bluuebunny/crossref_metadata_2025 huggingface dataset:
Interference of GTP hydrolysis in the mechanism of microtubule assembly: an experimental study.
Authors: Carlier M F , Hill T L , Chen Y
Published: May 2006

This paper reports an experimental study of the interference of GTP hydrolysis in the mechanism of microtubule assembly, following the model and theory previously published [Hill, T. L. & Carlier, M.-F. (1983) Proc. Natl. Acad. Sci. USA 80, 7234-7238]. Results from dilution experiments show that microtubules depolymerize faster below the critical concentration than expected with a reversible polymerization model. The experimental plot of flux versus tubulin concentration exhibits a slope discontinuity at the critical concentration, in agreement with the theory. Theoretical points calculated by the Monte Carlo method can be fitted qualitatively to the data. A consequence of this peculiar dynamic behavior of microtubules is that the ratio of tubulin dissociation and association rate constants measured, respectively, below and above the critical concentration does not yield the true value of the critical concentration. It is emphasized that the presence of GTP at microtubule ends is necessary to maintain the stability of the polymer.
Monoclonal antibodies detect a spectrin-like protein in normal and dystrophic human skeletal muscle.
Authors: Appleyard S T , Dunn M J , Dubowitz V , Scott M L , Pittman S J , Shotton D M
Published: May 2006

Spectrin is the major protein of the erythrocyte membrane skeleton, which is bound to the cytoplasmic surface of the membrane's lipid bilayer and is responsible for cell shape and membrane elasticity. Inability to identify spectrin in other cell types led to the assumption that this protein was unique to erythrocytes. However, spectrin-like proteins have been demonstrated recently in a variety of cell types, including skeletal and cardiac muscle, in several species. We used monoclonal antibodies against human erythrocyte spectrin subunits in an immunocytochemical study to detect related proteins in normal and diseased human skeletal muscle. Six of seven monoclonal antibodies against beta-spectrin determinants were bound at the cytoplasmic surface of muscle fiber plasma membranes, whereas none of six monoclonal antibodies against alpha-spectrin determinants was bound. Muscle fibers of patients with neuromuscular diseases showed similar distribution and specificity of antibody binding to those of normal subjects, but the intensity of binding was increased. In contrast, probable regenerating fibers in muscle of patients with muscular dystrophies showed reduced binding of antibodies, but reduced binding was not seen in fetal muscle fibers nor in those of a patient with a myotubular myopathy. We conclude that human skeletal muscle fibers possess a spectrin-related protein associated with their plasma membrane that shows extensive beta-chain similarities to erythrocyte spectrin but differs significantly with respect to the alpha-subunit. Its function may be associated with the maintenance of membrane and myofibril integrity during contraction, and the increased antibody binding in diseased muscle may reflect a structural rearrangement of spectrin or a compensatory increase in spectrin abundance in response to increased stress on these systems.
Induction of mouse metallothionein-I mRNA by bacterial endotoxin is independent of metals and glucocorticoid hormones.
Authors: Durnam D M , Hoffman J S , Quaife C J , Benditt E P , Chen H Y , Brinster R L , Palmiter R D
Published: May 2006

++Induction of metallothionein-I (MT-I) mRNA by bacterial endotoxin (LPS) was examined. A single injection of LPS induced MT-I mRNA accumulation in both liver and kidney comparable to that induced by heavy metals. Maximal message levels were achieved 6 hr after LPS administration, prior to any increase in either hepatic or renal Zn or Cu. Experiments in which LPS was administered to transgenic mice harboring recombinant genes made by fusing the MT-I gene promoter to the herpes simplex virus thymidine kinase structural gene revealed that the response to LPS is independent of glucocorticoid hormones. These experiments begin to define the region of the MT-I gene promoter required for the LPS response.
Induction of two genes by glucose starvation in hamster fibroblasts.
Authors: Lin A Y , Lee A S
Published: May 2006

The coordinated expression of two genes specifically induced by glucose starvation is demonstrated in a hamster fibroblast cell line, K12. Using two cDNA plasmids, p4A3 and p3C5, as hybridization probes, we examine the kinetics of induction of these genes when the cells are grown in medium deprived of glucose. The results show that (i) after a lag period of about 8 hr, there is a rapid and simultaneous increase of the p4A3 and p3C5 mRNA levels and (ii) the elevation of the mRNA levels for p4A3 and p3C5 is largely due to new transcription. In addition, we compare the mRNA transcripts encoded by these glucose-regulated genes in culture cells and phosphoenolpyruvate carboxykinase, the enzyme that catalyzes the rate-limiting step in gluconeogenesis in fasted rats. Our results indicate that the expression of phosphoenolpyruvate carboxykinase is not inducible by glucose starvation in our culture cells.
Formation of IgE-binding factors by human T-cell hybridomas.
Authors: Huff T F , Ishizaka K
Published: May 2006

Normal human T cells that proliferated in the presence of interleukin 2 (IL-2) formed IgE-binding factors when incubated with human IgE. These cells were then fused with a mutant of the human T-cell line CEM. Incubation of five hybridomas with human IgE or culture of the cells in IgE-coated wells resulted in the formation of IgE-binding factors. One hour of incubation with 10 micrograms of human IgE per ml was sufficient to induce the hybridomas to form IgE-binding factors. Polymerized IgE was much more efficient than monomeric IgE for the induction of the factor formation. As little as 10 ng of IgE dimer per ml was sufficient to induce factor formation. The IgE-binding factors produced by the hybridomas bound to human IgE-coated Sepharose and were recovered from the beads by elution at acid pH. The factors had low affinity for rat IgE but failed to bind to human IgG. The IgE-binding factors formed by four hybridomas had a Mr between 25,000 and 30,000, whereas one hybridoma formed IgE-binding factors of Mr 30,000 and Mr 15,000. The IgE-binding formed by all of the hybridomas had affinity for concanavalin A, indicating that the factors are glycoproteins.
Construction of shuttle vectors capable of conjugative transfer from Escherichia coli to nitrogen-fixing filamentous cyanobacteria.
Authors: Wolk C P , Vonshak A , Kehoe P , Elhai J
Published: May 2006

Wild-type cyanobacteria of the genus Anabaena are capable of oxygenic photosynthesis, differentiation of cells called heterocysts at semiregular intervals along the cyanobacterial filaments, and aerobic nitrogen fixation by the heterocysts. To foster analysis of the physiological processes characteristic of these cyanobacteria, we have constructed a family of shuttle vectors capable of replication and selection in Escherichia coli and, in unaltered form, in several strains of Anabaena. Highly efficient conjugative transfer of these vectors from E. coli to Anabaena is dependent upon the presence of broad host-range plasmid RP-4 and of helper plasmids. The shuttle vectors contain portions of plasmid pBR322 required for replication and mobilization, with sites for Anabaena restriction enzymes deleted; cyanobacterial replicon pDU1, which lacks such sites; and determinants for resistance to chloramphenicol, streptomycin, neomycin, and erythromycin.
ATP-dependent degradation of ubiquitin-protein conjugates.
Authors: Hershko A , Leshinsky E , Ganoth D , Heller H
Published: May 2006

Previous studies have indicated that the ATP-requiring conjugation of ubiquitin with proteins plays a role in the energy-dependent degradation of intracellular proteins. To examine whether such conjugates are indeed intermediates in protein breakdown, conjugates of 125I-labeled lysozyme with ubiquitin were isolated and incubated with a fraction of reticulocyte extract that lacks the enzymes that carry out ubiquitin-protein conjugation. ATP markedly stimulated degradation of the lysozyme moiety of ubiquitin conjugates to products soluble in trichloroacetic acid. By contrast, free 125I-labeled lysozyme was not degraded under these conditions, unless ubiquitin and the three enzymes required for ubiquitin conjugation were supplemented. Mg2+ was absolutely required for conjugate breakdown. Of various nucleotides, only CTP replaced ATP. Nonhydrolyzable analogs of ATP were not effective. In the absence of ATP, free lysozyme is released from ubiquitin-lysozyme conjugates by isopeptidases present in the extract. Thus, ATP is involved in both the formation and the breakdown of ubiquitin-protein conjugates.
Molecular model for elongation of the murein sacculus of Escherichia coli.
Authors: Burman L G , Park J T
Published: May 2006

Labeling experiments are presented that suggest that new (radioactive) strands of murein are initially inserted adjacent to old strands. After 8 min, new strands start to be inserted adjacent to the previously inserted radioactive strands. Analysis of these data suggests that, for Escherichia coli to double the length of the sacculus in each generation, about 90 separate membrane-bound enzyme complexes travel unidirectionally around the circumference of the cell. They travel at a constant rate, six times each generation, synthesizing, inserting, and crosslinking two strands of murein at a time, thereby doubling the length of the sacculus.
Evidence from bond lengths and bond angles for enneacovalence of cobalt, rhodium, iridium, iron, ruthenium, and osmium in compounds with elements of medium electronegativity
Authors: Pauling Linus
Published: May 2006

Enneacovalence of neutral atoms can be achieved for Co, Rh, and Ir by promoting some electrons from the
nd
orbital to the (
n
+ 1)
s
and (
n
+ 1)
p
orbitals and for Fe, Ru, and Os by a similar promotion together with the addition of an electron, which may be provided by an electron pair from a singly bonded carbonyl group or other group. The bond lengths and bond angles are predicted by the theory of enneacovalence to be significantly different for the different transition metals. Recently reported experimental values are shown to be in good agreement with the predicted values, providing support for the theory of enneacovalence and the theory of hybrid
sp
3
d
5
bond orbitals.
Embryonic----Fetal Hb switch in humans: studies on erythroid bursts generated by embryonic progenitors from yolk sac and liver.
Authors: Peschle C , Migliaccio A R , Migliaccio G , Petrini M , Calandrini M , Russo G , Mastroberardino G , Presta M , Gianni A M , Comi P
Published: May 2006

The synthesis of embryonic (zeta, epsilon), fetal (alpha, gamma), and adult (beta) globin was evaluated in human yolk sacs (YS) and livers at different ontogenic stages (i.e., from 6 through 10-12 wk of age) by means of analytical isoelectric focusing. Globin production was comparatively evaluated in vivo (i.e., in directly labeled erythroblasts from YS and liver) and in vitro [i.e., in erythroid bursts generated in culture by erythroid burst-forming units (BFU-E) from the same erythropoietic tissues]. Erythroid bursts generated in vitro by BFU-E from 6-wk livers and YS show essentially a "fetal" globin synthetic pattern: this is in sharp contrast to the "embryonic" pattern in corresponding liver and YS erythroblasts directly labeled in vivo. The invitro phenomenon suggests that (i) 6-wk BFU-E constitute a new generation of progenitors, which have already switched from an embryonic to a fetal program, and/or (ii) expression of their fetal program is induced by unknown in vitro factor(s), which may underlie the in vivo switch at later ontogenic stages. It is emphasized that 6- to 7-wk BFU-E are endowed with the potential for in vitro synthesis of not only epsilon- and gamma-chains but also some beta-globin. In general, we observed an inverse correlation between the levels of epsilon- and beta-chain synthesis. These results, together with previous studies on fetal, perinatal, and adult BFU-E, are compatible with models suggesting that in ontogeny the chromatin configuration is gradually modified at the level of the non-alpha gene cluster, thus leading to a 5'----3' activation of globin genes in a balanced fashion.
DNA polymorphic loci mapped to human chromosomes 3, 5, 9, 11, 17, 18, and 22.
Authors: Naylor S L , Sakaguchi A Y , Barker D , White R , Shows T B
Published: May 2006

Using the techniques of Southern filter hybridization and somatic cell genetics, seven genomic DNA fragments recognizing DNA polymorphic loci were mapped to specific chromosomes and regions of chromosomes. The seven probes, isolated from human genomic libraries, lacked repetitive sequences and were hybridized to DNA isolated from a set of human-rodent somatic cell hybrids segregating human chromosomes. These probes detected DNA sequences on human chromosomes 3, 5, 9, 11, 17, 18, and 22. These DNA polymorphic sites, which occur in 10% or greater of the population, will serve as markers for linkage studies with known polymorphic loci as well as to establish linkage with disease loci.
MAL6 of Saccharomyces: a complex genetic locus containing three genes required for maltose fermentation.
Authors: Needleman R B , Kaback D B , Dubin R A , Perkins E L , Rosenberg N G , Sutherland K A , Forrest D B , Michels C A
Published: May 2006

The MAL6 locus is one of five closely related unlinked loci, any one of which is sufficient for fermentation of maltose in Saccharomyces. Previous genetic analysis indicated that this locus is defined by two complementation groups, MALp and MALg. MALp reportedly is a regulatory gene required for inducible synthesis of the two enzymatic functions needed for fermentation: maltose permease and maltase. We have investigated the physical and genetic structure of the MAL6 locus, which has been isolated on a recombinant DNA plasmid. One subclone of the region, pDF-1, was found to encode a single transcribed region and to contain the MALp gene. A second subclone, p1, was shown to contain the MALg function but surprisingly had not one but two maltose-inducible transcripts. Subclones having only one of these transcribed regions lacked MALg activity. The three transcribed regions have been named MAL61 and MAL62, which correspond to MALg, and MAL63, which corresponds to MALp. This clustered arrangement of a regulatory gene adjacent to the sequences it controls has not previously been described in eukaryotes and is reminiscent of bacterial operons except that the messenger RNA molecules are not polycistronic.
Testicular peritubular cells secrete a protein under androgen control that modulates Sertoli cell functions.
Authors: Skinner M K , Fritz I B
Published: May 2006

Peritubular cells of the seminiferous tubule synthesize component(s) that stimulate Sertoli cells in culture to increase the production of androgen-binding protein and testicular transferrin. The active peritubular cell component(s) are trypsin-sensitive, heat-sensitive, acid-stable molecule(s) having a molecular weight between 50,000 and 100,000. These specific factors(s) are referred to as P Mod-S to designate protein(s), produced by peritubular cells (P), that modulate the functions of Sertoli cells (S). The degree of stimulation by P Mod-S is comparable to that obtained by maximal hormonal stimulation of the synthesis of ABP and transferrin by Sertoli cells. Levels of P Mod-S secreted into the medium by primary cultures of peritubular cells are increased in the presence of testosterone. Comparable concentrations of 17 beta-estradiol do not stimulate peritubular cells to synthesize P Mod-S. Data are interpreted to indicate that androgens act on testicular peritubular cells to increase the formation of P Mod-S and that P Mod-S may modulate the properties of adjacent Sertoli cells. Findings are discussed in relation to the nature of mesenchymal-epithelial cell interactions in the seminiferous tubule and to the possible role of P Mod-S as a mediator of androgen actions of Sertoli cells.
Glucose increases the synthesis of lipoxygenase-mediated metabolites of arachidonic acid in intact rat islets.
Authors: Metz S A
Published: May 2006

Previous studies suggested that products of a 12-lipoxygenase pathway in the pancreatic islet may promote insulin release. To determine whether glucose augments the production of such metabolites, intact rat islets prelabeled with [3H]arachidonate were stimulated with glucose, and 12-hydroxy-5,8,10,14-icosatetraenoic acid (12-HETE) release was measured by using HPLC. D-Glucose (16.7 mM) augmented the enzymatic synthesis of 12-HETE by 271% above that seen with 0-1.7 mM glucose. The glucose effect was stereospecific and preferential for the alpha anomer; it was modestly potentiated by the cyclo-oxygenase inhibitor ibuprofen. Glucose-stimulated 12-HETE accumulation was abrogated by mannoheptulose and was reproduced by the trioses glyceraldehyde or dihydroxyacetone, suggesting that the metabolism of glucose to glucose 6-phosphate or triose phosphates (or both) is critical. Glucose also augmented [3H]arachidonate labeling of islets, suggesting an action at the level of substrate release or re-uptake (or both). These features of islet 12-HETE synthesis accord well with other known effects of glucose on beta cell function and suggest that lipoxygenase-mediated metabolites of arachidonate may be suitable candidates to mediate or amplify glucose's effects on insulin release.
Thioredoxin is required for filamentous phage assembly.
Authors: Russel M , Model P
Published: May 2006

Sequence comparisons show that the fip gene product of Escherichia coli, which is required for filamentous phage assembly, is thioredoxin. Thioredoxin serves as a cofactor for reductive processes in many cell types and is a constituent of phage T7 DNA polymerase. The fip-1 mutation makes filamentous phage and T7 growth temperature sensitive in cells that carry it. The lesion lies within a highly conserved thioredoxin active site. Thioredoxin reductase (NADPH), as well as thioredoxin, is required for efficient filamentous phage production. Mutant phages defective in phage gene I are particularly sensitive to perturbations in the fip-thioredoxin system. A speculative model is presented in which thioredoxin reductase, thioredoxin, and the gene I protein interact to drive an engine for filamentous phage assembly.
Reovirus hemagglutinin mRNA codes for two polypeptides in overlapping reading frames.
Authors: Ernst H , Shatkin A J
Published: May 2006

Human reovirus s1 mRNA, which codes for the viral hemagglutinin, also directs the synthesis of a previously unrecognized polypeptide of molecular mass 14 kDa in reticulocyte and wheat germ extracts. Hybrid-arrest of translation by selected restriction fragments of cloned S1 DNA indicated that synthesis of the 14-kDa polypeptide initiates at the second AUG. This was confirmed by NH2-terminal sequence analyses. The coding sequence for the 14-kDa polypeptide thus lies entirely within the hemagglutinin gene but in a different reading frame. Although not found in virions, the 14-kDa polypeptide apparently is formed in virus-infected mouse L cells, as demonstrated by comparison of [35S]methionine-labeled polypeptides in cell extracts with the corresponding in vitro products.
Organization of the human transferrin gene: direct evidence that it originated by gene duplication.
Authors: Park I , Schaeffer E , Sidoli A , Baralle F E , Cohen G N , Zakin M M
Published: May 2006

We present the characterization of two overlapping human transferrin genomic clones isolated from a liver DNA library. The two clones represent a total length of 24 kilobase pairs and code for 70% of the protein. The organization of this gene region was elucidated by restriction mapping and DNA sequencing. It contains 12 exons, ranging from 33 to 181 base pairs, separated by introns of 0.7-4.9 kilobase pairs. This gene can be divided into two unequal parts corresponding to the known domains of the protein. Each part is essentially composed of an equal number of exons; introns interrupt the coding sequences, creating homologous exons of similar size in each moiety. Moreover, the pattern of intron interruption of the codon sequence is identical for all the analyzed homologous exon pairs. Comparison with the organization of the ovotransferrin gene shows an identical exon size distribution. These data confirm, at the gene level, the hypothesis that transferrins originated by a gene-duplication event. A model accounting for the origin of the human transferrin gene is presented.
Enzyme-catalyzed processes in organic solvents.
Authors: Zaks A , Klibanov A M
Published: May 2006

Three different lipases (porcine pancreatic, yeast, and mold) can vigorously act as catalysts in a number of nearly anhydrous organic solvents. Various transesterification reactions catalyzed by porcine pancreatic lipase in hexane obey Michaelis-Menten kinetics. The dependence of the catalytic activity of the enzyme in organic media on the pH of the aqueous solution from which it was recovered is bell-shaped, with the maximum coinciding with the pH optimum of the enzymatic activity in water. The catalytic power exhibited by the lipases in organic solvents is comparable to that displayed in water. In addition to transesterification, lipases can catalyze several other processes in organic media including esterification, aminolysis, acyl exchange, thiotransesterification, and oximolysis; some of these reactions proceed to an appreciable extent only in nonaqueous solvents.
Generation of a dominant 8-MDa deletion at the left terminus of vaccinia virus DNA.
Authors: Paez E , Dallo S , Esteban M
Published: May 2006

Vaccinia virus mutants were obtained in high frequency from mouse Friend erythroleukemia (FEL) cells persistently infected with this virus, which contains a large (122-MDa) DNA. During long-term cell passages viral particles with deletions of the DNA are generated in FEL cells. These mutants have a major 8-MDa deletion starting between 2.2 and 3.2 MDa from the left terminus of the viral genome. More than half of the left end terminal repetition is deleted. These mutants have reduced infectivity compared to wild-type virus. The ease with which vaccinia virus mutants are obtained in FEL cells should provide a suitable system for generating mutants with other poxvirus and permit study of the genetic basis of virulence for this group of viruses.
The postsynaptic density: a possible role in long-lasting effects in the central nervous system.
Authors: Siekevitz P
Published: May 2006

A theory is proposed that biochemical changes at the synapse that occur as a result of stimulation of specific neuronal circuits can lead to long-term changes only if alterations occur in synaptic structures in these circuits. The main synaptic structure that is thought to undergo this alteration is the postsynaptic density (PSD). There are many reports in the literature of overall structural changes at the synapse, including the PSD, resulting from various neuronal stimuli. These structural changes are here envisaged to include those of concentration and conformation of PSD proteins, changes that could alter the neural physiology of dendritic spines and even that of the presynaptic terminal.
An RNA-DNA copolymer whose synthesis is correlated with the transcriptional requirement for chromosomal initiation in
Bacillus subtilis
contains ribosomal RNA sequences
Authors: Séror-Laurent S.J. , Henckes G.
Published: May 2006

During synchronous replication induced in a temperature-sensitive initiation mutant of
Bacillus subtilis
, we previously isolated an RNA covalently linked to DNA. This molecule was synthesized at specific times, correlated with the period of transcription that is required to initiate a new round of replication. In this paper, we show that both RNA and DNA components of the RNA-DNA molecule hybridized with the coding strand for ribosomal RNA. Competition hybridization experiments also demonstrated that ribosomal RNA sequences represent the great majority of the RNA that is linked to DNA. Both RNA and DNA components of the RNA-DNA molecule also hybridized with a region close to the origin of replication, in particular with E19 and E22, two restriction fragments that are replicated early. These fragments in fact form part of the ribosomal operon
rrnO
. The role of the RNA-linked DNA molecule in initiation in
B. subtilis
and the possibility that this molecule emanates directly from
rrnO
rather than from other ribosomal RNA genes are discussed.
Structural effects of substitutions on the p21 proteins.
Authors: Pincus M R , Brandt-Rauf P W
Published: May 2006

The conformational effects of different amino acid substitutions (lysine, serine, proline, and D-valine) for glycine at position 12 in the p21 oncogene-encoded proteins have been investigated by using conformational energy calculations. The normal cellular gene codes for a glycine at position 12 in the amino acid sequence, in the middle of a hydrophobic p21-(6-15)-decapepetide from Leu-6 to Gly-15. Mutations that cause amino acid substitutions for Gly-12 result in a protein product that produces malignant transformation of cells. We now find that not only are the preferred structures for the lysine- and serine-containing peptides more restricted and more helical than those for the glycine-containing peptide, but the lowest-energy structure for each substituted peptide is exactly the same as that previously found for the peptide with Val-12, suggesting the existence of a "malignancy-causing" conformation. None of the preferred conformations for the valine-, lysine-, and serine-containing peptides contain chain reversals at positions 11 and 12. However, we find that proline, unlike these residues but like glycine, at position 12 causes helix termination at positions 11 and 12, a result that suggests that the p21 protein product with proline at position 12 may exhibit lowered transforming potential, in agreement with the results of recent genetic recombination experiments.
Bacterial peptide chain release factors: conserved primary structure and possible frameshift regulation of release factor 2.
Authors: Craigen W J , Cook R G , Tate W P , Caskey C T
Published: May 2006

Escherichia coli peptide chain release factors are proteins that direct the termination of translation in response to specific peptide chain termination codons. The mechanisms of codon recognition and peptidyl-tRNA hydrolysis are unknown. We have characterized the genes encoding release factor 1 (RF-1) and release factor 2 (RF-2) to study the structure-function relationships of the proteins and their regulation in the bacterium. In this report, we present the gene structure of RF-1 and RF-2, and a partial peptide sequence of RF-2. RF-1 and RF-2 are highly homologous in their primary structure. In addition, an in-frame premature opal (UGA) termination codon is located within the RF-2 coding region at amino acid position 26. This region of the protein was sequenced by automated Edman degradation to confirm the predicted reading frame, and a second independent isolate of the RF-2 gene was identified and sequenced to confirm the DNA sequence. These results imply that a frameshift occurs prior to the premature termination codon, thus allowing for translation of RF-2 to be completed. This may represent a mechanism of translational control of RF-2 expression. An alternative possible means of translational regulation is discussed.
Reduction of mammalian ferritin.
Authors: Watt G D , Frankel R B , Papaefthymiou G C
Published: May 2006

Mammalian ferritin from horse spleen undergoes an electrochemical or chemical reduction reaction in which each iron atom present is reduced by one electron (2300 electrons per ferritin molecule containing 2300 Fe3+ ions). Midpoint potentials of -190 mV, -310 mV, and -416 mV were determined at pH 7.0, 8.0, and 9.0. This variation of potential with pH indicates that approximately 2 H+ are transferred to the core for each Fe3+ reduced to Fe2+. Mössbauer measurements of partially reduced ferritin give spectra that consist of a ferric quadrupole doublet with a superposed ferrous quadrupole doublet. The relative intensities of these doublets are consistent with the electrochemically determined degree of reduction.
The alpha-spectrin gene is on chromosome 1 in mouse and man.
Authors: Huebner K , Palumbo A P , Isobe M , Kozak C A , Monaco S , Rovera G , Croce C M , Curtis P J
Published: May 2006

By using alpha-spectrin cDNA clones of murine and human origin and somatic cell hybrids segregating either mouse or human chromosomes, the gene for alpha-spectrin has been mapped to chromosome 1 in both species. This assignment of the mouse alpha-spectrin gene to mouse chromosome 1 by DNA hybridization strengthens the previous identification of the alpha-spectrin locus in mouse with the sph locus, which previously was mapped by linkage analysis to mouse chromosome 1, distal to the Pep-3 locus. By in situ hybridization to human metaphase chromosomes, the human alpha-spectrin gene has been localized to 1q22-1q25; interestingly, the locus for a non-Rh-linked form of elliptocytosis has been provisionally mapped to band 1q2 by family linkage studies.
Inhibition of Ca2+ conductance in identified leech neurons by benzodiazepines.
Authors: Johansen J , Taft W C , Yang J , Kleinhaus A L , DeLorenzo R J
Published: May 2006

Benzodiazepines (BZs) in micromolar concentrations inhibit Mn2+- and Co2+-sensitive regenerative divalent cation potentials, which are revealed in the presence of tetraethylammonium ion, in leech nociceptive neurons (N cells). This BZ effect is reversible and dose-dependent. The BZs, like Mn2+ and Co2+, inhibit the maximum rate of depolarization (Vmax) and duration of divalent cation potentials at concentrations that do not significantly affect resting membrane potential or Vmax of the Na+-dependent action potential. Ultraviolet-induced BZ binding to micromolar-affinity sites in ganglia and isolated cells irreversibly blocks Ca2+ conductance in neurons without significantly affecting resting membrane potentials. BZ binding studies with leech neuronal membrane show saturable, specific binding in the micromolar concentration range that was similar to BZ binding to synaptosomal membrane fractions. The apparent Kd obtained from the micromolar-affinity BZ binding curve for leech ganglionic membrane preparations agrees well with the apparent Ki estimated from the dose-response curve measuring BZ inhibition of Vmax of the divalent cation potentials. These findings indicate that BZs act like Ca2+-channel antagonists in intact neuronal preparations and are consistent with the hypothesis that BZ binding to micromolar-affinity receptors modulates voltage-gated Ca2+ channels.
Monoclonal antibody 18B8 detects gangliosides associated with neuronal differentiation and synapse formation.
Authors: Grunwald G B , Fredman P , Magnani J L , Trisler D , Ginsburg V , Nirenberg M
Published: May 2006

Mouse monoclonal antibody 18B8 detects developmentally regulated antigens in chicken retina and brain. The antigens detected by immunofluorescence appear initially on cell bodies in retinas of 6-13 day embryos. In older embryos during synapse formation and in adults, the antigen is localized in discrete laminae within the inner synaptic layer of retina and also is present in the outer synaptic layer and the outer segments of photoreceptor cells. The antigens from retina and brain were purified partially and were shown to be gangliosides of unknown structure that contain at least two sialic acid residues. Gangliosides that are recognized by antibody 18B8 change both qualitatively and quantitatively during neuronal development. These changes were correlated with the spatial and temporal changes in antigen expression detected histochemically.
Activation of expression of latent Epstein-Barr herpesvirus after gene transfer with a small cloned subfragment of heterogeneous viral DNA.
Authors: Countryman J , Miller G
Published: May 2006

We previously found that a form of Epstein-Barr virus with rearranged DNA induces replication of latent Epstein-Barr virus. We now have found that one of three fragments of this rearranged DNA, when cloned in recombinant plasmids and used to transfect cells, can activate expression of several polypeptides from a latent viral genome. The 33-kDa protein that is the product of the active fragment is likely to be responsible for disruption of latency.
Two-photon spectroscopy of locked-11-cis-rhodopsin: evidence for a protonated Schiff base in a neutral protein binding site.
Authors: Birge R R , Murray L P , Pierce B M , Akita H , Balogh-Nair V , Findsen L A , Nakanishi K
Published: May 2006

We studied the nature of the protein binding site of rhodopsin, using two-photon spectroscopy to assign the location of the low-lying "covalent" 1Ag*- -like pi pi * state in a model rhodopsin containing a locked-11-cis chromophore. The two-photon thermal lens maximum is observed at 22,800 cm-1, approximately equal to 2000 cm-1 above the one-photon absorption maximum, indicating that the protein environment has induced a level ordering reversal of the low-lying pi pi * states relative to that observed in retinyl Schiff bases in solution. The spectroscopic results clearly indicate that the chromophore is protonated and that the binding site is uncharged. Electrostatic energy contour maps of the binding site are calculated, showing possible locations for the external counterion(s). Two models of the binding site are proposed that accommodate the available spectroscopic data. One model involves a protonated Schiff base chromophore stabilized by a single negatively charged aspartic or glutamic acid residue. A more complicated model involving two residues (one charged, the other neutral) is also proposed. The latter model is interesting because it also accommodates the observed deuterium isotope effect in the form of a proton translocation between the two residues. The translocation is assumed to be a ground state process, initiated subsequent to the photoisomerization of the chromophore and energetically driven via destabilization of the counterion environment as a result of isomerization-induced charge separation.
Isolation and properties of soybean [
Glycine max
(L.) Merr.] mutants that nodulate in the presence of high nitrate concentrations
Authors: Carroll Bernard J. , McNeil David L. , Gresshoff Peter M.
Published: May 2006

Soybean seeds [
Glycine max
(L.) Merr. ev. Bragg] were mutagenized with ethyl methanesulfonate. The M
2
progeny (i.e., the first generation after mutagenesis) of these seeds were screened for increased nodulation under high nitrate culture conditions. Fifteen independent nitrate-tolerant symbiotic (
nts
) mutants were obtained from 2500 M
2
families. In culture on sand with KNO
3
, nodule mass and nodule number in mutant lines were several-fold those of the wild type cultured under the same conditions. Inheritance of the nts character through to subsequent generations was observed in the 10 mutants tested. Mutant
nts
382 also nodulated more than the wild type in the absence of nitrate. Furthermore, nitrate stimulated growth in both the wild type and
nts
382, and these lines had similar nitrate reductase activity. These results indicate that
nts
382 is affected in a nodule-development regulatory gene and not in a gene related to nitrate assimilation.
Characterization of a continuous T-cell line susceptible to the cytopathic effects of the acquired immunodeficiency syndrome (AIDS)-associated retrovirus.
Authors: Folks T , Benn S , Rabson A , Theodore T , Hoggan M D , Martin M , Lightfoote M , Sell K
Published: May 2006

We have developed a continuous human T-cell line (A3.01) for the study of acquired immunodeficiency syndrome (AIDS)-associated retrovirus that mimics normal peripheral blood lymphocytes in susceptibility to viral cytopathic effect without the need for cell activation or conditioned medium. Following infection, substantial quantities of virus are produced during a 3- to 5-day period; the associated killing of cells can be monitored in a microtiter assay as a function of virus input. Southern blot hybridization of infected cellular DNAs indicated that no gross alteration occurred in the restriction maps of the proviral DNA during the transfer of virus to and its passage in A3.01 cells. This cell system offers an alternative to other AIDS retrovirus cell systems because it permits the monitoring of viral cytopathic effects.
Reactivation by a trans-acting factor of human major histocompatibility complex Ia gene expression in interspecies hybrids between an Ia-negative human B-cell variant and an Ia-positive mouse B-cell lymphoma.
Authors: Accolla R S , Carra G , Guardiola J
Published: May 2006

Raji, a human B-lymphoma cell line, expresses high levels of class II (Ia) antigens. The expression of Ia antigens is totally abolished at the level of specific mRNA accumulation in RJ 2.2.5, a variant cell line derived from Raji after mutagenesis and immunoselection. We report here that the human Ia antigen expression can be restored in interspecies somatic cell hybrids between RJ 2.2.5 cells and BALB/c-derived M12.4.1 (I-A+, I-E+) B-lymphoma cells. Two hybrid clones were studied in detail. In both clones Ia molecules of the DR and NG2 type were easily detected by cell surface immunofluorescence and specific immunoprecipitation. In contrast, the DQ1 molecules were not detected with the above technique. DNA hybridization experiments using specific probes indicated that alpha-chain DR and beta-chain DQ genes were present in the hybrids. However, RNA hybridization experiments revealed that beta-chain DQ mRNA was present in the hybrids at very low amount compared to alpha-chain DR-specific mRNA. These results indicate that at least several genes of the class II gene cluster are coordinately regulated by trans-acting factor(s) that operate across species barriers. The basis of the mechanisms controlling the expression of class II antigens in these human-mouse hybrids might be related to the extinction (lack of expression) or activation of tissue-specific traits that take place when genomes of cells of dissimilar developmental potentials are brought together.
Identification of a nucleic acid helix-destabilizing protein from rat liver as lactate dehydrogenase-5.
Authors: Williams K R , Reddigari S , Patel G L
Published: May 2006

A rat liver DNA helix-destabilizing protein (HDP) that has previously been proposed to play a role in transcription has been identified as M chain lactate dehydrogenase (LDH-5; L-lactate:NAD+ oxidoreductase, EC 1.1.1.27). Tryptic peptides accounting for 157 amino acids in the rat liver HDP have been characterized and then matched to the published sequence for the M chain of porcine LDH. Based on amino acid compositions and direct solid-phase protein sequencing, at least 148 of the 157 residues that were compared are identical in both proteins. In addition, both porcine LDH and the rat liver HDP have blocked amino termini and similar amino acid compositions and molecular weights. Rat liver HDP and LDH-5 that were purified to molecular homogeneity had similar specific activities in both single-stranded DNA (ss DNA) binding and LDH assays. HPLC tryptic peptide maps were also identical for both the rat liver HDP and LDH proteins. Since preincubation of HDP in NADH prevents its binding to ss DNA, both NADH and ss DNA may be binding at the same site. Further support for this latter idea derives from chemical-modification studies which demonstrate that tyrosine-238, which is located near the coenzyme binding site of LDH, seems to be essential for the ability of HDP to bind ss DNA. These results indicate caution in ascribing in vivo roles solely on the basis of binding to ss DNA. Alternatively, they suggest that a single protein may play more than one biological role.
Immunoglobulin gene rearrangements and deletions in human Epstein-Barr virus-transformed cell lines producing different IgG and IgA subclasses.
Authors: Webb C F , Cooper M D , Burrows P D , Griffin J A
Published: May 2006

During differentiation B lymphocytes may switch from the expression of surface IgM to the synthesis of IgG, IgA, or IgE isotypes by using a different heavy chain constant region (CH) gene. The molecular mechanisms by which switching occurs remain controversial. Rearrangements and deletions of CH genes 5' to the expressed gene have often been observed in the mouse and, more recently, in human cells that have switched isotypes. We have used human JH, C micro, C gamma, and C alpha probes to examine the extent of the deletions and rearrangements in clones of Epstein-Barr virus-transformed human cells that produce IgG1, IgG3, IgG4, or IgA1. Though deletions of CH genes 5' to the expressed CH gene were consistently observed, the rearrangement process appeared to be highly variable for the nonproductive CH gene locus: deletion or persistence of 5' CH genes, combinations of deletion and duplication of 5' genes, and deletions extending to 3' CH genes. Our results reveal an unexpected lack of specificity in the DNA deletions in cells that have undergone isotype switching.
Expression of genes transferred into monocot and dicot plant cells by electroporation.
Authors: Fromm M , Taylor L P , Walbot V
Published: May 2006

We have developed a general method for electrically introducing DNA into plant cells. Gene transfer occurs when a high-voltage electric pulse is applied to a solution containing protoplasts and DNA. Carrot protoplasts were used as a model system to optimize gene-transfer efficiency, which was measured 24-48 hr after electroporation by the amount of chloramphenicol acetyltransferase activity resulting from the expression of the introduced chimeric plasmids. Gene-transfer efficiency increased with the DNA concentration and was affected by the amplitude and duration of the electric pulse as well as by the composition of the electroporation medium. Our optimized gene-transfer conditions were effective when applied to tobacco and maize protoplasts, demonstrating that the method is applicable to both monocot and dicot protoplasts.
A third viral nuclear protein in lymphoblasts immortalized by Epstein-Barr virus.
Authors: Hennessy K , Fennewald S , Kieff E
Published: May 2006

Most sera from patients with rheumatoid arthritis as well as some sera from normal Epstein-Barr virus (EBV)-infected people detect a 140-kDa protein on immunoblots of EBV-infected lymphoblasts. The 140-kDa protein is a nuclear protein characteristic of latent EBV infection. Sera reactive with this protein identify a distinctive globular nuclear antigen. Although the 140-kDa protein is encoded by EBV, it is not encoded by genes that encode the two previously described EBV nuclear antigens (EBNA) or the latent-infection membrane protein. The 140-kDa protein is therefore designated EBNA3. The EBV genes, including the gene encoding EBNA3, that are characteristically expressed in latent infection are likely to play a role in the maintenance of persistent latent viral infection or in the cell proliferation caused by virus infection.
Acetylcholine and phorbol esters inhibit potassium currents evoked by adenosine and cAMP in Xenopus oocytes.
Authors: Dascal N , Lotan I , Gillo B , Lester H A , Lass Y
Published: May 2006

In Xenopus laevis oocytes, adenosine and other purinergic agonists induce a K+-conductance increase that is fully mimicked by intracellular application of cAMP. Acetylcholine suppresses the K+-conductance increase caused by adenosine, by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, or by intracellular injection of cAMP. This effect of acetylcholine is not mimicked by intracellular injection of Ca2+ or of the Ca-mobilizing agent inositol 1,4,5-trisphosphate. However, adenosine and cAMP responses are inhibited by 4 beta-phorbol 12,13-dibutyrate and 4 beta-phorbol 12-myristate 13-acetate. These results suggest that, in Xenopus oocytes, the muscarinic inhibition of purinergic and cAMP responses is mediated through the activation of the phospholipid-dependent, Ca-activated protein kinase (protein kinase C).
Pure brain-derived acidic fibroblast growth factor is a potent angiogenic vascular endothelial cell mitogen with sequence homology to interleukin 1.
Authors: Thomas K A , Rios-Candelore M , Giménez-Gallego G , DiSalvo J , Bennett C , Rodkey J , Fitzpatrick S
Published: May 2006

Pure bovine brain-derived acidic fibroblast growth factor is a very potent mitogen for vascular endothelial cells in culture and, in the presence of heparin, induces blood vessel growth in vivo. Partial amino acid sequence determinations confirm that this mitogen is a unique protein having amino acid sequence homology with human interleukin 1.
RNase T is responsible for the end-turnover of tRNA in Escherichia coli.
Authors: Deutscher M P , Marlor C W , Zaniewski R
Published: May 2006

A mutant strain deficient in RNase T was isolated and used to study the role of this enzyme in Escherichia coli. Strains lacking as much as 70% of RNase T activity, alone or in combination with the absence of other RNases, display normal growth properties. However, in cca strains, which lack tRNA nucleotidyltransferase, RNase T-deficient derivatives accumulate lower levels of defective tRNA and grow at increased rates compared to their RNase T+ parents. Slow-growing cca strains revert to a faster-growing form that contains less defective tRNA but which is still cca. All of these strains have decreased levels of RNase T. These data indicate that RNase T is responsible for nucleotide removal during the tRNA end-turnover process and that the amount of defective tRNA in cells is determined by the relative levels of RNase T and tRNA nucleotidyltransferase.
Aleurain: a barley thiol protease closely related to mammalian cathepsin H.
Authors: Rogers J C , Dean D , Heck G R
Published: May 2006

We have isolated and sequenced a 1400-base-pair cDNA derived from gibberellic acid-treated aleurone cell mRNA. This sequence contains an open reading frame that would code for a protein of 361 amino acids. The carboxyl-terminal two-thirds of the predicted amino acid sequence is closely related to that of the rat lysosomal thiol protease cathepsin H; the initial 143 amino acids may code for a secretory peptide plus a prosegment. The expression of this aleurone thiol protease mRNA is unusual in that, in aleurone cells, its abundance is regulated by the plant hormones gibberellic acid and abscisic acid, but it is also expressed at high levels in leaf and root tissue. This protease may represent the equivalent of a plant lysosomal thiol protease.
UV-induced mutagenesis of phage S13 can occur in the absence of the RecA and UmuC proteins of Escherichia coli.
Authors: Tessman I
Published: May 2006

The UV-induced mutagenesis of phage S13 that accompanies Weigle repair is known to require the products of the recA and umuDC genes, as does the UV-induced mutagenesis of the Escherichia coli chromosome. I found that UV-induced mutagenesis of phage S13 occurred in the absence of both the RecA and UmuC functions when the irradiated phage was photoreactivated. Furthermore, UV-induced phage mutations were produced in a recA- umuC- cell even without photoreactivation and in the absence of any other known UV repair mechanism, at a frequency 29% of that found after photoreactivation and 7% of that found after Weigle repair, implying that DNA synthesis can proceed past a dimer at an unexpectedly high frequency even when unaided by the UmuC-RecA SOS repair functions. The unaided DNA synthesis appears capable of producing mutations in the vicinity of a pyrimidine dimer; by aiding synthesis past a dimer, a repair mechanism may disclose a mutation without having any active role in producing it.