bluuebunny/crossref_metadata_2025 huggingface dataset:
Search for mutations affecting protein structure in children of atomic bomb survivors: preliminary report.
Authors: Neel J V , Satoh C , Hamilton H B , Otake M , Goriki K , Kageoka T , Fujita M , Neriishi S , Asakawa J
Published: May 2006

A total of 289,868 locus tests, based on 28 different protein phenotypes and using one-dimensional electrophoresis to detect variant proteins, has yielded one probable mutation in the offspring of "proximally exposed" parents, who received an estimated average gonadal exposure of 31 to 39 rem in the atomic bombings of Hiroshima and Nagasaki. There were no mutations in 208,196 locus tests involving children of "distally exposed" parents, who had essentially no radiation exposure.
HLA antigen-related restriction of T lymphocyte cytotoxicity to Epstein-Barr virus.
Authors: Misko I S , Moss D J , Pope J H
Published: May 2006

The specificity of cytotoxic T cells generated in Epstein-Barr virus (EBV)-infected lymphocyte cultures was investigated, using a 51Cr release assay. Potent cytotoxic T cells with preferred specificity directed to antigens expressed on autologous lymphoblastoid cell line (LCL) target cells were present in 14-day cultures of lymphocytes from EBV-seropositive donors and not from seronegative donors. Moreover, the cytotoxic patterns obtained with a panel of HLA antigen-related and unrelated LCL target cells, supported by unlabeled target inhibition tests, strongly indicate that T cell cytotoxicity to EBV is restricted to HLA antigens.
Processing of human beta-globin mRNA precursor to mRNA is defective in three patients with beta+-thalassemia.
Authors: Maquat L E , Kinniburgh A J , Beach L R , Honig G R , Lazerson J , Ershler W B , Ross J
Published: May 2006

Nucleated bone marrow cells from normal individuals and from three patients with homozygous beta+-thalassemia were pulse-labeled with tritiated nucleosides. The processing of the newly synthesized globin mRNA precursors was monitored by inhibiting additional transcription with actinomycin D for 30 min. Human beta-globin mRNA is derived from its precursor via a series of reactions that generate processing intermediates. In nonthalassemic cells the precursor is processed efficiently to mature mRNA during the chase. In contrast, in beta+-thalassemic cells the processing of beta-globin RNA is defective. In one patient the beta-globin mRNA precursor turns over during the chase, but some of the intermediate RNAs accumulate and are not processed to mRNA. In two other patients a large fraction of the precursor and intermediate RNAs is not processed to mRNA. The alpha-globin mRNA precursor and intermediates are processed efficiently to mRNA-sized molecules in thalassemic and normal cells. The reduction in the rate of beta-globin but not alpha-globin RNA processing accounts for the alpha/beta globin mRNA imbalance in thalassemic erythroid cells. We discuss the possibility that the genetic lesions in beta+-thalassemia are at splicing signal sites within intervening sequences of the beta-globin gene.
Isoprotein specificity in the hepatic uptake of apolipoprotein E and the pathogenesis of familial dysbetalipoproteinemia.
Authors: Havel R J , Chao Y , Windler E E , Kotite L , Guo L S
Published: May 2006

The uptake and catabolism of lamellar complexes of rat 125I-labeled apolipoprotein (apo) E with egg lecithin were increased severalfold in perfused livers of rats given large amounts of 17 alpha-ethynylestradiol for 5 days. Estrogen-stimulated uptake of lamellar complexes of human apo E that contained all of the major normally occurring "isoforms" of the protein (apo E-1, E-2, E-3, and E-4) was comparable to that of rat apo E. By contrast, uptake of complexes containing apo E from individuals with familial dysbetalipoproteinemia (dys beta LP; characterized by a lack of apo E-3 and E-4) was stimualted to a much smaller extent. With complexes containing individual isoforms of human apo E, it was shown that estrogen-stimulated hepatic uptake was largely confined to apo E-3 and E-4; uptake of apo E-2 from normolipoproteinemic or dys beta LP individuals, or of apo E-1 from the latter, was stimulated little or not at all. When considered in the light of available information about the hepatic uptake of lipoproteins and the metabolic defect in familial dys beta LP, these results are consistent with the following hypothesis: (i) apo E comprises an essential component of the site for normal recognition of remnants of triglyceride-rich lipoproteins by a specific hepatic receptor; (ii) accumulation of remnant lipoproteins in familial dys beta LP is caused by lack of apo E-3 and E-4, which are the forms of this protein that are primarily recognized by the receptor; and (iii) the normal conversion of remnants of very low density lipoproteins to low density lipoproteins is mediated by the hepatic remnant receptor.
Separation of random fragments of DNA according to properties of their sequences.
Authors: Fischer S G , Lerman L S
Published: May 2006

The separation of DNA fragments by electrophoresis at high temperature in a denaturing gradient is independent of the length of the fragments. We have suggested that the basis of fragment separation is that each DNA molecule undergoes partial melting as it encounters a concentration of denaturants sufficient to melt its least stable sequence, while other sequences remain double stranded; in the partially melted configuration, DNA can continue migration only slowly. This model is consistent with the observation that fragments of lambda phage DNA cleaved by different restriction endonucleases reach the same final depth in the gel if they contain the same least-stable sequence. A unique set of bands is produced from the electrophoresis of randomly fragmented DNA; this would be expected if there were a limited number of melting centers occupying discrete genetic loci. An intact DNA molecule penetrates about as deeply into the gel as the uppermost band after fragmentation; this would be expected only if the least-stable sequence controls the final depth of the whole molecule.
Isolation of a heparan sulfate-containing proteoglycan from basement membrane.
Authors: Hassell J R , Robey P G , Barrach H J , Wilczek J , Rennard S I , Martin G R
Published: May 2006

We have isolated a unique, basement membrane proteoglycan from the Engelbreth-Holm-Swarm (EHS) sarcoma. This proteoglycan, estimated to be 0.75 X 10(6) daltons, was found to contain about equal amounts of protein and covalently linked heparan sulfate. Antibody prepared against this proteoglycan reacts with the basement membrane matrix in the tumor and with the basement membranes in skin, kidney, and cornea. These studies indicate that the heparan sulfate proteoglycan is a normal constituent of basement membranes that presumably plays an important role in the organization of basement membrane components and that also may determine the permeability of basement membranes to acidic molecules.
Association of microtubules and intermediate filaments in chicken gizzard cells as detected by double immunofluorescence.
Authors: Geiger B , Singer S J
Published: May 2006

By double indirect immunofluorescence, using guinea pig and rabbit antibodies to tubulin and to desmin, we have simultaneously labeled microtubules and intermediate filaments in cultured chicken embryo gizzard cells. At the resolution of the light microscope there was extensive but not complete superposition of the labeling patterns for the two filamentous structures within cells in interphase and an essentially complete dissociation of the two labeling patterns in cells in mitosis. These results indicate that there is an extensive association of microtubules and intermediate filaments in these interphase muscle cells and suggest that this association is regulated metabolically.
Composition of native and reconstituted chromatin particles: direct mass determination by scanning transmission electron microscopy.
Authors: Woodcock C L , Frado L L , Wall J S
Published: May 2006

Chromatin particles reconstituted from 145-base-pair lengths of DNA and either the arginine-rich histones H3 and H4 only or all four nucleosomal core histones have been compared with native nucleosomes in terms of their ultrastructure and mass distribution, as determined by scanning transmission electron microscopy (STEM). The mass of the nucleosome derived from STEM analysis was very close to that calculated for its DNA and histone components. The reconstituted particles showed a broader mass distribution, but it was clear that the majority contained at least eight histone molecules. This was to be expected for structures reconstituted from all four core histones, but in the case of H3H4-DNA complexes clearly showed that an octamer rather than tetramer of these histones was required to fold nucleosomal DNA into a stable compact particle. The significance of the H3H4 octamer complex with respect to nucleosomal structure is discussed, and the evidence that nucleosomal DNA can accept even greater numbers of histones is considered.
Genetic recombination of bacterial plasmid DNA: electron microscopic analysis of in vitro intramolecular recombination.
Authors: Kolodner R
Published: May 2006

a tetramer of pMB9 DNA containing a single EcoRI site per tetramer was used to investigate intramolecular recombination in Escherichia coli. When transformed into wild-type E. coli strains, the tetramer was converted into dimers and a small proportion of trimers and monomers. The conversion was blocked in recA strains and rec B recC recF strains but not in recB recC strains or recF strains. Extracts of E. coli converted the tetramer into dimers, trimers, and monomers. Figure of 8 molecules and catenanes were minor products. The proportion of recombinant molecules ranged from 7% to 14%. Intramolecular recombination in vitro was blocked in extracts of recA strains and recB recC recF strains but not significantly blocked in extracts of recB recC strains and recF strains. recA protein restored activity to recA extracts; activity in recB recC recF extracts was restored by purified exonuclease V (recBC nuclease) or a recF protein donor extract. Novobiocin and oxolinic acid inhibited the reaction by 70-80%.
Mouse peritoneal macrophages release leukotriene C in response to a phagocytic stimulus.
Authors: Rouzer C A , Scott W A , Cohn Z A , Blackburn P , Manning J M
Published: May 2006

Mouse peritoneal macrophages that had ingested zymosan particles released a polar metabolite of arachidonic acid possessing slow-reacting substance activity in the guinea pig ileum assay. The metabolite was purified by solvent extraction, Sephadex G-25 column chromatography. The purified metabolite absorbed light at 280 nm and contained a free amino group. When macrophages were preincubated overnight with [3H]arachidonic acid, [35H]cysteine, or [14C]glutamic acid, each radiolabel was incorporated into the compound. Direct amino acid analysis revealed glycine, glutamic acid, and cysteine at molar ratios of 0.97:1.00:0.82. The above data were consistent with the structure of leukotriene C, an adduct of arachidonic acid and glutaathione. Quantification of the leukotriene C based on incorporation of [3H]arachidonic acid or amino acid analysis indicated that 6 X 10(7) macrophages (3.6 mg of cell protein) released 7.5 nmol after a maximal phagocytic stimulus. The purified leukotriene C had a slow reacting substance activity of 11,500 units/nmol (1 unit has the activity of 5 ng of histamine in a guinea pig ileum contraction assay).
Enzymatic reduction of alloxan by thioredoxin and NADPH-thioredoxin reductase.
Authors: Holmgren A , Lyckeborg C
Published: May 2006

Alloxan reacts with certain sulfhydryl groups either by chemical modification or reduction to dialuric acid. The effects of the drug on NADPH-thioredoxin oxidoreductase, EC 1.6.4.5] and thioredoxin-(SH)2, a ubiquitous thiol-dependent disulfide reductase system, are described. Alloxan was a direct substrate for a nearly homogenous preparation of calf thymus NADPH-thioredoxin reductase with an apparent Km of 330 microM and a Kcat of 1000 min-1 at pH 7.0 and 25 degrees C. Alloxan was not a substrate for the corresponding Escherichia coli NADPH-thioredoxin reductase. However, E. coli and calf thymus thioredoxin-(SH)2 both efficiently reduced alloxan. Thus, alloxan showed an apparent Km of 70 microM in the presence of 3.4 microM E. coli thioredoxin, 0.2 microM thioredoxin reductase, and 0.4 mM NADPH. The insulin disulfide reductase activity of the complete calf thymus thioredoxin system was inhibited by alloxan, as predicted from the reaction of the drug with both thioredoxin-(SH)2 and thioredoxin reductase. The toxic action of alloxan on animal cells, particularly the beta cells of pancreas, may be caused by rapid oxidation of cellular NADPH and generation of cytotoxic dialuric acid catalyzed by the thioredoxin system.
Expression of the human fibroblast interferon gene in Escherichia coli.
Authors: Taniguchi T , Guarente L , Roberts T M , Kimelman D , Douhan J , Ptashne M
Published: May 2006

We applied the method of Guarente et al. [Guarente, L., Lauer, G., Roberts, T.M. & Ptashne, M. (1980) Cell 20, 543-553] to construct plasmids that direct expression in Escherichia coli of the human fibroblast interferon (F-IF) gene. Two plasmids were recovered. One directs efficient synthesis of a protein whose primary sequence is that of pre-F-IF and the other, that of mature F-IF. Extracts of bacteria synthesizing mature F-IF display antiviral activity characteristic of human F-IF. This activity is lower than that expected from the differential rate of synthesis of the protein. We have detected no such activity in extracts of bacteria synthesizing pre-F-IF.
Clonal analysis of proliferation and differentiation of paired daughter cells: action of granulocyte-macrophage colony-stimulating factor on granulocyte-macrophage precursors.
Authors: Metcalf D
Published: May 2006

Mouse granulocyte-macrophage progenitor cells were stimulated to divide by the granulocyte-macrophage colony-stimulating factor (GM-CSF). The two daughter cells were separated; one daughter was transferred to medium containing a high concentration of GM-CSF, the other to medium containing a low concentration. Daughter cell-derived clones in the presence of 2500 units of GM-CSF had average cell cycle times 3.5 +/- 2.5 (SEM) hr shorter than clones derived from the paired daughter cell stimulated by 50 units of GM-CSF. Final colony size achieved after stimulation by 50 units of GM-CSF was always smaller than that of colonies stimulated by 2500 units of GM-CSF. In 8 of 41 instances, colonies stimulated by 50 units of GM-CSF developed, or were composed only of, macrophage populations in contrast to the granulocytic composition of colonies derived from the paired daughter cell growing in the presence of 2500 units of GM-CSF. The regulator GM-CSF appears able to directly influence cell cycle times and the pathway of differentiation entered by many bipotential granulocyte-macrophage precursor cells.
Determination of deoxyhemoglobin S polymer in sickle erythrocytes upon deoxygenation.
Authors: Noguchi C T , Torchia D A , Schechter A N
Published: May 2006

We have used 13C/1H magnetic double-resonance spectroscopy to measure the amount of sickle hemoglobin polymer within sickle erythrocytes as a function of oxygen saturation. We previously showed that the methods of cross-polarization and scalar decoupling could be used to measure accurately the polymer fraction in deoxygenated sickle hemoglobin solutions [Noguchi, C.T., Torchia, D.A. & Schechter, A.N. (1979) Proc. Natl. Acad. Sci. USA 76, 4936-4940]. Our measurements show that the amount of intracellular deoxyhemoglobin S polymer increases monotonically with decreasing oxygen saturation. Polymer can be detected at oxygen saturation values above 90%. This result can be theoretically explained by the excluded volume effect of the oxyhemoglobin S in the cell. The very high total intracellular hemoglobin concentration (34 g/dl) reduces the amount of soluble deoxyhemoglobin S to about 3 g/dl at 90% oxygen saturation. The agreement between theory and experiment indicates that the equilibrium properties of intracellular polymerization can be described by the analyses resulting from studies of concentrated sickle hemoglobin solutions. The curve for polymer formation as a function of oxygen saturation is roughly hyperbolic whereas that for cell sickling is sigmoidal; the difference is most apparent for measurements at pH 7.65. Intracellular polymer formation may in general have a different relationship to oxygen saturation than cell sickling and may be a more meaningful parameter of the pathophysiological process in sickle cell anemia than cell morphology. In addition, measurements of intracellular polymer should be useful in evaluating potential therapeutic agents.
Brain regulation of gastric secretion: influence of neuropeptides.
Authors: Taché Y , Vale W , Rivier J , Brown M
Published: May 2006

Several neuropeptides injected intracisternally were assessed for their effects on gastric secretion in rats. Bombesin (1 microgram) completely suppressed gastric acid secretion, produced the volume of gastric secretion, and partially blocked insulin- or 2-deoxy-D-glucose-induced stimulation of gastric acid output. The inhibitory effect of this peptide is dose-dependent, long-acting, reversible, and specific. Bombesin response appears to be central nervous system-mediated; its expression is not dependent on the vagus nerve or the adrenal glands, and does not rely on a decrease in gastrin secretion. Among seven other peptides tested, only beta-endorphin and a potent gonadotropin releasing-factor (gonadoliberin) agonist significantly reduced gastric acid secretion, with an activity ca. 100 times less than that of bombesin. The presence of bombesin-like material in rat brain and the high potency of bombesin to inhibit gastric secretion suggest that this peptide may be of physiologic significance as a chemical messenger involved in brain modulation of gastric secretion.
Biosynthesis of vitamin B12: identity of fragment extruded during ring contraction to the corrin macrocycle.
Authors: Battersby A R , Bushell M J , Jones C , Lewis N G , Pfenninger A
Published: May 2006

Incorporation experiments with labeled sirohydrochlorin and trimethylisobacteriochlorin demonstrate that ring contraction in vivo to the corrin macrocycle of vitamin B12 liberates acetic acid. The C-20 atom of the precursors becomes the acetate carboxyl carbon.
Restoration of glucagon responsiveness in spontaneously transformed rat hepatocytes (RL-PR-C) by fusion with normal progenitor cells and rat liver plasma membranes.
Authors: Reilly T M , Blecher M
Published: May 2006

Spontaneously transformed RL-PR-C rat hepatocytes, unlike their normal differentiated progenitor cells, are insensitive to glucagon, although seemingly possessing large numbers of glucagon receptors and although retaining guanyl nucleotide regulatory protein-adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] system that responds to catecholamines, cholera toxin, and fluoride ions. Biochemical fusions between normal RL-PR-C hepatocytes or purified rat liver plasma membranes (whose adenylate cyclases were previously irreversibly inactivated with N-ethylmaleimide) with spontaneously transformed hepatocytes produced hybrids whose basal and fluoride-stimulated adenylate cyclase activities reflected those of the parental transformed cells but that now responded to glucagon. Using cholera toxin-catalyzed ADP-riboxylation of transformed hepatocytes to mark their guanyl nucleotide regulatory protein, fusiong such cells with N-ethylmaleimide-treated normal hepatocytes, and examining glucagon stimulation of adenylate cyclase activity in fusion hybrids produced results suggesting that the regulatory protein of the transformed cells is functionally normal. In fusion experiments between N-ethylmaleimide-treated hepatocytes and igeon erythrocytes, we found that normal, but not transformed, hepatocytes were effective in conferring glucagon sensitivity upon erythrocytes. Glucagon binding data revealed that, whereas normal RL-PR-C hepatocytes have two independent classes of binding sites, one of higher and the other of lower affinity, transformed cells possess only the low-affinity receptors. From these and previous observations, it is possible to conclude that the insensitivity of spontaneously transformed RL-PR-C hepatocytes to glucagon is due to the loss, during the transformation process, of the high-affinity glucagon receptor.
Formation of hybrid complexes between Ca
2+
and the ionophores bromolasalocid (Br-X537A) and A23187
Authors: Deleers Michel , Gelbcke Michel , Malaisse Willy J.
Published: May 2006

The ionophores A23187 and bromolasalocid (Br-X537A) acted synergistically in translocating Ca
2+
from an aqueous into an organic immiscible phase or in mediating Ca
2+
transport across the organic phase, the effects obtained in the simultaneous presence of both ionophores being greater than those expected from a summation of the individual effects of each ionophore. The nuclear magnetic resonance spectrum obtained when the complexation of Ca
2+
occurred in the presence of both ionophores differed strikingly from the individual spectra obtained with the A23187-Ca and Br-X537A-Ca complexes. These findings indicate that the two ionophores are able to form hybrid complexes with Ca
2+
.
H-2-restricted antigen binding by a hybridoma clone that produces antigen-specific helper factor.
Authors: Lonai P , Puri J , Hämmerling G
Published: May 2006

Somatic cell hybrids were prepared by fusing the AKR mouse lymphoma BW-5147 with splenic T cells from mice immunized with 4-hydroxy-3-nitrophenylacetic acid (NP) conjugated to chicken serum globulin (CG). From 500 fusion lines 11 were selected on the basis of binding radioiodinated NP-CG. The autoradiographic binding assay was based on previous findings which showed that Lyt-1+ T cells need a lymphokine, lymphocyte-activating factor (LAF), for optimal antigen binding and that they bind preferentially a self-Ia-associated antigen complex, IAC, which is released by adherent cells upon incubation with antigen. Six of the 11 antigen-binding positive lines were tested for helper activity and specific helper factor production in vitro. All of them were found to be positive. One clone was characterized in more detail. It secretes a CG-specific helper factor that contains immunoglobulin heavy chain variable region and I-A determinants. The hybridoma cells bind Ia-containing CG complexes specifically. For binding they need to be treated with LAF, and the binding is restricted to syngenicity in H-2 between the adherent cells used to produce IAC and the antigen-binding hybridoma cells. Regular CG does not bind significantly and does not compete even at high excess with the binding of CG-IAC. These data are interpreted to suggest that the antigen is bound by cells of a clone functional helper T-cell hybridoma line in conjunction with products controlled by H-2I and that the receptor of these cells may have considerably higher affinity for Ia-associated than for regular antigen.
Mg and Mc: mutations within the amino-terminal region of glycophorin A.
Authors: Furthmayr H , Metaxas M N , Metaxas-Bühler M
Published: May 2006

M and N are the two common ("normal") alleles at the MN locus of the MNSs blood group system. The antigens M and N that they determine are located within the amino-terminal region of glycophorin A. In the serologically active and glycosylated (*) fragment of glycophorin AN the sequence is Leu-Ser*-Thr*-Thr*-Glu-, and in that of glycophorin AM it is Ser-Ser*-Thr*-Thr*-Gly-. Mg and Mc are very rare ("variant") alleles of M and N; as to the corresponding antigens, Mg is serologically quite distinct from M and N, while Mc is a compound of both. Erythrocytes of genotypes MgN, MgM, MgMg, and McM, which were the object of the present study, contain normal amounts of glycophorin A in their membrane. In glycophorin AMg the amino-terminal sequence is related to that of glycophorin AN by substitution of asparagine for threonine in position 4, and it is nonglycosylated: Leu-Ser-Thr-Asn-Glu-. The corresponding structure of glycophorin AMc is Ser-Ser*-Thr*-Thr*-Glu-; it is thus closely related to that of glycophorin AN and AM, by substitution of the amino acids in positions 1 or 5, respectively. All of these substitutions can be explained by single base changes. The distinctions in chemical structure not only confirm the location of M and N in this region of glycophorin A, because they are the only differences observed, but also indicate, because they are correlated with the distinctions in antigenic specificity, that M and N are structural genes coding for amino acid sequences. The finding that Mc contains structural features of both M and N suggests that these two forms of glycophorin A have evolved from a common ancestral gene by single base substitutions at sites in the genome coding for amino acids in positions 1 and 5 of the sequence. Carbohydrate structures, however, are also necessary for full expression of antigens M and N. Glycosylation during biosynthesis of residues within the polypeptide appears to depend on a particular protein structure.
Molecular cloning of the genome of poliovirus type 1.
Authors: van der Werf S , Bregegere F , Kopecka H , Kitamura N , Rothberg P G , Kourilsky P , Wimmer E , Girard M
Published: May 2006

Poliovirus cDNA.RNA hybrids were prepared from the Mahoney strain of poliovirus type 1 by using reverse transcriptase (RNA-dependent DNA nucleotidyltransferase) and cloned in the Escherichia coli plasmid pBR322. Bacteria colonies carrying recombinant plasmids were selected by in situ hybridization with virus-specific RNase T1-resistant oligonucleotides. Analysis of the cDNA inserts by restriction mapping and electron microscopy showed that the cloned cDNAs, the longest of which was 3.2 kilobase pairs, originated from various parts of the viral RNA, covering at least 99% of the genome length. Due to overlapping of the clones, the restriction map of the poliovirus genome could be reconstructed. The complete 5' end of the genome was successfully cloned in at least one of the recombinant plasmids, pPV1-366.
Isolation of the Escherichia coli leader peptidase gene and effects of leader peptidase overproduction in vivo.
Authors: Date T , Wickner W
Published: May 2006

The only covalent modifications known to accompany protein insertion into membranes or protein secretion are glycosylation and the proteolytic removal of an NH2-terminal leader (signal) sequence. This latter reaction is catalyzed by leader peptidase, a constitutive, membrane-bound proteinase. We now report the identification of a plasmid-bearing strain of Escherichia coli that overproduces leader peptidase 4- to 6-fold. This strain grows normally and shows an unaltered polypeptide composition of inner ad outer membranes. The leader peptidase gene has been subcloned and transferred from this plasmid to the multicopy plasmid pBR322, yielding a new plasmid (pTD101). Strains transformed by pTD101 have a 30-fold increase in leader peptidase. We have studied the effect of leader peptidase overproduction on the insertion of newlymade M13 phage coat protein into the plasma membrane of infected cells. The overproducer strain, when infected by M13 phage, shows a dramatic acceleration in the conversion of procoat (a cytoplasmic precursor form) to coat (an integral, transmembrane protein). Thus the leader peptidase that converts M13 procoat to coat in vitro can catalyze this reaction in vivo as well.
32P-labeling test for DNA damage.
Authors: Randerath K , Reddy M V , Gupta R C
Published: May 2006

Covalent adducts formed by the reaction of DNA with chemical carcinogens and mutagens may be detected by a 32P-labeling test. DNA preparations exposed to chemicals known to bind covalently to DNA [N-methyl-N-nitrosourea, dimethyl sulfate, formaldehyde, beta-propiolactone, propylene oxide, streptozotocin, nitrogen mustard, and 1,3-bis(2-chloroethyl)-1-nitrosourea] were digested to a mixture of deoxynucleoside 3'-monophosphates by incubation with micrococcal endonuclease (EC 3.1.31.1) and spleen exonuclease (EC 3.1.16.1). The digests were treated with [gamma-32P]ATP and T4 polynucleotide kinase (ATP:5'-dephosphopolynucleotide 5'-phosphotransferase, EC 2.7.1.78) to convert the monophosphates to 5'-32P-labeled deoxynucleoside 3',5'-bis-phosphates. These compounds were then separated on polyethyleneimine-cellulose thin layers in ammonium formate and ammonium sulfate solutions. Autoradiograms of the chromatograms obtained by this high-resolution procedure showed the presence of nucleotides derived from chemically altered, as well as normal, DNA constituents. Maps from DNA exposed to any of the chemicals used exhibited a spot pattern typical for the particular chemical. This method detected a single adduct in 10(5) DNA nucleotides without requiring that the compound under investigation be radioactive and thus provides a useful test to screen chemicals for their capacity to damage DNA by covalent binding.
Microinjection of a rabbit beta-globin gene into zygotes and its subsequent expression in adult mice and their offspring.
Authors: Wagner T E , Hoppe P C , Jollick J D , Scholl D R , Hodinka R L , Gault J B
Published: May 2006

We have transferred a gene coding for rabbit beta-globin into the male pronucleus of mouse zygotes by direct microinjection. Some of these zygotes developed into mature mice which contained this gene and appeared to be producing a rabbit globin. Evidence for the presence of the gene in these animals was provided by Southern blot hybridization analysis. Evidence for the expression of the rabbit gene in these transformed mice and their offspring was provided by hemoglobin isoelectric focusing analysis and specific serological reactivity between mouse anti-rabbit hemoglobin antiserum and a hemolysate from the mice that developed from the microinjected zygotes. The use of this zygote transformation may allow the introduction and expression of a broad range of genetic elements in mammals.
Herpes simplex type 1 DNA in human brain tissue.
Authors: Fraser N W , Lawrence W C , Wroblewska Z , Gilden D H , Koprowski H
Published: May 2006

Herpes simplex virus type 1 (HSV-1) is known to reside latently in the trigeminal ganglia of man. Reactivation of this virus causes skin lesions and may occasionally infect other tissues, including the brain. To determine whether the brain tissue of humans free of clinical signs of HSV-1 infection contains any trace of HSV-1, we examined the DNA from brain tissue by endonuclease digestion, separation of the fragments by gel electrophoresis, and hybridization with labeled HSV-1 DNA probes. Hybrid bands were detected autoradiographically in experiments using cloned and virion-purified fragments of the HSV-1 genome. HSV-1 DNA sequences were found in 6 of 11 human brain DNA samples tested. In some cases, these bands corresponded to the bands expected for the complete viral genome, whereas others contained bands representing only a part of the genome. In some cases, the terminal fragments could be found, suggesting that the DNA was in a linear, nonintegrated form.
Viable deletions of the M13 complementary strand origin
Authors: Kim Myoung Hee , Hines Jane C. , Ray Dan S.
Published: May 2006

The single-stranded DNA of bacteriophage M13 is converted to a duplex replicative form by a mechanism involving RNA-primed initiation at a single unique site on the viral DNA. The DNA sequence that specifies the RNA primer is contained largely within one of two adjacent hairpin structures protected from DNase degradation by RNA polymerase. We have used
in vitro
techniques to construct a series of M13 mutants having deletions in the region of the complementary strand origin. Deletions of the duplex replicative form DNA range in size from 54 to 201 base pairs. The largest deletions remove both of the RNA polymerase-protected hairpins and the entire sequence specifying the primer RNA. Mutants lacking one or both hairpins form faint plaques, give reduced phage yields, and show a lag in phage production of >30 min. The rate of conversion of the single-stranded viral DNA to the parental replicative form is reduced both
in vivo
and
in vitro
. These results indicate that both the RNA polymerase-protected hairpins and the RNA primer-coding sequence are important, but not essential, for replication. Other sequences within the origin region, or possibly elsewhere in the genome, may play a role in complementary strand initiation in these mutant phages. The M13 viral strand is initiated by extension of the 3′ terminus generated by site-specific nicking of the viral strand of the replicative form DNA by the M13 gene II protein. This specific nicking site is retained in all of the M13 deletion mutants. Deletion end points do not extend into a 13-nucleotide sequence preceding the viral strand nicking site. We propose that a sequence including these 13 nucleotides is required for gene II protein action at this site.
Fimbrin is a cytoskeletal protein that crosslinks F-actin in vitro.
Authors: Bretscher A
Published: May 2006

Fimbrin is a cytoskeletal protein associated with microfilaments in microvilli, microspikes, stereocilia, membrane ruffles, and cell--substratum attachment sites. Fimbrin purified from intestinal epithelial cell brush borders was found to be a monomeric protein of molecular weight 68,000. In a sedimentation assay, fimbrin bound to F-actin in a salt-dependent manner, with binding being optimal in 30 mM KCl and inhibited in greater than 100 mM KCl. In 50 mM KCl, which allows efficient polymerization of actin, the interaction was stabilized by the presence of polyethylene glycol. Under these conditions, binding was unaffected by the inclusion of up to 5 mM Ca2+ but was inhibited by greater than 0.5 mM Mg2+. Electron microscopy revealed that fimbrin crosslinked F-actin into relatively straight bundles with shorter bundles being formed at high fimbrin-to-actin ratios. The results suggest that fimbrin crosslinks F-actin in such a way as to confer some rigidity on the bundle formed. This proposed function for fimbrin is consistent with its in vivo localization in straight, highly organized, microfilament bundles such as microvilli, microspikes, and stereocilia.
Direct effect of insulin on the synthesis of specific plasma proteins: biphasic response of hepatocytes cultured in serum- and hormone-free medium.
Authors: Liang T J , Grieninger G
Published: May 2006

Monolayers of chicken embryo hepatocytes. cultured in chemically defined medium, retain the ability to synthesize a wide spectrum of plasma proteins for several days in the absence of added hormones. Addition of insulin to the medium elicited a biphasic stimulation of plasma protein synthesis: a rapid response of the synthesis of a limited number of plasma proteins (e.g., albumin and alpha 1-globulin "M"), then, after prolonged exposure to the hormone, the involvement of additional plasma proteins (e.g., fibrinogen and lipoproteins). Synthesis of transferrin and a few other plasma proteins was not affected by the presence of insulin. The degree of stimulation for the most response plasma proteins ranged between 2- to 4-fold during the early phase and 10- and even 30-fold during the late phase of the cells' response t insulin. Stimulated synthesis in the early phase was detected within 1 hr and was rapidly reversible. Plasma protein synthesis in culture was sensitive to concentrations of insulin below 0.35 nM, well within the physiological range. The delayed response was elicited only at higher hormone levels. Parallels between the control of synthesis of plasma proteins in this system and that observed in diabetic animals suggest that the embryonic chicken hepatocytes may be a useful model for studying liver function in diabetes as well as insulin action in general.
Stable cycling in discrete-time genetic models.
Authors: Hastings A
Published: May 2006

Examples of stable cycling are discussed for two-locus, two-allele, deterministic, discrete-time models with constant fitnesses. The cases that cycle were found by using numerical techniques to search for stable Hopf bifurcations. One consequence of the results is that apparent cases of directional selection may be due to stable cycling.
Localization of cholesteryl sulfate in human spermatozoa in support of a hypothesis for the mechanism of capacitation.
Authors: Langlais J , Zollinger M , Plante L , Chapdelaine A , Bleau G , Roberts K D
Published: May 2006

Cholesteryl sulfate is a normal constituent of human spermatozoa. The in vitro uptake of tritiated cholesteryl sulfate resulted in the labeling of all spermatozoa as demonstrated by light-microscope radioautography. The binding of the sterol sulfate was localized mainly in the head and midpiece. Radioautography at the level of the electron microscope revealed that the sterol sulfate is localized on the plasma membrane, mostly in the region of the acrosome. Further proof of this localization was obtained by selective dissolution of the plasma membrane and acrosome of the spermatozoa with low concentrations of Triton X-100. This treatment resulted in the simultaneous removal of tritiated cholesteryl sulfate bound to the spermatozoa. A hypothesis is presented concerning the role of cholesteryl sulfate as a membrane stabilizer and enzyme inhibitor during the maturation of spermatozoa in the epididymis. According to this hypothesis, the cleavage of the sulfate moiety within the female reproductive tract triggers a cascade of events leading to sperm capacitation and fertilization.
Evidence for presence of an arginine residue in the coenzyme A binding site of choline acetyltransferase.
Authors: Mautner H G , Pakula A A , Merrill R E
Published: May 2006

Choline acetyltransferase (acetyl-CoA:choline O-acetyltransferase, EC 2.3.1.6) may be inactivated by arginine-specific reagents such as butanedione, phenylglyoxal, and camphorquinone-10-sulfonic acid. The enantiomers of the latter compound were prepared, but inactivation was not stereospecific. Protection against inactivation by the arginine-specific reagents was provided by CoA and, to a lesser extent, by 3'-dephospho-CoA. No protection was provided by choline, NAD+, NADH, NADP+, or NADPH. Sodium chloride could protect, to some extent, against inactivation by arginine-specific reagents; this protection showed no cation or anion specificity. The data are compatible with the postulate that the salt anion competes with the attachment of the 3'-phospho group of CoA to an active site arginine residue.
Transmembranous incorporation of photoelectrically active bacteriorhodopsin in planar lipid bilayers.
Authors: Bamberg E , Dencher N A , Fahr A , Heyn M P
Published: May 2006

Various methods to incorporate bacteriorhodopsin in black lipid membranes are reported. Both purple membrane patches and monomeric bacteriorhodopsin were used as starting material. The incorporation of bacteriorhodopsin into planar lipid bilayers was achieved by the following methods. (i) Purple membrane patches were transferred from water to solutions of lipids in n-alkanes. Black membranes were formed from such organic suspensions. (ii) Lipid layers containing solvent and purple membranes were spread on an air/water interface. These layers were used to form planar bilayers. (iii) Vesicles containing purple membranes or monomeric bacteriorhodopsin were spread on an air/water interface and, from the resulting layer, bilayers were formed. On illumination, steady-state photocurrents were observed in all three cases, indicating that these methods lead to functional transmembranous integration of the protein in the planar black lipid membrane. The influence of an applied electric field on the pumping process was studied on membranes formed by using method i. At approximately 200 mV, the photocurrent tends to zero. Furthermore, it was possible to make planar lipid bilayers photoelectrically active by adding vesicles containing monomeric bacteriorhodopsin to the bathing solution. Because, in this case, only transient photocurrents were observed, it can be concluded that the vesicles are attached to but not fused with the black lipid membrane.
Monoclonal antibodies against receptor for epidermal growth factor induce early and delayed effects of epidermal growth factor.
Authors: Schreiber A B , Lax I , Yarden Y , Eshhar Z , Schlessinger J
Published: May 2006

Mice were immunized with human epidermoid carcinoma cells (A-431 cell line) that possess an unusually high number of membrane receptors for epidermal growth factor (EGF). Spleen cells from these mice were fused with NSI cells, a nonsecreting murine myeloma. The immunoglobulins secreted by the obtained hybridomas were screened for specific binding to A-431 cells and selected according to their ability to inhibit the binding of radiolabeled EGF to the membrane of A-431 cells. Several antibodies secreted by cloned hybrid lines were found to inhibit the binding of radiolabeled EGF to membrane receptors of living A-431 cells, human foreskin fibroblasts, and mouse 3T3 fibroblasts and also to membrane preparations from A-431 cells. These monoclonal antibodies induced the early and delayed biological effects mediated by EGF. Like EGF, the antibodies induced morphological changes in A-431 cells and enhanced the phosphorylation of endogenous membrane proteins in membranes from these cells. They also stimulated DNA synthesis in human foreskin fibroblasts. These observations support the notion that the biological information of the EGF-receptor complex resides in the membrane receptor. Furthermore, the antibodies offer a powerful tool to study the structure, processing, and mode of action of EGF receptors.
Identification of a spectrin-like protein in nonerythroid cells.
Authors: Goodman S R , Zagon I S , Kulikowski R R
Published: May 2006

We have demonstrated the existence of a spectrin-like protein in a variety of nonerythroid cultured cells. Indirect immunofluorescence studies with monospecific antispectrin IgG indicated the presence of proteins that have common antigenic determinants to spectrin in embryonic chicken cardiac myocytes, mouse fibroblast lines (3T3, simian virus 4-transformed 3T3), and rat hepatoma lines (HTC, HMOA). Two spectrin-like peptides of 240,000 and 230,000 daltons were immunoprecipitated from octyl glucoside-solubilized embryonic chicken cardiac myocytes, along with associated cytoskeletal proteins. Immunoautoradiographic characterization of the myocyte immunoprecipitate showed that only the spectrin-like 240,000- and 230,000-dalton peptides were stained with monospecific antispectrin IgG and 125I-labeled protein A. One-dimensional partial proteolytic mapping of the myocyte 240,000- and 230,000-dalton peptides showed that these peptides share substantial sequence homology with embryonic chicken erythrocyte spectrin 240,000- and 220,000-dalton peptides.
Ultrastructure of chicken cardiac muscle as studied by double immunolabeling in electron microscopy.
Authors: Tokuyasu K T , Dutton A H , Geiger B , Singer S J
Published: May 2006

The ultrastructural localization of alpha-actinin and vinculin in chicken cardiac muscle was studied by double indirect immunoelectron microscopy, using ferritin and iron-dextran (Imposil) as the electron-dense markers conjugated to the secondary antibodies, on ultrathin frozen sections of fixed tissue. Fixation and immunolabeling procedures were developed that permitted maximal retention of the two proteins at their natural sites as well as their adequate labeling. alpha-Actinin was found both on the Z-bands, as expected, and near the fascia adherens of the intercalated discs, whereas vinculin was confined to the latter sites. At the fascia adherens, the double labeling results clearly showed that vinculin was situated closer to the membrane than was alpha-actinin. These results, coupled with earlier observations, suggest that vinculin may participate in the linkage of actin-containing microfilament bundles to membranes in a variety of cell types.
Calcitonin: regional distribution of the hormone and its binding sites in the human brain and pituitary.
Authors: Fischer J A , Tobler P H , Kaufmann M , Born W , Henke H , Cooper P E , Sagar S M , Martin J B
Published: May 2006

Immunoreactive calcitonin (CT), indistinguishable from human CT-(1-32) and its sulfoxide, has been identified in extracts of the hypothalamus, the pituitary, and the thyroid obtained from human subjects at autopsy. DCT concentrations were highest in a region encompassing the posterior hypothalamus, the median eminence, and the pituitary; intermediate in the substantia nigra, the anterior hypothalamus, the globus pallidus, and the inferior colliculus; and low in the caudate nucleus, the hippocampus, the amygdala, and the cerebral and cerebellar cortices. Specific CT binding measured with 125I-labeled salmon CT was highest in homogenates of the posterior hypothalamus and the median eminence, shown to contain the highest concentrations of endogenous CT in the brain; CT binding was less than 12% of hypothalamic binding in all of the other regions of the brain examined and was negligible in the pituitary. Half-maximal binding was achieved with 0.1 nM nonradioactive salmon CT-(1-32), and the binding was directed to structural or conformational sites, or both, in the COOH-terminal half of salmon CT. The rank order of the inhibition of the binding by CT from different species and analogues of the human hormone was the same as in receptors on a human lymphoid cell line (Moran, J., Hunziker, W. & Fischer, J. A. (1978) Proc. Natl. Acad. Sci. USA 75, 3984-3988). The functional role of CT and of its binding sites in the brain remains to be elucidated.
Simple method for constructing phylogenetic trees from distance matrices.
Authors: Li W H
Published: May 2006

A simple method is proposed for constructing phylogenetic trees from distance matrices. The procedure for constructing tree topologies is similar to that of the unweighted pair-group method (UPG method) but makes corrections for unequal rates of evolution among lineages. The procedure for estimating branch lengths is the same as that of the Fitch and Margoliash method (F-M method) except that it allows no negative branch lengths. The performance of the present procedure for the construction of tree topologies is compared with that of the UPG method, the F-M method, Farris' method, and the modified Farris method by using Tateno's simulation outputs for nucleotide sequence divergence and his results for the performances of the latter four methods [Tateno, Y. (1978) Dissertation (Univ. Texas, Houston, TX). In this limited comparison, the present method performs considerably better than the UPG method and the F-M method and about equally well as the last two methods. The present method appears to be preferable to the UPG method for analysis of data from populations that have not differentiated much. Indeed, an application of the present method to gene frequency data from some Amerindian populations gives a tree topology far more reasonable than that obtained by the UPG method.
Structure and chemistry of a metal cluster with a four-coordinate carbide carbon atom
Authors: Davis Jimmy H. , Beno M. A. , Williams Jack M. , Zimmie JoAnn , Tachikawa M. , Muetterties E. L.
Published: May 2006

Molecular metal clusters with carbide carbon atoms of low coordination number have been prepared; they are the anionic [HFe
4
C(CO)
12
-
] and [Fe
4
C(CO)
12
2-
] clusters. An x-ray crystallographic analysis of a tetraaminozinc salt of the latter has established a butterfly array of iron atoms with the carbide carbon atom centered above the wings of the Fe
4
core. Each iron atom was bonded to three peripheral carbonyl ligands. The distances from the carbide carbon to iron were relatively short, particularly those to the apical iron atoms (1.80 Å average). Protonation of the anionic carbide clusters reversibly yielded HFe
4
(CH)(CO)
12
, and methylation of the dianion gave {Fe
4
[CC(O)CH
3
](CO)
12
-
}. Oxidation of [Fe
4
C(CO)
12
2-
] yielded the coordinately unsaturated Fe
4
C(CO)
12
cluster, which was extremely reactive. Hydrogen addition to this iron cluster was rapid below 0°C, and a C—H bond was formed in this transformation.