bluuebunny/crossref_metadata_2025 huggingface dataset:
Charge movements during the Na+-Ca2+ exchange in heart sarcolemmal vesicles.
Authors: Caroni P , Reinlib L , Carafoli E
Published: May 2006

The Na+-Ca2+ exchange was studied in a highly purified vesicular preparation derived from heart sarcolemma. The initial velocity of the Na+-driven Ca2+ influx, which was monitored continuously with a specific electrode, was 15 nmol/mg of protein per sec; the total Ca2+-accumulation capacity was 80 nmol/mg of protein. The Na+-Ca2+ exchange generated a current that was compensated for by the uptake of tetraphenylphosphonium in (Ph4P+) (when the latter was present in the medium), the influx of K+, and the efflux of Cl-. The movements of Ph4P were followed with a specific electrode. Ca2+ in the concentration range 3-50 microM induced an increase in the permeability of the sarcolemmal membrane to K+. Under conditions of optimal charge neutralization by K+ (i.e., in the presence of valinomycin), the Km (Ca2+) of the exchanger was 1.5 microM. The Na+-Ca2+ exchange was inhibited by chlorpromazine and was not inhibited by vanadate.
Calcium control of the intestinal microvillus cytoskeleton: its implications for the regulation of microfilament organizations.
Authors: Glenney J R , Bretscher A , Weber K
Published: May 2006

The microvillus core-filament bundle from intestinal epithelial cells is a highly ordered structure containing actin and four major associated proteins. Two of these, villin and calmodulin, bind calcium ions (Kd approximately 10(-6) M) in the physiologically important range. Because ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid is present throughout the purification and the isolated cores contain levels of calcium substoichiometric to calmodulin, the protein is bound in the structure without calcium saturation. 10-[3-(4-Methyl-1-piperazinyl)propyl]-2-trifluoromethylphenothiazine, a calmodulin-specific drug, removes the protein from the cores without visibly affecting their ultrastructure. Calmodulin-depleted cores rebind exogenously supplied brain calmodulin. Although the core filaments are stable when the calcium level is less than 10(-7) M, they dissassemble when it is greater than 10(-6) M. This appears to be due to the calcium-sensitive allosteric transition of villin from an F-actin bundling protein to an F-actin severing protein. The actions of the two calcium-binding proteins, villin and calmodulin, are discussed in terms of the calcium sensitivity of the filament bundle. We suggest that villin may act as a calcium-sensitive factor regulating microfilament assembly and disassembly and that calmodulin serves as a buffer modulating the free calcium concentration. This hypothesis may explain some aspects of the physiological process of calcium uptake in the intestine and of the effects of calcium fluxes on the submembranous organization of microfilaments in other cells and tissues.
Isolation and characterization of the mouse metallothionein-I gene.
Authors: Durnam D M , Perrin F , Gannon F , Palmiter R D
Published: May 2006

Double-stranded cDNA was synthesized from a mouse liver mRNA fraction enriched for metallothionein mRNA activity, ligated to restriction site linkers, inserted into pBR322, and used to transform Escherichia coli chi 1776. The sequence of the largest plasmid containing DNA that hybridized to metallothionein mRNA was determined and shown to contain a 380-base-pair insert that includes the entire coding region and 3' untranslated region of metallothionein-I. The metallothionein-I insert was nick-translated and used to screen both a mouse myeloma and a mouse embryo DNA library in bacteriophage lambda. A metallothionein-I genomic clone containing 13-15 kilobase pairs of mouse DNA was isolated from each library. Both contain a 3.8-kilobase-pair EcoRI fragment that hybridizes to the metallothionein-I probe. The location, size, and orientation of the metallothionein-I gene within the 3.8-kilobase-pair fragment were determined by heteroduplex and restriction mapping. The gene spans 1.1 kilobase pairs and contains at least two introns.
Expression of a DNA strand initiation sequence of ColE1 plasmid in a single-stranded DNA phage.
Authors: Nomura N , Ray D S
Published: May 2006

In order to investigate initiation of H-strand (lagging strand) replication of the plasmid ColE1, the origin region fragment (Hae II-E) of ColE1 was inserted into the intergenic region of filamentous DNA phage M13 and cloned. A site capable of promoting DNA strand initiation on a single-stranded DNA template has been detected on the L-strand (leading strand) of the cloned fragment. The site, named rri-1 rifampicin-resistant initiation), directs conversion of chimeric phage single-stranded DNA to parental replicative form in the presence of rifampicin, which blocks the function of the complementary strand origin of M13. The function of rri-1 is dependent on both the dnaG and dnaB gene products. It is postulated that rri-1 might be an initiation site for synthesis of the lagging DNA strand during unidirectional replication of ColE1 DNA.
Proton longitudinal relaxation investigation of histidyl residues of normal human adult and sickle deoxyhemoglobin: evidence for the existence of pregelation aggregates in sickle deoxyhemoglobin solutions.
Authors: Russu I M , Ho C
Published: May 2006

Proton nuclear magnetic resonance longitudinal-relaxation-rate measurements have been used to investigate the molecular events that occur during the early stages of the polymerization process of sickle hemoglobin. The longitudinal relaxation rates (T1-1) of the C2 protons of 11 observable surface histidyl residues in normal human adult and sickle hemoglobin in the deoxy state were measured in 0.1 M bis[(2-hydroxyethyl)imino]tris(hydroxymethyl)methane (pH 6.8) in 2H2O. These proton resonances in hemoglobin occur at a position 1.5-5.0 ppm downfield from that of residual water in 2H2O. The T1-1 values for the C2 protons of several surface histidyl residues in sickle hemoglobin in the deoxy state were sensitive to the temperature and the concentration of hemoglobin, factors known to have a profound effect on the polymerization process of sickle hemoglobin. For hemoglobin concentrations of 13.5% or less and temperatures of 25 degrees C or less, the T1-1 values in sickle hemoglobin solutions were the same as the corresponding values in normal hemoglobin, except for the C2 proton of beta 2 histidine, which had a larger T1-1 value. When the temperature or the hemoglobin concentration was increased (i) several additional histidine resonances in sickle hemoglobin solutions had larger T1-1 values than the corresponding ones in normal hemoglobin and (ii) the differences between the T1-1 values (sickle versus normal hemoglobin) of these histidine resonances as well as that of the beta 2 histidine resonance gradually increased. It is proposed that these results reflect the formation of small aggregates in the deoxygenated sickle hemoglobin solutions before gelation. In this model, the histidyl residues for which the T1-1 values are greatly increased in sickle hemoglobin solutions as compared with those in normal hemoglobin are viewed as being located in or near the "contact" areas between sickle hemoglobin molecules within the pregelation aggregates. Thus, this magnetic resonance technique can also be used to identify the intermolecular contacts in the polymerization of sickle hemoglobin.
Endogenous electrical currents in the water mold Blastocladiella emersonii during growth and sporulation.
Authors: Stump R F , Robinson K R , Harold R L , Harold F M
Published: May 2006

We have explored the pattern of electrical currents generated by single cells of the water mold Blastocladiella emersonii at several stages of its life cycle. Extracellular currents were measured with a vibrating probe constructed after the design of Jaffe and Nuccitelli [Jaffe, L. F. & Nuccitelli, R. (1974) J. Cell Biol. 63, 614-628]. In growing cells positive current, of the order of 1 microA/cm2, enters the rhizoid and leaves from the thallus; circumstantial evidence suggests that protons carry much of the current. Sporulation is associated with reversal of the current pattern, such that positive current enters the thallus and leaves from the rhizoidal region; the ions that carry the current have not been identified. These current patterns appear to play a role in the spatial localization of fungal growth and development.
Circadian rhythm mutations in Drosophila melanogaster affect short-term fluctuations in the male's courtship song.
Authors: Kyriacou C P , Hall J C
Published: May 2006

Courtship song in Drosophila is produced by the male's wing vibration and consists of pulses of tone produced at intervals of approximately 34 msec in D. melanogaster and 48 msec in D. simulans. We have observed that the intervals between these pulses are not constant but fluctuate rhythmically with periods of approximately 1 min in D. melanogaster and 0.5 min in D. simulans. In D. melanogaster, three allelic per mutations have been isolated which affect the periodicity of the circadian oscillators affecting both eclosion and locomotor activity [Konopka, R. & Benzer S. (1971) Proc. Natl. Acad. Sci. USA 68, 2112-2116]. Each of the per alleles--pers, which shortens the circadian period, perI, which lengthens it, and perO, which abolishes it--strikingly affects the 60-sec song rhythm in a parallel fashion. Therefore, both circadian rhythms and a very short, noncircadian oscillation appear to be influenced by the same gene.
High molecular weight antigens present on human T cells.
Authors: Judd W , Poodry C A , Broder S , Friedman S M , Chess L , Strominger J L
Published: May 2006

A series of eight high molecular weight (140,000-220,000) glycoproteins on human peripheral T cells were recognized by radioimmunoprecipitation with a rabbit antiserum. The pattern of antigens present on each of eight human T cell lines studied was unique, and no line displayed the range of antigens present on peripheral T cells. The pattern of bands on peripheral T cells changed after allogeneic or lectin stimulation. Adsorption/elution experiments with antiserum showed that some of these proteins were antigenically related, and at least three different groups of proteins were present. Two of these groups could be partially distinguished by their ability to bind to ricin or lentil lectin and by their reactivity with two additional rabbit antisera. On some cell lines, it was found that proteins bound by lentil lectin but not ricin were precursors of higher molecular weight material recognized by ricin. Taken together, the data suggest that these proteins may be the products of a multigenic or multiallelic system, probably equivalent to the murine Ly 5 antigens.
Strains and clones of Trypanosoma cruzi can be characterized by pattern of restriction endonuclease products of kinetoplast DNA minicircles.
Authors: Morel C , Chiari E , Camargo E P , Mattei D M , Romanha A J , Simpson L
Published: May 2006

A simple method was developed for the characterization of different strains of Trypanosoma cruzi. T. cruzi stocks isolated from vectors or by hemoculture from patients with Chagas disease could be grouped in subpopulations having similar patterns of restriction endonuclease products of kinetoplast DNA minicircles. We designate such subpopulations by the term "schizodemes." Furthermore, it is shown that, from a given T. cruzi strain, clones with different biological properties can be isolated and identified by their restriction patterns.
Studies with [35S]methionine indicate that the 22,000-dalton [Met]enkephalin-containing protein in chromaffin cells is a precursor of [Met]enkephalin.
Authors: Rossier J , Trifaró J M , Lewis R V , Lee R W , Stern A , Kimura S , Stein S , Udenfriend S
Published: May 2006

It has been shown that [35S]methionine is incorporated into the [Met]enkephalin sequences of a 22,000-dalton enkephalin-containing protein in the adrenal medulla. Pulse-chase experiments indicate that label is incorporated into the large polypeptide before it appears in free [Met]enkephalin and a smaller [Met]enkephalin-containing peptide. These findings provide direct evidence of a precursor-product relationship of these structurally related polypeptides.
Biochemical characterization of putative central purinergic receptors by using 2-chloro[3H]adenosine, a stable analog of adenosine.
Authors: Williams M , Risley E A
Published: May 2006

After pretreatment of rat brain synaptic membranes with adenosine deaminase to remove endogenous adenosine, 2-chloro[3H]adenosine, a stable analog of adenosine, binds to two sites with Kd values of 1.3 and 16 nM and corresponding Bmax values of 207 and 380 fmol/mg of protein. Binding is reversible, and the highest density of sites occurs in enriched synaptosomal fractions. In peripheral tissue, negligible binding is observed in heart, kidney, and liver, while testicle has 11 fmol of binding sites/mg of protein. In brain, caudate and hippocampus have the highest density of sites, and spinal cord and hypothalamus have the lowest. This high-affinity binding is stereospecific; the L diasteromer of N6-phenylisopropyladenosine is approximately 30-times more potent as a displacer of 2-chloro[3H]adenosine than the D isomer and is also sensitive to theophylline (IC50 = 8.8 microM) and other purine-related compounds. Several putative neurotransmitters, neurotransmitter antagonists, and other centrally active compounds have no effect on binding. The data are consistent with the hypothesis that 2-chloro[3H]adenosine is binding to central purinergic receptors.
Quantization of wave equations and hermitian structures in partial differential varieties
Authors: Paneitz S. M. , Segal I. E.
Published: May 2006

Sufficiently close to 0, the solution variety of a nonlinear relativistic wave equation—e.g., of the form □ϕ +
m
2
ϕ +
g
ϕ

p

= 0—admits a canonical Lorentz-invariant hermitian structure, uniquely determined by the consideration that the action of the differential scattering transformation in each tangent space be unitary. Similar results apply to linear time-dependent equations or to equations in a curved asymptotically flat space-time. A close relation of the Riemannian structure to the determination of vacuum expectation values is developed and illustrated by an explicit determination of a perturbative 2-point function for the case of interaction arising from curvature. The theory underlying these developments is in part a generalization of that of M. G. Krein and collaborators concerning stability of differential equations in Hilbert space and in part a precise relation between the unitarization of given symplectic linear actions and their full probabilistic quantization. The unique causal structure in the infinite symplectic group is instrumental in these developments.
Cell-free coupled transcription-translation system for investigation of linear DNA segments.
Authors: Yang H L , Ivashkiv L , Chen H Z , Zubay G , Cashel M
Published: May 2006

Heretofore the DNA-directed coupled transcription-translation system, most useful in gene expression analysis, has been limited to the use of circular or long linear DNAs. Linear DNAs are degraded in this system by an exonucleolytic activity that can be eliminated by making the synthetic extracts from a suitable recB mutant of Escherichia coli. Using these extracts, we have examined the gene expression of a variety of linear DNAs. In particular, the complex pattern of expression of ribosomal protein genes and RNA polymerase genes in the rpoBC-rplLJ region has been analyzed by comparing the protein products obtained when using lambda rifd18 DNA with the product obtained when using the same DNA segmented with various restriction enzymes. The results obtained confirm the conclusions of others obtained by much more elaborate in vivo techniques. It seems highly likely that this cell-free system will have extensive applications in the area of analysis of gene expression.
Calcium-dependent protein kinase: widespread occurrence in various tissues and phyla of the animal kingdom and comparison of effects of phospholipid, calmodulin, and trifluoperazine.
Authors: Kuo J F , Andersson R G , Wise B C , Mackerlova L , Salomonsson I , Brackett N L , Katoh N , Shoji M , Wrenn R W
Published: May 2006

A widespread occurrence of Ca2+-dependent protein kinase was shown in various tissues and phyla of the animal kingdom. Phosphatidylserine appeared to be more effective than calmodulin in supporting the Ca2+-dependent phosphotransferase activity. The phospholipid-sensitive Ca2+-dependent protein kinase activity, distributed in both the cytosolic and particulate fractions, was not inhibited by trifluoperazine, a specific inhibitor of calmodulin-sensitive, Ca2+-dependent reactions or processes. The enzyme activity levels, compared to those of cyclic AMP-dependent and cyclic GMP-dependent protein kinases, were exceedingly high in certain tissues (such as brain and spleen) and exhibited a much greater disparity among tissues. The Ka for Ca2+ was about 100 microM in the presence of phosphatidylserine; the value was as low as 2 microM in the presence of phosphatidylserine and diolein. It is suggested that phospholipid-sensitive Ca2+-dependent protein kinase may mediate certain actions of Ca2+ in tissues, acting independently or in a complementary manner with other protein phosphorylation systems stimulated by calmodulin-Ca2+, cyclic AMP, or cyclic GMP.
Regulation of expression from the glnA promoter of Escherichia coli in the absence of glutamine synthetase.
Authors: Rothstein D M , Pahel G , Tyler B , Magasanik B
Published: May 2006

One of the suspected regulators of glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2] in enteric bacteria is glutamine synthetase itself. We isolated Escherichia coli strains carrying fusions of the beta-galactosidase structural gene to the promoter of the glutamine synthetase gene, with the aid of the Casadaban Mud1 (ApR, lac, cts62) phage. Some aspects of regulation were retained in haploid fusion strains despite the absence of glutamine synthetase, whereas other aspects required glutamine synthetase catalytic or regulatory activity or both. The direction of transcription of the glutamine synthetase gene was also determined.
Genetic transformation of mouse embryos by microinjection of purified DNA.
Authors: Gordon J W , Scangos G A , Plotkin D J , Barbosa J A , Ruddle F H
Published: May 2006

A recombinant plasmid composed of segments of herpes simplex virus and simian virus 40 viral DNA inserted into the bacterial plasmid pBR322 was microinjected into pronuclei of fertilized mouse oocytes. The embryos were implanted in the oviducts of pseudopregnant females and allowed to develop to term. DNA from newborn mice was evaluated by the Southern blotting technique for the presence of DNA homologous to the injected plasmid. Two of 78 mice in one series of injections showed clear homology, though the injected sequences had been rearranged. Band intensities from the two positive mice were consistent with the presence of donor DNA in most or all of the cells of the newborns. These results demonstrate that genes can be introduced into the mouse genome by direct insertion into the nuclei of early embryos. This technique affords the opportunity to study problems of gene regulation and cell differentiation in a mammalian system by application of recombinant DNA technology.
Posttranslational modification of elongation factor 2 in diphtheria-toxin-resistant mutants of CHO-K1 cells.
Authors: Moehring J M , Moehring T J , Danley D E
Published: May 2006

We have identified two types of mutants of Chinese hamster ovary cells in which the unique ADP-ribose attachment site in elongation factor 2 (EF-2) is altered, thereby rendering them resistant to diphtheria and Pseudomonas toxins (TOXR). The first is mutant in the gene for EF-2 and possesses a permanently altered, TOXR gene product. The second lacks a component of a posttranslational modification system that converts TOXR EF-2 to the toxin-sensitive (TOXS) state. We postulate that this modification system is involved in the conversion of a single histidine residue in EF-2 to the specific target of toxin-catalyzed ADP-ribosylation, the novel amino acid X. We have designated the second type MOD- mutants. The missing of nonfunctional component in the MOD- mutants can be restored by hybridizing them with either normal TOXS cells or with EF-2 structural gene mutants. The TOXR EF-2 from MOD- mutants is also converted to toxin sensitivity in vitro by incubation with extracts of TOXS or EF-2 gene mutant cells in the presence of an energy-generating system. Our results demonstrate that EF-2 can be synthesized and released from ribosomes in a toxin-resistant form and then converted to toxin sensitivity by posttranslational modification.
Enzyme null alleles in natural populations of
Drosophila melanogaster
: Frequencies in a North Carolina population
Authors: Voelker Robert A. , Langley Charles H. , Brown Andrew J. Leigh , Ohnishi Seido , Dickson Barbara , Montgomery Elizabeth , Smith Sandra C.
Published: May 2006

A Raleigh, NC, population of
Drosophila melanogaster
was sampled for the presence of enzyme null alleles at 25 loci. No nulls were found at any of five X-linked loci. Nulls were recovered at 13 of 20 autosomal loci; the weighted mean frequency for all 20 autosomal loci was estimated to be 0.0025. A consideration of the effects of these null alleles on viability strongly suggests that, although they may contribute to so-called polygenic variation, they are not representative of the entire genome.
Hybridoma proteins expressing the predominant idiotype of the antiazophenylarsonate response of A/J mice.
Authors: Marshak-Rothstein A , Siekevitz M , Margolies M N , Mudgett-Hunter M , Gefter M L
Published: May 2006

Hybridoma cell lines that secrete monoclonal antiazophenylarsonate antibodies were isolated from the fusion of A/J splenic lymphocytes with a myeloma cell line. A small percentage of these hybridoma proteins were recognized by rabbit antisera that detect the crossreactive idiotype characteristics of the antiazophenylarsonate response of A/J mice. The isotype, pI value, and amino-terminal sequences of four independently derived idiotype-positive hybridoma proteins were determined. These proteins were either of the IgG1 or IgG2a heavy chain class. For two mice tested, the majority of the idiotype in the immune serum was shown to be of the same isotype as the fusion-derived monoclonal antibodies. The pI values of the hybridoma proteins differed from one another and ranged from 6.9 to 7.6. Amino acid sequences of the heavy chains showed a significant degree of homology with each other, but each chain was unique in the framework or the first complementarity determining region (or both). A comparable pattern of sequence variation was evident for the light chains. The azophenylarsonate idiotype, therefore, appears to consist of a family of nonidentical but closely related molecules that are the product of more than one germline gene or the result of somatic mutation of a single germline gene.
Comparison of rotational barriers of related diphenylmethyl anions and cations studied by
13
C NMR spectroscopy: Mechanistic and structural considerations
Authors: Olah George A. , Watkins Michael I.
Published: May 2006

The rotational barriers of diphenylmethyl anions and cations were measured through their temperature-dependent
13
C NMR spectra. The ground state structures were found to possess a symmetrical propeller nature. The mechanism for phenyl rotation involves a nonsynchronous process, in which in the transition state one ring is coplanar with, and the other is perpendicular to, the plane formed by the central carbon and its bonds. The barriers of rotation in the carbanions vary with the strength of chelation of the potassium gegenion, whereas the
13
C NMR shifts remain unaffected. Changing substituents in the
para
position of the aromatic rings allowed both the elucidation of structure and mechanism and the observation of effects of changes in electron distribution on the height of the barrier and the
13
C NMR shifts.
In vitro synthesis of the major lens membrane protein.
Authors: Ramaekers F C , Selten-Versteegen A M , Benedetti E L , Dunia I , Bloemendal H
Published: May 2006

The biosynthetic activity of a polyribosomal fraction isolated from the lens fiber plasma membrane-cytoskeleton complex by DNase I treatment has been assayed. After translation of these polyribosomes in a reticulocyte cell-free system and analysis of the products by electrophoresis in sodium dodecyl sulfate gels, the preferential synthesis of a protein with an apparent molecular weight of 26,000 was observed. By means of immunochemical characterization we showed that this protein, which seems not to be synthesized by "free" polyribosomes, is identical with the major intrinsic plasma membrane protein MP26 of lens fibers. Upon storage, the molecular weight of the newly synthesized protein decreases to about 22,000, a phenomenon that has previously been observed for MP26 in isolated plasma membranes and that may be caused by the presence of a specific proteolytic cleaving site in the protein.
Sequence determination of protected oligodeoxyribonucleotides containing phosphotriester linkages by californium-252 plasma desorption mass spectrometry.
Authors: McNeal C J , Narang S A , Macfarlane R D , Hsiung H M , Brousseau R
Published: May 2006

A mass spectrometric method for determining the sequence and molecular weight of protected oligodeoxyribonucleotides is described. By using the method of 252Cf plasma desorption mass spectrometry [Macfarlane, R. D. & Torgerson, D. F. (1976) Science 191, 920--925], positive-ion mass spectra were obtained for a series of protected oligonucleotides extending to a decanucleotide; the spectra were dominated by the presence of the oligonucleotide molecular ion. The negative-ion mass spectra were characterized by a nested set of fragment ions extending from the 3'- or 5'-terminal nucleotide to the opposite terminal nucleotide, thereby identifying the sequence. The utility of this method has been demonstrated by the sequence determination of protected tetra-, penta-, and hexanucleotides synthesized by the improved phosphotriester method.
Purification of a factor that restores translation of vesicular stomatitis virus mRNA in extracts from poliovirus-infected HeLa cells.
Authors: Trachsel H , Sonenberg N , Shatkin A J , Rose J K , Leong K , Bergmann J E , Gordon J , Baltimore D
Published: May 2006

It was previously shown that the poliovirus-induced inhibition of translation of capped mRNAs can be reversed by a protein found in preparations of the eukaryotic initiation factor eIF-4B [Rose, J. K., Trachsel, H., Leong, K. & Baltimore, D. (1978) Proc. Natl. Acad. Sci. USA 75, 2732--2736]. This "restoring factor" has now been purified from a high-salt wash of rabbit reticulocyte ribosomes by taking advantage of its tight association with factor eIF-3 at low salt concentrations. It did not copurify with the major Mr 80,000 polypeptide of eIF-4B preparations but did copurify with a Mr 24,000 polypeptide previously shown to bind to the cap structures of mRNAs [Sonenberg, N., Rupprecht, K. M., Hecht, S. M. & Shatkin, A. J. (1979) Proc. Natl. Acad. Sci. USA 76, 4345--4349]. Both the electrophoretic mobility and the tryptic peptide pattern of the restoring factor were indistinguishable from those of the cap-binding protein, and the restoring factor could be crosslinked to the 5'-terminal cap on mRNA. Thus, is appears that poliovirus inhibits cellular protein synthesis by inactivation of some crucial property of the cap-binding protein.
Molecular mechanism of acetylcholine receptor-controlled ion translocation across cell membranes
Authors: Cash Derek J. , Hess George P.
Published: May 2006

Two molecular processes, the binding of acetylcholine to the membrane-bound acetylcholine receptor protein and the receptor-controlled flux rates of specific inorganic ions, are essential in determining the electrical membrane potential of nerve and muscle cells. The measurements reported establish the relationship between the two processes: the acetylcholine receptor-controlled transmembrane ion flux of
86
Rb
+
and the concentration of carbamoylcholine, a stable analog of acetylcholine. A 200-fold concentration range of carbamoylcholine was used. The flux was measured in the millisecond-to-minute time region by using a quench flow technique with membrane vesicles prepared from the electric organ of
Electrophorus electricus
in eel Ringer's solution at pH 7.0 and 1°C. The technique makes possible the study of the transmembrane transport of specific ions, with variable known internal and external ion concentrations, in a system in which a determinable number of receptors is exposed to a known concentration of ligand. The response curve of ion flux to ligand was sigmoidal with an average maximum rate of 84 sec
-1
. Carbamoylcholine induced inactivation of the receptor with a maximum rate of 2.7 sec
-1
and a different ligand dependence so that it was fast relative to ion flux at low ligand concentration but slow relative to ion flux at high ligand concentration. The simplest model that fits the data consists of receptor in the active and inactive states in ligand-controlled equilibria. Receptor inactivation occurs with one or two ligand molecules bound. For channel opening, two ligand molecules bound to the active state are required, and cooperativity results from the channel opening process itself. With carbamoylcholine, apparently, the equilibrium position for the channel opening step is only one-fourth open. The integrated rate equation, based on the model, predicts the time dependence of receptor-controlled ion flux over the concentration range of carbamoylcholine investigated. The values of the constants in the rate equation form the basis for predicting receptor-controlled changes in the transmembrane potential of cells and the conditions leading to transmission of signals between cells.
Immunoelectron microscopic localization of the site of photo-induced affinity labeling of the small ribosomal subunit with puromycin.
Authors: Olson H M , Grant P G , Glitz D G , Cooperman B S
Published: May 2006

Ribosomes from Escherichia coli strain TPR201 (which lack N6,N6-dimethyladenosine) have been photoaffinity labeled with [3H]puromycin in the presence of chloramphenicol. Puromycin-modified 30S ribosomal subunits appear to be identical to untreated subunits in electron micrographs and are efficiently precipitated by antibodies to the puromycin analog N6,N6-dimethyladenosine. Electron micrographs of subunit-antibody complexes show ribosomal subunits to which an individual antibody molecule is bound and pairs of 30S subunits which appear to be crosslinked by a single IgG molecule. A predominant site of puromycin photoaffinity labeling has been identified from the apparent point of contact of antibody and ribosomal subunit. The puromycin site is localized to the small upper portion of the particle on the side opposite to the subunit platform. This location is close to that reported for ribosomal protein S14, the major puromycin-labeled protein in the small ribosomal subunit.
Insulin and glucose 6-phosphate stimulation of Ca2+ uptake by skinned muscle fibers.
Authors: Brautigan D L , Kerrick W G , Fischer E H
Published: May 2006

Calcium uptake by skinned muscle fibers is stimulated by physiological concentrations of insulin. These fibers, which lack a functional plasma membrane, are permeable to macromolecules but retain extensive portions of their sarcolemma in the form of transverse tubules intercalated between the myofibrils. They have an active sarcoplasmic reticulum that removes 45Ca2+ from solution at concentrations below the threshold that initiates contraction (less than 1 microM). The Ca2+ uptake activity is stimulated by insulin, presumably in response to its binding to those receptors located in the transverse tubules. Addition of glucose 6-phosphate, whose intracellular concentration increases in response to insulin, also stimulates Ca2+ uptake, a unique property of this preparation. These data indicate that insulin and glucose 6-phosphate act in concert to stimulate the sarcoplasmic reticulum. The resulting decrease in myoplasmic Ca2+ and the increase in glucose 6-phosphate would serve to mediate some of the anabolic effects of the hormone.
Independent lines of evidence suggesting a major gap junctional protein with a molecular weight of 26,000.
Authors: Finbow M , Yancey S B , Johnson R , Revel J P
Published: May 2006

Several polypeptides have been described in the past as components of gap junction fractions. Of these, a peptide of Mr 26,000 is found in gap junctions isolated from livers of different species under conditions that minimize proteolysis. Tryptic digestion of purified "intact" junctions causes the rapid disappearance of this peptide with a concomitant appearance of a band of Mr 10,000, which has previously been found to be characteristic of junctional fractions isolated with the aid of proteolytic treatment. Both the peptides of Mr 26,000 and 10,000 are missing from gap junction preparations after partial hepatectomy, when gap junctions are absent from the surface of the hepatocytes. They have reappeared in fractions from the livers of animals killed 3 days postoperatively, when gap junctions are again present in vivo.
Deposition of fibronectin in the course of reverse transformation of Chinese hamster ovary cells by cyclic AMP.
Authors: Nielson S E , Puck T T
Published: May 2006

The Chinese hamster ovary (CHO) cell, like other transformed cells, has lost the fibronectin deposit around its membrane. Treatment with cyclic AMP derivatives restores the typical fibroblastic deposit of fibronectin. Thus, the reverse transformation process induced by cyclic AMP (cAMP) in the CHO cell restores this important property as well as other morphological, biochemical, and growth behavioral characteristics of the normal fibroblastic state. The fibronectin deposit occurs significantly later in time than do other characteristics of the reverse transformation reaction and may therefore reflect a metabolic action that requires other cAMP effect to precede it. The restoration of fibronectin deposition in response to cAMP derivatives is also exhibited by vole cells transformed by avian sarcoma virus, but it is not by HeLa cell. Addition of Colcemid, which disrupts microtubules, to CHO cells containing a fibronectin deposit induced by cAMP derivatives causes little or no erosion of the deposit, but cytochalasin B, which disrupts 5-nm microfilaments, eliminates it completely. Thus, various features of the action of cAMP derivatives on CHO and related cells require integrity of the cellular microfibrils--in some cases microtubules only, in some cases 5-nm microfilaments only, and in some cases both classes of fibrils.
Generic Galois extensions
Authors: Saltman David J.
Published: May 2006

We define the notion of a generic Galois extension with group
G
over a field
F
. Let
R
be a communtative ring of the form
F
[
x
1
,...,

x
n

](1/
s
) and let
S
be a Galois extension of
R
with group
G
. Then
S/R
is generic for
G
over
F
if the following holds. Assume
K/L
is a Galois extension of fields with group
G
and such that
L
⊇
F
. Then there is an
F
algebra map
f
:
R
→
L
such that
K
≅
S
[unk]

R

L
. We construct generic Galois extensions for certain
G
and
F
. We show such extensions are related to Noether's problem and the Grunwald-Wang theorem. One consequence is a simple proof of known counter examples to Noether's problem. On the other hand, we have an elementary proof of a chunk of the Grunwald-Wang theorem, and in a more general context. In fact, we have a Grunwald-Wang-type theorem whenever there is a generic extension for a group
G
over a field
F
.
Molecular structure and intermolecular interactions of N1'-methoxycarbonylbiotin methyl ester: a model for carboxybiotin.
Authors: Stallings W C , Monti C T , Lane M D , DeTitta G T
Published: May 2006

The crystal structure of N1'-methoxycarbonylbiotin methyl ester, a model for N1'-carboxybiotin, has been determined. The ureido carbonyl bond has more double bond (keto) character than does the corresponding bound in free biotin, which has single bond (enolate) character. In addition, there is an interesting intermolecular interaction between the ureido carbonyl oxygen and a methyl group. Comparison of the molecular structure and crystal packing with those of free biotin suggests that the coenzyme may have evolved with the incorporation of the ureido moiety because the electronic configuration of this region of the molecule is sensitive to N1' carboxylation. On decarboxylation, the ureido carbonyl bond becomes more polarized (C-O-), thereby facilitating the deprotonation of N1' and increasing its nucleophilicity. As a result, carboxylation can occur readily. On carboxylation, the carbonyl bond is depolarized (C = O), allowing the carboxylated coenzyme to interact with nonpolar groups and carboxylate them. Thus, the carboxylation and decarboxylation of biotin appear to act as a mechanistic switch, turning off and on the polarization of the ureido carbonyl bond as well as modulating the nucleophilicity of N1'.
Could poly(A) align the splicing sites of messenger RNA precursors?
Authors: Bina M , Feldmann R J , Deeley R G
Published: May 2006

In general, poly(A)-mRNA appears to be derived from larger nuclear RNA precursors. The maturation of these precursors involves excision of sequences of variable length from within the molecule and splicing of the remaining structural and coding sequences. The mechanism by which this process occurs is not known. It does not appear to operate solely through the recognition of a defined primary sequence or through the formation of a consistent secondary structure. We propose an alternative model in which poly(A) facilitates the splicing event by promoting the formation of triple-stranded structures within the mRNA precursor.
Induction of pyruvate carboxylase apoenzyme and holoenzyme in 3T3-L1 cells during differentiation
Authors: Freytag Svend O. , Utter Merton F.
Published: May 2006

The specific activity of pyruvate carboxylase [pyruvate:carbon-dioxide ligase (ADP-forming); EC 6.4.1.1] in 3T3-L1 cells increases approximately 20-fold when these cells differentiate to an adipocyte-like form [Mackall, J. C. & Lane, M. D. (1977)
Biochem. Biophys. Res. Commun.
79, 720-725]. A specific antibody to the purified rat liver enzyme quantitatively precipitated pyruvate carboxylase from 3T3-L1 crude homogenates. Use of this immunological technique permitted us to demonstrate that the increase in pyruvate carboxylase activity is due to an increase in the intracellular concentration of the enzyme. The content of pyruvate carboxylase in differentiated 3T3-L1 cells is sufficiently high (1-2% of total protein) that the increase in this large protein (subunit
M
r
= 130,000) can be visualized when 3T3-L1 crude extracts are subjected to electrophoresis on sodium dodecyl sulfate/polyacrylamide gels. When 3T3-L1 cells differentiated in the presence of avidin, they contained less than 5% of the pyruvate carboxylase activity of cells that differentiated in the absence of avidin. However, the immunoprecipitable pyruvate carboxylase content of the avidin-treated cells was essentially the same as that of cells that differentiated without avidin. Full activity of the enzyme was rapidly restored in the avidin-treated cells upon the addition of excess biotin. The recovery of activity was closely correlated with the incorporation of [
14
C]biotin into immunoprecipitable pyruvate carboxylase. The rapidity with which the activity was restored and the insensitivity of the process to inhibitors of protein synthesis strongly suggest that the apoenzyme of pyruvate carboxylase accumulates during differentiation in the presence of avidin.
Initiation of protein synthesis in bacteria at a translational start codon of mamalian cDNA: effects of the preceding nucleotide sequence.
Authors: Chang A C , Erlich H A , Gunsalus R P , Nunberg J H , Kaufman R J , Schimke R T , Cohen S N
Published: May 2006

Plasmids containing a mouse cDNA sequence encoding the enzyme dihydrofolate reductase (DHFR; tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3) have been used to study the efficiency of initiation of protein synthesis at an ATG (AUG) translational start codon indigenous to the eukaryotic CDNA. differences in DHFR production assayed phenotypically, enzymatically, and immunologically were correlated with the primary structure of the DNA segment that precedes the translational start codon. Our results indicate that initiation of a structurally discrete and biologically functional eukaryotic protein can occur in bacteria on a fused mRNA molecule, and that the efficiency of expression is strongly affected by: (i) the extent of homology of the translational control region with the 3'-OH end of 16S ribosomal RNA, and (ii) the distance between the protein start codon and the ribosome-binding sequence on the mRNA.
Role of G0-G1 arrest in the inhibition of tumor cell growth by interferon.
Authors: Creasey A A , Bartholomew J C , Merigan T C
Published: May 2006

We report here that human leukocyte interferon preparations are capable of influencing the transition of human melanoma cells from the "A" state to the "B" phase. Human melanoma cells that enter a quiescent stage at high cell density are more sensitive to the cytostatic action of interferon than those that continue to proliferate under similar conditions. Cell cycle perturbations caused by interferon in these cells include a decreased transition rate out of G0-G1 ("A" state) into S ("B" phase) and a prolongation of S. These findings support the idea that some metabolic event is required for progress through the G0-G1 phase of the cell cycle and is susceptible to interferon action.
Progesterone induces a rapid increase in [Ca2+]in of Xenopus laevis oocytes.
Authors: Wasserman W J , Pinto L H , O'Connor C M , Smith L D
Published: May 2006

Progesterone causes a rapid increase in the intracellular free calcium level in fully grown amphibian oocytes. When albino Xenopus laevis oocytes were microinjected with the Ca-specific photoprotein aequorin, the calcium-induced luminescence from this protein increased at 40-60 sec after the addition of progesterone and returned to the control level within 5-6 min. No further change in the aequorin glow could be detected through the remainder of the maturation period. This transient increase in cytoplasmic free calcium may be involved in controlling the resumption of oocyte maturation.
Introduction of transposon Tn901 into a plasmid of Anacystis nidulans: preparation for cloning in cyanobacteria.
Authors: van den Hondel C A , Verbeek S , van der Ende A , Weisbeek P J , Borrias W E , van Arkel G A
Published: May 2006

We have used the TEM beta-lactamase transposon Tn901, located on Escherichia coli plasmid pRI46, to introduce in vivo a genetic marker into plasmid pUH24, present in the cyanobacterial strain Anacystis nidulans R-2. Restriction enzyme analysis and heteroduplex studies of the 8.3 x 10(6)-dalton plasmids pCH1-pCH5, present in the ampicillin-resistant A. nidulans R-2 colonies obtained after transformation with pRI46, demonstrated that these plasmids consist of the complete sequence of Tn901 inserted at different places into plasmid pUH24. The pUH24::Tn901 recombinant plasmids transform A. nidulans R-2 with a frequency of 10(-4)--10(-5) per microgram of plasmid DNA and contain a single cleavage site for the restriction enzyme Xho I. From pCH1 a plasmid of 5.5 x 10(6) daltons,pUC1, was constructed with only a part of the Tn901 sequence and an additional single cleavage site for the restriction enzyme BamHI. This plasmid, as well as plasmids pCH1-pCH5, are potentially useful as vectors for cloning genes in cyanobacteria and for studying cyanobacterial plasmid biology.
Prediction of peptide retention times in high-pressure liquid chromatography on the basis of amino acid composition
Authors: Meek James L.
Published: May 2006

Analysis of peptides by reverse-phase high-pressure liquid chromatography would be simplified if retention times could be predicted by summing the contribution to retention of each of the peptide's amino acid side chains. This paper describes the derivation of values (“retention coefficients”) that represent the contribution to retention of each of the common amino acids and end groups. Peptide retention times were determined on a Bio-Rad “ODS” column at room temperature with a linear gradient from 0.1 M NaclO
4
, pH 7.4 or 2.1, at 0 min to 60% acetonitrile/0.1 M NaclO
4
at 80 min. The NaclO
4
, a chaotropic agent, was added to improve peak shape and to minimize conformational effects. Retention coefficients for the amino acids were computed by using a Hewlett-Packard 9815A calculator programmed to change the retention coefficients for all amino acids sequentially to obtain a maximum correlation between actual and predicted retention times. Correlations of 0.999 at pH 7.4 and 0.997 at pH 2.1 were obtained for 25 peptides including glucagon, oxytocin, [Met]enkephalin, neurotensin, and somatostatin. This high degree of correlation suggests that, for peptides containing up to 20 residues, retention is primarily due to partition processes that involve all the residues. Although steric or conformational factors do have some effect on retention, the data suggest that under the above chromatographic conditions the retention of peptides containing up to 20 residues can be predicted solely on the basis of their amino acid composition. This possibility was tested by using data taken from the literature.
Self-assembled chlorophyll
a
systems as studied by californium-252 plasma desorption mass spectroscopy
Authors: Hunt J. E. , Macfarlane R. D. , Katz J. J. , Dougherty R. C.
Published: May 2006

Self-assembled chlorophyll
a
and pheophytin
a
systems in thin solid films have been studied by
252
Cf plasma desorption mass spectrometry (PDMS). The
252
Cf-PDMS spectra of these films show monomer cation and anion molecular ions, ions of molecular aggregates, and positive and negative ion fragmentation patterns arising from the loss of various aliphatic side chains from the chlorin ring. Chlorophyll
a
films cast from dry carbon tetrachloride solution, in which chlorophyll
a
is known to occur as the dimer, produced an abundant dimer ion. The highest degree of chlorophyll
a
self-assembly was observed in chlorophyll
a
films cast from
n
-octane solutions. Oligomer ions extending upwards in size to the heptamer were detected in this system.
Fatty acid-requiring mutant of Saccharomyces cerevisiae defective in acetyl-CoA carboxylase.
Authors: Roggenkamp R , Numa S , Schweizer E
Published: May 2006

The isolation and biochemical properties of a Saccharomyces cerevisiae mutant (acc1-167) defective in acetyl-CoA carboxylase [acetyl-CoA:carbon-dioxide ligase (ADP forming), EC 6.4.1.2] activity are described. The mutant is deficient in de novo biosynthesis of long-chain fatty acids and specifically requires a saturated fatty acid of chain length 14-16 C atoms for growth. Fatty acid synthetase levels were normal, but the acetyl-CoA carboxylase specific activity of the purified enzyme was reduced to approximately 5% compared to wild-type yeast. Upon sodium dodecyl sulfate/polyacrylamide gel electrophoresis the purified mutant enzyme migrated as a single band and was essentially indistinguishable from the wild-type enzyme. The study of acetyl-CoA carboxylase partial activities revealed that the biotin incorporation capacity and the transcarboxylase partial activity were unaffected whereas the biotin carboxylase component enzyme exhibited less than 10% of wild-type specific activity. This biotin carboxylase mutational deficiency could be ascribed to a more than 90% reduction of Vmax and to a comparable increase in the Km value for ATP, which was accompanied by an increased requirement for Mg2+. It is concluded that acc1-167 contains a structural gene mutation in the biotin carboxylase domain of acetyl-CoA carboxylase.