adjacent actin monomers in F-actin. This information may be crucial in establishing the orientation of actin monomers within F-actin.
Amino acid sequence of bovine heart coupling factor 6.
Authors: Fang J K , Jacobs J W , Kanner B I , Racker E , Bradshaw R A
Published: May 2006

The amino acid sequence of bovine heart mitochondrial coupling factor 6 (F6) has been determined by automated Edman degradation of the whole protein and derived peptides. Preparations based on heat precipitation and ethanol extraction showed allotypic variation at three positions while material further purified by HPLC yielded only one sequence that also differed by a Phe-Thr replacement at residue 62. The mature protein contains 76 amino acids with a calculated molecular weight of 9006 and a pI of approximately equal to 5, in good agreement with experimentally measured values. The charged amino acids are mainly clustered at the termini and in one section in the middle; these three polar segments are separated by two segments relatively rich in nonpolar residues. Chou-Fasman analysis suggests three stretches of alpha-helix coinciding (or within) the high-charge-density sequences with a single beta-turn at the first polar-nonpolar junction. Comparison of the F6 sequence with those of other proteins did not reveal any homologous structures.
Transcriptional induction of two genes in human cells by beta interferon.
Authors: Larner A C , Jonak G , Cheng Y S , Korant B , Knight E , Darnell J E
Published: May 2006

The binding of interferons to distinct cell surface receptors leads to the induction of synthesis of several unique polypeptides and their corresponding mRNAs (1-6). We have isolated two cDNAs that are complementary to nuclear RNA whose synthesis is induced from undetectable levels to maximal rates of transcription within 30-60 min after the addition of beta interferon to human fibroblasts or to HeLa cells. These results prove that a single polypeptide can, by binding to a specific plasma membrane receptor, promptly activate the transcription of a defined set of genes.
Movement of scallop myosin on Nitella actin filaments: regulation by calcium.
Authors: Vale R D , Szent-Gyorgyi A G , Sheetz M P
Published: May 2006

In order to determine if Ca2+ regulates scallop myosin movement on actin, we have measured motility of scallop myosin along actin filaments using a direct visual assay. This procedure consists of covalently linking myosin to 1-micron beads and pipetting them onto a parallel array of actin filaments located on the cytoplasmic face of a Nitella internodal cell. In the absence of Ca2+, scallop myosin-coated beads exhibit no directed motion; however, in the presence of pCa2+ of greater than 5.84, these beads undergo linear translocations with average velocities of 2.0 micron/s. This Ca2+ -sensitive motility requires the presence of regulatory light chains on the scallop myosin. Removal of regulatory light chains with 10 mM EDTA produces a "desensitized" myosin, no longer sensitive to Ca2+, which moves at rates of 0.09-0.3 micron in the presence or absence of Ca2+. Readdition of regulatory light chains to preparations of desensitized myosin once again confers Ca2+-sensitive motility. The Ca2+ dependence of scallop-myosin motility shows a sharp transition, consistent with the Ca2+ activation sensitivity of the actin-activated ATPase. Furthermore, relative rates of movement of calcium-regulated myosins from various molluscan species are consistent with their respective rates of ATP hydrolysis. Thus, myosin motility along actin filaments provides a sensitive and direct assay of myosin activity and is suitable for studying myosin regulation.
Characterization and regulation in the expression of a gene coding for the intermediate filament protein desmin.
Authors: Capetanaki Y G , Ngai J , Lazarides E
Published: May 2006

Using synthetic oligonucleotide probes, we have isolated chicken cDNA clones for the intermediate filament protein desmin. We show that the gene for this protein probably exists as a single copy in the haploid chicken genome and is transcribed into one mature mRNA species of approximately equal to 2.4 kilobases. Expression of this mRNA is tissue-specific, as it is present in high abundance in smooth and skeletal muscle but is absent from erythrocytes, spinal cord, and lens cells. A 10- to 20-fold increase in desmin mRNA is observed in myogenic cells upon fusion, which suggests that the level of expression of the desmin gene and the accumulation of desmin filaments during muscle differentiation is regulated at the transcriptional and/or posttranscriptional level but not at the translational level. Hybridization studies and nucleotide sequence comparison of the cDNAs specific for desmin and for other intermediate filament subunits reveal a region that is highly conserved (80%) among different members of the intermediate filament protein gene superfamily, with the exception of the chicken vimentin gene; this gene appears to be less homologous to the genes for the other chicken intermediate filament subunits than the mammalian vimentin gene is to the genes for other mammalian intermediate filament proteins and to the chicken desmin gene.
Two regulatory elements for immunoglobulin kappa light chain gene expression.
Authors: Bergman Y , Rice D , Grosschedl R , Baltimore D
Published: May 2006

By using internal deletions within a rearranged immunoglobulin kappa light chain gene, the presence of an intron regulatory sequence (enhancer) has been confirmed. Its presence is required for high-level transcription from a plasmid after transfection into myeloma cells. Transfection efficiency was monitored by the activity of a deleted H4 histone gene included in the plasmid. The intron element could be moved upstream of the gene in both orientations, fulfilling the definition of an enhancer. By using 5' deletions, a second regulatory element was located upstream of the "TATA" box, between positions -69 and -104. These two elements both are required for efficient kappa chain gene expression.
Order of free energy couplings between ligand binding and protein subunit association in hemoglobin.
Authors: Weber G
Published: May 2006

The number of protein subunits that must be liganded to effect changes in subunit interaction may be characterized by defining an order for the free energy couplings between these two processes. From available data on the chemical equilibrium of stripped hemoglobin A with oxygen, I show that couplings are unequivocally of first order. The two-state model of cooperative binding is shown to be incompatible with the results of this analysis, as the Monod-Wyman-Changeux parameters derived from the same experimental data demand free energy couplings of an order higher than the second.
Activation and fusion induced by 1 alpha,25-dihydroxyvitamin D3 and their relation in alveolar macrophages.
Authors: Abe E , Shiina Y , Miyaura C , Tanaka H , Hayashi T , Kanegasaki S , Saito M , Nishii Y , DeLuca H F , Suda T
Published: May 2006

1 alpha,25-Dihydroxyvitamin D3 [1 alpha,25-(OH)2D3] induces fusion of murine alveolar macrophages. This effect was observed in growth medium containing 5% human serum but not in the medium with 5% fetal bovine serum. Unlike 1 alpha,25-(OH)2D3, bacterial lipopolysaccharides (LPS) did not induce fusion of alveolar macrophages. However, both 1 alpha,25-(OH)2D3 and LPS activated alveolar macrophages, as measured by glucose consumption, increase in Fc receptors, and induction of cytotoxicity. The number of Fc receptors on the surface of multinucleated giant cells induced by 1 alpha,25-(OH)2D3 was much smaller than that on the surface of mononuclear macrophages treated with the hormone. These results indicate that 1 alpha,25-(OH)2D3 induces both fusion and activation of alveolar macrophages, whereas LPS elicits activation only.
Gene transfer in lymphoid cells: expression of the Thy-1.2 antigen by Thy-1.1 BW5147 lymphoma cells transfected with unfractionated cellular DNA.
Authors: Berman J W , Basch R S , Pellicer A
Published: May 2006

We have transferred the gene coding for the Thy-1.2 alloantigen into a Thy-1.1-bearing T-cell lymphoma. BALB/c thymocyte DNA, precipitated with calcium phosphate, was used to effect the transfer. We report the stable transformation of lymphoid cells by total cellular DNA. To our knowledge, this has not been previously reported. Transient expression of the transfected gene could be detected by flow cytofluorometry, and 5% (range, 1.5%-11%) of the recipient cells had Thy-1.2 antigen detectable on their surface 48 hr after transfer. "Stable" transformants were isolated by repeatedly selecting for cells expressing the Thy-1.2 antigen, by use of fluorescence-activated cell sorting or "panning." No metabolic selection was required. The transferred gene, detected by Southern blotting, encoded a product that is indistinguishable from the normal antigen by immunoprecipitation and NaDod-SO4/PAGE.
Evidence for inclusion of regions of nonhomology in heteroduplex products of bacteriophage lambda recombination.
Authors: Lichten M , Fox M S
Published: May 2006

Total intracellular DNA was isolated from replication-restricted bacteriophage lambda crosses in which the infecting parents were heteroallelic for wild-type and deletion mutant alleles. This DNA was examined for the presence of heteroduplex DNA molecules that contained wild-type sequences in one strand and deletion-mutant sequences in the other. Molecules hybrid for a 689-nucleotide deletion in the immunity region of lambda were detected at significant levels only in crosses in which both the red recombination system of lambda and the rec recombination system of Escherichia coli were active. Molecules hybrid for a 1300-nucleotide deletion in the central portion of the lambda genome were detected at significant levels in DNA isolated from both red+ and red- crosses in which recA function was present.
A placental polypeptide activator of a membranous protein kinase and its relation to histone 1.
Authors: Abdel-Ghany M , Riegler C , Racker E
Published: May 2006

Crude transforming growth factor preparations of placenta contain a polypeptide that is required for the activity of a protein kinase that has been purified from plasma membrane preparations of Ehrlich ascites tumor cells. The kinase activator has been separated from transforming growth factor beta by reversed-phase HPLC and affinity chromatography. Like the transforming growth factor, it is heat stable and trypsin labile, but it is not inactivated by dithiothreitol. In sodium dodecyl sulfate/polyacrylamide gel electrophoresis the purified preparation shows a major double band at about 31,000 daltons. Comparisons of electrophoretic mobility, protein kinase stimulatory activity, and cross-reactivity with an antibody against histone 1 suggest that the placental activator is identical with histone 1.
In vitro translocation of bacterial proteins across the plasma membrane of Escherichia coli.
Authors: Müller M , Blobel G
Published: May 2006

Precursors to two periplasmic proteins and one outer membrane protein were synthesized in a membrane-free extract from Escherichia coli programmed with plasmid DNA. In the presence of inverted plasma membrane vesicles from E. coli up to 25% of the precursor molecules were converted into their mature forms. Using externally added proteinase K as a probe, we found the processed proteins segregated within the membrane vesicles. By the same criteria, a small amount of each precursor also proved to be translocated, indicating that translocation and signal sequence cleavage are not necessarily coupled processes. Furthermore, we present conclusive evidence that the translocation step can occur post-translationally even as late as 60 min after the beginning of translation.
Induction of a Ca2+, Mg2+-dependent endonuclease activity during the early stages of murine erythroleukemic cell differentiation.
Authors: McMahon G , Alsina J L , Levy S B
Published: May 2006

A Ca2+, Mg2+-dependent endonuclease activity was detected in erythroleukemic cells undergoing differentiation in vitro in response to induction by dimethyl sulfoxide (Me2SO) or hexamethylene-bis-acetamide (HMBA). The endonuclease activity was demonstrated in isolated nuclei within 6 hr after the addition of inducer, reached maximum levels between 24 and 48 hr, and returned to control levels within 72 hr. The activity caused single strand breaks in high molecular weight native DNA, which could be labeled at exposed 3'-OH termini with Escherichia coli DNA polymerase I and radiolabeled nucleotides. Alkaline elution studies revealed DNA fragmentation that appeared coincident with the presence of the endonuclease activity. The detection and levels of single strand DNA breakage correlated with induction of terminal differentiation by Me2SO or HMBA. Induction of the endonuclease activity was reversible: depletion of Me2SO from the growth medium after treatment for 6 and 18 hr led to a rapid decrease in the level of activity. Removal of the inducer prevented terminal differentiation, a finding that strongly suggests the endonuclease activity is present during the precommitment phase of differentiation. DNA fragmentation was not observed in cells incubated with hemin, which has been shown previously to increase the cytoplasmic level of globin mRNA without causing commitment to terminal maturation. Me2SO did not induce the endonuclease activity or DNA fragmentation in an uninducible Friend cell line.
Infection of human endothelial cells by human T-cell leukemia virus type I.
Authors: Hoxie J A , Matthews D M , Cines D B
Published: May 2006

The effects of the human T-cell leukemia virus type I (HTLV-I) on cultured human endothelial cells were evaluated. Coculture of endothelial monolayers with either irradiated HTLV-producing lymphocytes or cell-free virus resulted in the production of multinucleated syncytia. The development of syncytia was inhibited by sera from patients with adult T-cell leukemia/lymphoma (ATLL). HTLV antigens were present on endothelial syncytia passaged in culture for greater than 3 months as detected by an anti-p19 monoclonal antibody, which detects a core protein of HTLV-I, and by ATLL sera. Moreover, these HTLV-infected endothelial cells were then able to infect and transform normal cord blood T lymphocytes with HTLV. These studies demonstrate that human endothelial cells are susceptible to productive HTLV-I infection in vitro and may have relevance for the spectrum of human disease associated with this family of retroviruses.
Essential site for growth rate-dependent regulation within the Escherichia coli gnd structural gene.
Authors: Baker H V , Wolf R E
Published: May 2006

Expression of gnd of Escherichia coli, which encodes the hexose monophosphate shunt enzyme, 6-phosphogluconate dehydrogenase (6PGD; EC 1.1.1.44), is subject to growth rate-dependent regulation: the level of the enzyme is directly proportional to growth rate under a variety of growth conditions. Previous results obtained with strains carrying transcriptional fusions of gnd to the structural genes of the lactose operon suggested that the growth rate-dependent regulation of gnd expression is at the post-transcriptional level. To characterize the regulation further, we prepared with phage MudII a set of eight independent gnd-lac gene (protein) fusions. We showed through genetic analysis and DNA sequencing that each fusion joint was located within the 6PGD-coding sequence between the first and second base pair of a codon, the reading frame required for production of a hybrid 6PGD-beta-galactosidase. Strains harboring the gnd-lac fusion plasmids produced proteins whose mobility in a NaDodSO4/polyacrylamide gel agreed with the molecular weights predicted from the DNA sequence for the respective hybrid proteins. The level of beta-galactosidase was high and relatively growth rate-independent in the fusion whose fusion joint was at codon 48. The level of beta-galactosidase in the other seven fusion strains whose fusion joints were located further downstream in the 6PGD-coding sequence showed the same dependence on growth rate as 6PGD in a normal strain. beta-Galactosidase levels were not affected by the presence of a gnd+ gene in trans to any of the fusions. The results suggest that all sites necessary for growth rate-dependent regulation of 6PGD level lie in gnd upstream from codon 118 and that an essential site of negative control lies within the coding sequence, between codons 48 and 118.
Kinetic circular dichroism shows that the S-peptide alpha-helix of ribonuclease S unfolds fast and refolds slowly.
Authors: Labhardt A M
Published: May 2006

It is shown that circular dichroism (CD) can distinguish between the S-peptide and the S-protein fragments of RNase S at 225 nm and 235 nm. The conformational source for the strong CD at 225 nm is the S-peptide alpha-helix. The structural assignment of the CD at 235 nm is not clear but it is shown to be largely due to the S-protein moiety. This situation is utilized to monitor the kinetics of pH-induced unfolding and refolding of the two moieties. It is observed that major changes occur both in the fast and slow phases of unfolding as well as refolding. Specifically, the S-peptide alpha-helix unzippering is a fast reaction, followed by slow kinetics only at 235 nm. These latter kinetics parallel the appearance of the slow-folding species commonly attributed to the accumulation of non-native proline isomers. In refolding, a large fraction of the CD of S-protein at 235 nm recovers rapidly. The S-peptide alpha-helix zippers up last. These results are unexpected and their implications for the folding mechanism of ribonuclease are discussed.
The 64-kilodalton membrane protein of Bacillus subtilis is also present as a multiprotein complex on membrane-free ribosomes.
Authors: Caulfield M P , Horiuchi S , Tai P C , Davis B D
Published: May 2006

The 64-kDa membrane protein of Bacillus subtilis is evidently involved in the attachment of secreting ribosomes to membrane. On immunoprecipitation with antibody to this protein, the solubilized particulate fraction, with or without prior chemical cross-linking, yields a complex of four proteins (64, 60, 41, and 36 kDa). This "S complex" was found to be associated with membrane-free ribosomes rather than with membrane, but the 64-kDa protein is also present, without the other proteins of the S complex, in the membrane-ribosome fraction and in the cytosol. Only the form present in the membrane-ribosome fraction is protected from protease. These findings suggest a cycle in which the complex participates in initiation of secretion but not in the later stages. It is not yet clear whether the 64-kDa protein found in the membrane-ribosome complexes is retained from the S complex after initiation and later recycled via the cytosol or whether it is a separate pool.
Identification of a higher molecular weight DNA polymerase alpha catalytic polypeptide in monkey cells by monoclonal antibody.
Authors: Karawya E , Swack J , Albert W , Fedorko J , Minna J D , Wilson S H
Published: May 2006

A monoclonal antibody against purified calf DNA polymerase alpha (deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) was used to immunoprecipitate proteins from a crude soluble extract of growing monkey BSC-1 cells. Immunoprecipitates contained familiar DNA polymerase alpha catalytic polypeptides of Mrs approximately equal to 115,000 and 70,000 and also a Mr 40,000 catalytic polypeptide; the major component in the immunoprecipitates, however, was a polypeptide of Mr approximately equal to 190,000 not previously identified as a DNA polymerase. This protein was capable of DNA polymerase activity after electroelution from NaDodSO4/polyacrylamide gels and renaturation. The highly purified enzyme so obtained was active with poly(dT).oligo(rA) as template.primer, resistant to dideoxy TTP (ddTTP), and inhibited by aphidicolin and butylphenyldeoxyguanosine 5'-triphosphate, thus identifying it as a DNA polymerase alpha. The results indicate that a polypeptide of Mr approximately equal to 190,000 is an abundant component among DNA polymerase alpha catalytic polypeptides in growing monkey cells.
15N electron nuclear double resonance of the primary donor cation radical P+.865 in reaction centers of Rhodopseudomonas sphaeroides: additional evidence for the dimer model.
Authors: Lubitz W , Isaacson R A , Abresch E C , Feher G
Published: May 2006

Four 15N hyperfine coupling constants, including signs, have been measured by electron nuclear double resonance (ENDOR) and electron nuclear nuclear triple resonance (TRIPLE) for the bacteriochlorophyll a radical cation, BChla+., in vitro and for the light-induced primary donor radical cation, P+.865, in reaction centers of Rhodopseudomonas sphaeroides R-26. A comparison of the data shows that the hyperfine coupling constants have the same sign in both radicals and are, on the average, smaller by a factor of 2 in P+.865. These results provide additional evidence that P+.865 is a bacteriochlorophyll dimer and are in contradiction with the monomer structure of P+.865 recently proposed by O'Malley and Babcock. The reduction factors of the individual 15N couplings, together with the evidence from proton ENDOR data and molecular orbital calculations, indicate a dimer structure in which only two rings (either I and I or III and III) of the bacteriochlorophyll macrocycles overlap.
Necrosin: purification and properties of a cytotoxin derived from a murine macrophage-like cell line.
Authors: Kull F C , Cuatrecasas P
Published: May 2006

An acidic proteinaceous cytotoxin was isolated from the serum-free, cell-free supernatants of J774.1 (a murine macrophage-like line) cells that had been treated with bacterial endotoxin. Cytotoxic activity was routinely monitored using the sensitive murine tumorigenic fibroblast line L-M. The toxin was purified 8000-fold by ion-exchange and electrophoretic procedures. It was purified to greater than 90% homogeneity, as assessed by photometric scanning of silver-stained NaDodSO4/polyacrylamide gels. The specific activity of the purified toxin was 32,000 units/microgram. The toxin was composed of self-aggregating non-sulfhydryl-linked multimers of a Mr 15,000 subunit. The pI of the monomer was 4.6. The active multimeric forms, as assessed by gel filtration and assayed using L-M cells, were of Mr 70,000 and 55,000. These forms were identical to those observed both in crude supernatants and in purified fractions that had not been subjected to denaturing agents. We call these forms "holotoxins" and conclude that they are aggregates of the Mr 15,000 protein. The purified toxin (1-1000 pM, 0.06-60 ng/ml) was active against a random assortment of tumorigenic and normal cell lines of murine, bovine, and human origin. For example, the diploid bovine endothelial line CPAE was nearly as sensitive as the L-M line. Similarities to other toxic macrophage products, lymphotoxin, and tumor necrosis factor are discussed.
gamma-Aminobutyric acid acts at axon terminals of turtle photoreceptors: difference in sensitivity among cell types.
Authors: Tachibana M , Kaneko A
Published: May 2006

It has been proposed that horizontal cells of the vertebrate retina have a negative feedback synapse with cone photoreceptors. gamma-Aminobutyric acid (GABA) has been suggested to be a neurotransmitter of monophasic horizontal cells (a subtype of horizontal cells), which have direct connections with red-sensitive and green-sensitive cones. We have examined the feedback hypothesis by measuring the GABA sensitivity of photoreceptors. To eliminate interaction with other cells, we dissociated photoreceptors from the turtle retina enzymatically. The subtype of photoreceptors was identified unequivocally on the bases of the shape of the cell and the color of the oil droplets, which are known to correlate with the spectral sensitivity. Cells were voltage-clamped using "Giga-ohm sealed" suction pipettes in the whole-cell recording configuration, and membrane currents were measured in response to GABA applied ionophoretically at various positions on the cell. It was found that red-sensitive and green-sensitive cones were highly sensitive to GABA and that the sensitivity was localized at the axon terminals. GABA-sensitivity in blue-sensitive cones and in rods was very low. GABA-induced current reversed its polarity near the equilibrium potential of chloride, suggesting that GABA increased chloride conductance. Thus, our findings are consistent with the negative feedback hypothesis.