bluuebunny/crossref_metadata_2025 huggingface dataset:
Dynamics of heme iron in crystals of metmyoglobin and deoxymyoglobin.
Authors: Bauminger E R , Cohen S G , Nowik I , Ofer S , Yariv J
Published: May 2006

The 57Fe gamma-ray resonance absorption spectra have been measured in crystals of metmyoglobin and deoxymyoglobin over a wide range of temperatures. Above a critical temperature common to both proteins (220 K), the dynamics of heme iron display a dramatic change, in that two kinds of thermal fluctuations come into play--a fast fluctuation associated with a steep decrease of the total fluctuation of characteristic time 10(-8) sec, associated with bounded diffusive motion. By using both discrete jump and continuous diffusion models, the latter based on the Brownian motion of an overdamped harmonic oscillator, the essential parameters of the iron motion (mean square displacement and jump frequency or diffusion constant) can be derived as a function of temperature. Thus, for deoxy Mb at 288 K, the mean square displacement for the fast fluctuation is about 6 X 10(-2) A2 and for the diffusive motion is 1.6 X 10(-2) A2; the diffusion constant is 4 X 10(-10) cm2/sec. The diffusive process is associated with an activation energy of about 0.75 kcal/mol. Although the same general kinds of phenomena are observed in crystals of MetMb and deoxy Mb, significant differences in behavior are found, which suggest that the main dynamical phenomenon observed reflects internal large-scale motions of the protein.
Human hypoxanthine (guanine) phosphoribosyltransferase: an amino acid substitution in a mutant form of the enzyme isolated from a patient with gout.
Authors: Wilson J M , Tarr G E , Kelley W N
Published: May 2006

We have investigated the molecular basis for a deficiency of the enzyme hypoxanthine (guanine) phosphoribosyltransferase (HPRT; IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) in a patient with a severe form of gout. We reported in previous studies the isolation of a unique structural variant of HPRT from this patient's erythrocytes and cultured lymphoblasts. This enzyme variant, which is called HPRTLondon, is characterized by a decreased concentration of HPRT protein in erythrocytes and lymphoblasts, a normal Vmax, a 5-fold increased Km for hypoxanthine, a normal isoelectric point, and an apparently smaller subunit molecular weight. Comparative peptide mapping experiments revealed a single abnormal tryptic peptide in HPRTLondon. Edman degradation of the aberrant peptide from HPRTLondon identified a serine-to-leucine amino acid substitution at position 109. This substitution can be explained by a single nucleotide change in the codon for serine-109 (UCA leads to UUA). Thus a mutation at the HPRT locus has now been defined at the molecular level.
Epithelial cell types and their evolution in the rat mammary gland determined by immunological markers.
Authors: Dulbecco R , Unger M , Armstrong B , Bowman M , Syka P
Published: May 2006

We have used immunological reagents for characterizing epithelial cell types in the adult rat mammary gland. Some were sera against purified proteins; others were monoclonal antibodies to mammary cells. On the basis of marker distribution we have identified 10 different cell types. We have established the developmental connection between these types by adopting the principle that cells displaying the same markers are directly related to each other. The results concur with those previously obtained by studying the growth response of mammary cells to the hormonal changes of the estrus cycle.
Isolation and characterization of the plasma membrane of human erythrocytes infected with the malarial parasite Plasmodium falciparum.
Authors: Gruenberg J , Sherman I W
Published: May 2006

Human erythrocytes infected with the malarial parasite Plasmodium falciparum were labeled metabolically with a mixture of 15 radioactive amino acids. When synchronously growing parasites were at the schizont stage of development infected cells were concentrated and purified by using a Percoll-Hypaque gradient. The plasma membrane of the infected erythrocyte, isolated by binding cells to a solid support (Affi-Gel 731, Bio-Rad), was less than 1% contaminated with parasite membranes. Erythrocyte membrane proteins were analyzed by polyacrylamide gel electrophoresis and autoradiography. Despite the high sensitivity of the procedure, there was no evidence for the insertion of parasite proteins into the infected host cell membrane. One possible exception is a Mr 230,000 parasite protein present maximally as 9,000 copies per infected erythrocyte membrane. Moreover, no differences in the membrane proteins were observed between a highly knobby clone and a knobless clone of the same strain of P. falciparum. These findings appear to rule out the presence of parasite protein(s) playing a structural role in the formation of knobs on the erythrocyte surface and question whether the antigenic determinants on the P. falciparum-infected erythrocyte are of parasite origin or whether such antigens represent newly exposed or chemically modified erythrocyte determinants.
Immunohistochemical distribution of dynorphin B in rat brain: relation to dynorphin A and alpha-neo-endorphin systems.
Authors: Weber E , Barchas J D
Published: May 2006

A specific antiserum was prepared against dynorphin B, an endogenous opioid peptide contained in a recently isolated 4,000-dalton dynorphin. The antiserum did not crossreact with dynorphin A, alpha-neo-endorphin, beta-neo-endorphin, dynorphin-(1-8), or [Leu]enkephalin. In immunohistochemical staining experiments on frozen sections through rat brains from normal and colchicine-treated animals, the antiserum labeled the same neuronal fiber systems previously described as containing both dynorphin A and alpha-neo-endorphin immunoreactive material. The alpha-neo-endorphin/dynorphin A immunoreactive perikarya in the hypothalamic magnocellular nuclei also were labeled by the dynorphin B antiserum. In addition, the dynorphin B antiserum revealed groups of immunoreactive neuronal cell bodies in several other hypothalamic and extrahypothalamic areas, including brain-stem, midbrain, central nucleus of amygdala, and in the dorsomedial, lateral, and anterior nuclei of hypothalamus. These perikarya had not been detected in previous studies that used dynorphin A and alpha-neo-endorphin antisera. The findings are in agreement with recent studies demonstrating a common precursor for dynorphin A, dynorphin B, and alpha-neo-endorphin. The apparently wider distribution of dynorphin B immunoreactive cell bodies compared to alpha-neo-endorphin/dynorphin A immunoreactive perikarya may be a reflection of differential processing of the precursor in different brain regions.
Secondary structures of proteins and peptides in amphiphilic environments. (A review).
Authors: Kaiser E T , Kézdy F J
Published: May 2006

Many peptides and proteins that act at lipid--water interfaces assume a unique amphiphilic secondary structure which is induced by the anisotropy of the interface. By using synthetic peptides in which these inducible amphiphilic structures have been optimized, one can show that the amphiphilic alpha helix is a functional determinant of representative apolipoproteins, peptide toxins, and peptide hormones. By increasing the amphiphilicity of the structurally important regions of the molecule, one can enhance the biological activity of the peptide even beyond that of the naturally occurring polypeptide. It is proposed that rigid amphiphilic secondary structures such as alpha helix, beta sheet, or pi helix will be found in most medium-sized peptides acting at membranes and lipid--water interfaces.
An intermediate step in translocation of lipopolysaccharide to the outer membrane of Salmonella typhimurium.
Authors: Mulford C A , Osborn M J
Published: May 2006

Evidence for transient localization of newly synthesized lipopolysaccharide at the periplasmic face of the inner membrane has been obtained by immunoelectron microscopic techniques. Salmonella typhimurium galE mutants in which O-antigen synthesis is dependent on addition of exogenous galactose were employed, and the distribution and fate of pulse-synthesized O antigen was examined by indirect ferritin labeling with anti-O-antigen IgG of spheroplasts prepared by treatment with lysozyme/EDTA. O-reactive lipopolysaccharide appeared rapidly at the exposed periplasmic face of the inner membrane after addition of galactose and was rapidly depleted upon termination of the pulse. Control experiments showed that secondary redistribution of lipopolysaccharide from outer membrane did not occur under the conditions employed for spheroplast formation and immunolabeling, and the pulse-chase kinetics were consistent with those expected for an intermediate in translocation of lipopolysaccharide to the outer membrane. In addition, undecaprenol-linked O antigen was detectable at the periplasmic face of the inner membrane within 30 sec after addition of galactose to a galE deep rough double mutant, and it accumulated stably in that location. The mutation in synthesis of the lipopolysaccharide core in the deep rough strain prevents transfer of O-antigen chains from undecaprenol phosphate to lipopolysaccharide. The result suggests that attachment of O antigen to lipopolysaccharide occurs on the extracytoplasmic side of the inner membrane and supports the conclusion that lipopolysaccharide is translocated to the outer membrane from the periplasmic, rather than the cytoplasmic, face of the inner membrane.
Potassium-dependent increases in cytosolic pH stimulate metabolism and motility of mammalian sperm.
Authors: Babcock D F , Rufo G A , Lardy H A
Published: May 2006

Sperm cytosolic pH, determined by the spectral properties of intracellular carboxyfluorescein, is decreased rapidly by the diffusion and subsequent dissociation of the uncharged weak acids pyruvic, lactic, or hydroxybutyric and is increased by diffusion and subsequent intracellular protonation of the weak base NH3. Metabolic and kinetic activity increases dramatically when intracellular pH is elevated above 6.8-6.9 by addition of 50 mM NH4Cl to sperm suspended in a 120 mM NaCl medium. Respiratory stimulation is not observed upon comparable additions of 50 mM Li+ or K+ or when the pH of the medium is increased from 6.5 to 8.2. However, increases of the external pH to 7.8-8.2 in medium employing 120 mM KCl result in increased metabolic and kinetic activity, comparable to the maximal stimulation induced by the phosphodiesterase inhibitor caffeine. An increase in cytosolic pH from 6.3-6.6 to 6.8 occurs concomitant with the respiratory stimulation induced by KCl in alkaline media. No change in cytosolic pH follows addition of caffeine. Cyclic AMP-dependent protein kinase activity ratios, determined in cellular extracts, are increased by caffeine treatment but are not elevated by 120 mM KCl, by alkaline pH, or by their combination. These observations indicate that cytosolic pH plays a role in the regulation of motility and metabolism of mammalian sperm that is not mediated by cyclic AMP but that may be under control of a plasma membrane voltage-dependent proton channel. However, H+ fluxes across vesicles prepared from sperm membranes are unaffected by variation in the magnitude of the transvesicular K+ concentration gradient.
Optimal sequence alignments
Authors: Fitch Walter M. , Smith Temple F.
Published: May 2006

Current theory is adequate to the task of finding an optimal alignment between two character strings such as nucleic acids. Most algorithms currently in use must fail to find the homologous alignment between a set of codons for the chicken α- and β-hemoglobin sequence when it is in fact discoverable by a more general treatment of gaps. Fundamental reasons for this are discussed.
Yeast is unable to excise foreign intervening sequences from hybrid gene transcripts.
Authors: Langford C , Nellen W , Niessing J , Gallwitz D
Published: May 2006

To investigate whether transcripts from foreign split genes are correctly processed in yeast cells we have constructed two hybrid genes by inserting into the split yeast actin gene an intron-containing fragment from either the Acanthamoeba actin I gene or the duck alpha D-globin gene. The hybrid genes were inserted into the autonomously replicating yeast plasmid YRp7, which was then used to transform yeast cells. It was found that the yeast but not the foreign intervening sequences were excised from the chimeric transcripts. This indicates that the recognition of intervening sequences or the splicing mechanism of RNA polymerase II transcripts is not universal.
Model for mammalian metallothionein structure.
Authors: Boulanger Y , Goodman C M , Forte C P , Fesik S W , Armitage I M
Published: May 2006

The results of physicochemical studies of mammalian metallothioneins are summarized and used to propose a model of the protein. The primary structures of all mammalian metallothioneins are very homologous; there are 38 invariant residues and 20 of them are cysteines. The results of UV and CD optical studies indicated that all 20 cysteines are involved in the ligation of 7 mol of metal per mol of metallothionein and that the protein does not contain any alpha-helix structure. A theoretical analysis by the Chou-Fasman method has predicted 11 beta-bends, each one involving at least one cysteine residue. The most significant structural data, provided by 113Cd NMR, demonstrated that the 7 mol of bound Cd2+ are arranged in two separate metal clusters, one containing four metal ions and the other containing three, with all Cd2+ tetrahedrally coordinated to cysteine thiolate ligands. The 11 cysteine residues of the carboxyl-terminal portion of the metallothionein chain (residues 30-61) are ligated to the 4-metal cluster as shown by 113Cd NMR of this enzymatically cleaved fragment. The remaining cysteine residues from the amino-terminal polypeptide portion (residues 1-29) form the 3-metal cluster. Such a division of the chain is consistent with the presence of an intron in the mouse metallothionein-1 gene corresponding to residue 32 in the polypeptide chain. A two-domain molecular model has been constructed based on an analysis of all the available data and is described in detail. The accuracy of this model was tested by 1H NMR at 500 MHz and the data are in agreement with our proposed structure.
Overproduction of a
M
r
92,000 protomer of 3-hydroxy-3-methylglutaryl-coenzyme A reductase in compactin-resistant C100 cells
Authors: Hardeman Edna C. , Jenke Hans-Stephan , Simoni Robert D.
Published: May 2006

We describe a cell line, designated C100, that displays a 100-fold increase in the major regulatory enzyme of the cholesterol biosynthetic pathway, 3-hydroxy-3-methylglutaryl-coenzyme A reductase [HMG-CoA; mevalonate:NADP
+
oxido-reductase (CoA-acylating), EC 1.1.1.34]. Immunoprecipitation of [
35
S]methionine-labeled enzyme from C100 microsomal membranes prepared in the presence of the protease inhibitors phenyl-methylsulfonyl fluoride and leupeptin revealed two up regulated proteins: a major band of
M
r
92,000 and a minor band of
M
r
63,000. We conclude that the
M
r
92,000 protein is probably the intact form of HMG-CoA reductase protomer based on the following criteria. (
i
) It is a highly up regulated microsomal membrane protein that coincides with the increase in HMG-CoA reductase specific activity in this cell line. (
ii
) It is recognized by a specific HMG-CoA reductase antiserum under a variety of stringencies. (
iii
) Isolation and solubilization of [
35
S]methionine-labeled C100 microsomal membranes in the absence of protease inhibitors resulted in the disappearance of the
M
r
92,000 protein and the appearance of two proteins of
M
r
52,000 and 38,000. (
iv
) Analysis of cells labeled for 30 min with [
35
S]methionine, well under the half-life of HMG-CoA reductase, revealed only the
M
r
92,000 protein to be present in total cell extract. (
v
) The previously reported single immunoprecipitation polypeptide for HMG-CoA reductase of
M
r
62,000 [Chin, D. J., Luskey, K. L., Anderson, R. G. W., Faust, J. R., Goldstein, J. L. & Brown, M. S. (1982)
Proc. Natl. Acad. Sci. USA
79, 1185-1189] can be isolated and appears to be the result of both proteolysis and sample preparation for NaDodSO
4
gel electrophoresis. Analysis of C100 cells labeled with [
35
S]methionine for 24 hr indicates that the predominant steady-state form of the enzyme is the
M
r
92,000, rather than the
M
r
63,000, protein, further suggesting that the two proteins do not have a classical precursor-product relationship.
Human coagulation factor Va is a cofactor for the activation of protein C.
Authors: Salem H H , Broze G J , Miletich J P , Majerus P W
Published: May 2006

During blood clotting in vitro, protein C is converted in part to protein Ca, Protein Ca, in turn, inactivates factor Va. This is evidenced by the rapid inactivation of factor Va coagulant activity after clot formation which is associated with the cleavage of the Mr 110,000 peptide of factor Va. When exogenous factor Va is added to serum, it is inactivated only after a lag of 10-20 min. Using purified coagulation factors in the presence of EDTA, we demonstrated that factor Va enhances the rate of protein C activation by thrombin by 50-fold. The Km for factor Va in the reaction is 14 nM, 100 times higher than its Km for accelerating platelet surface prothrombin activation by factor Xa. By this mechanism, factor Va can act as a procoagulant as well as limit dissemination of the coagulation process through the activation of protein C and the subsequent inactivation of both factor Va and factor VIIIa.
Structure of nuclear ribonucleoprotein: heterogeneous nuclear RNA is complexed with a major sextet of proteins in vivo.
Authors: Economidis I V , Pederson T
Published: May 2006

Mouse erythroleukemia cells were pulse-labeled with [3H]uridine and irradiated with 254-nm light to produce covalent crosslinks between RNA and proteins in close proximity to one another in vivo. Nuclear ribonucleoprotein particles containing heterogeneous nuclear RNA were isolated and digested with nucleases, and the resulting proteins were subjected to gel electrophoresis. Proteins carrying covalently crosslinked [3H]uridine nucleotides were identified by fluorography. The results demonstrate that heterogeneous nuclear RNA is complexed in vivo with a set of six major proteins having molecular weights between 32,500 and 41,500. Analysis of chromatin fractions indicates that nascent heterogeneous nuclear RNA chains assemble with these six proteins as a very early post-transcriptional event. These data, and other results [Nevins, J. R. & Darnell, J. E. (1981) Cell 15, 1477-1493], lead us to propose the usual order of post-transcriptional events to be: heterogeneous nuclear RNA-ribonucleoprotein particle assembly leads to poly(A) addition leads to splicing.
Drosophila melanogaster mutations suppressible by the suppressor of Hairy-wing are insertions of a 7.3-kilobase mobile element.
Authors: Modolell J , Bender W , Meselson M
Published: May 2006

Certain spontaneous mutations of Drosophila melanogaster are suppressed by su(Hw), the suppressor of Hairy-wing (3R-54.8). We find that mutations suppressible by su(Hw) result from insertions of a mobile element at the affected loci. The element, named gypsy, is approximately 7.3 kilobases long and includes 0.5-kilobase direct terminal repeats. It was first identified in DNA cloned from the bithorax chromosomal region of several Drosophila stocks carrying suppressible mutations of the bithorax complex. Cloned gypsy DNA was used as a probe to test for the association of gypsy with suppressible mutations at various other loci by hybridization in situ. Gypsy was found to be associated with 19 suppressible alleles at 10 different loci: yellow, Hairy-wing, scute, diminutive, cut, lozenge, forked, Beadex, hairy, and the bithorax complex. It was found with wild-type or nonsuppressible mutations at any of these loci. Gypsy DNA was also used as a probe to clone the element and adjacent unique DNA from the loci of some suppressible mutations. This confirmed the presence of the full-length element and also provided cloned DNA from the previously uncloned loci scute and cut. The suppressor of Hairy-wing is generally recessive and behaves as a null mutation. Thus, the disruption of normal gene function caused by the inserted gypsy element appears to require some product of the wild-type suppressor gene, su(Hw)+.
Multilocus genetic structure in natural populations of Escherichia coli.
Authors: Whittam T S , Ochman H , Selander R K
Published: May 2006

A survey of allozyme variation at 12 enzyme loci in 1,705 clones of the genetic species Escherichia coli (including four species of Shigella) from natural populations revealed 302 unique allele combinations (electrophoretic types). Single-locus diversity estimates fall within the range predicted by the neutral allele theory of molecular evolution, but the combination of alleles in electrophoretic types are highly nonrandom, as indicated by a test of association over all loci and by evidence of complex linkage disequilibria in several four-locus combinations. These linkage disequilibria reflect genetic differentiation of E. coli into three groups of strains. Because of restricted recombination, both the stochastic extinction of lines and selective differences between particular genetic combinations may have contributed to the evolution of subspecific structure in E. coli.
Interferon induces a unique protein in mouse cells bearing a gene for resistance to influenza virus.
Authors: Horisberger M A , Staeheli P , Haller O
Published: May 2006

Mouse cells carrying the dominant resistance gene Mx develop a more efficient antiviral state toward influenza viruses in response to interferon than do Mx-negative cells. We have identified an Mx gene-associated product by labeling cultured peritoneal macrophages and embryonic cells with [35S]methionine in the presence or absence of interferon. The radioactive proteins from unfractionated cytoplasmic extracts were separated electrophoretically in two dimensions and were revealed by fluorography. A protein with a Mr of 72,500 and an isoelectric point of 6.3 was induced by mouse interferon type I (a mixture of alpha and beta interferons) in cells carrying the gene Mx but not in cells lacking Mx. The induction of this protein could be blocked by actinomycin D. The maximal rate of synthesis was reached in embryonic cells 4-5 hr after treatment with 10(3) reference units of interferon per ml. When the allele Mx (present in the inbred mouse strain A2G) was repeatedly backcrossed on different genetic backgrounds (BALB/c, C57BL/6, A/J), a clear correlation between the inducibility by interferon of this protein and the presence of the allele Mx was observed. The results suggest that this protein induced by the interaction of interferon with Mx plays a role in the selective antiviral state against influenza viruses that is observed in interferon-treated Mx-bearing cells.
Cultured endothelial cells produce a platelet-derived growth factor-like protein
Authors: DiCorleto Paul E. , Bowen-Pope Daniel F.
Published: May 2006

The platelet-derived growth factor (PDGF) binds specifically to high-affinity receptors on the surface of bovine aortic smooth muscle cells and 3T3 cells. Conditioned medium from cultured bovine aortic endothelial cells (EC) prevents PDGF binding to these receptors in a dose-dependent manner at 4°C. The
125
I-labeled PDGF that is displaced by the conditioned medium shows no increase in trichloroacetic acid solubility or decrease in binding capability to fresh cells. The competitor activity was identified as a protein by ammonium sulfate precipitability and sensitivity to trypsin. The competitor protein also is found in the serum-free conditioned media from porcine aortic EC and human umbilical vein EC but not in media from bovine aortic smooth muscle cells, human neonatal foreskin fibroblasts, or the interleukin-producing thyoma cell line EL-4. The competitor protein, like PDGF, has no effect on the specific 4°C binding of either
125
I-labeled insulin to 3T3 cells or
125
I-labeled epidermal growth factor to human epidermoid A431 cells. Saturation curves of PDGF binding to smooth muscle cells that had been preincubated in the presence and absence of competitor indicate that the concentration for half-maximal binding of
125
I-labeled PDGF to its receptor (≈30 pM) is unchanged by the competitor, whereas the apparent number of available receptor sites or maximal level of binding is greatly diminished. The competitor activity produced by cultured human umbilical vein EC is completely inhibited by antiserum against pure human PDGF, whereas the same PDGF antiserum only partially inhibits the mitogenic activity of the conditioned media. In addition, ≈7-fold more crude endothelium-derived growth factor is required for half-maximal inhibition of
125
I-labeled PDGF binding as is required for half-maximal stimulation of DNA synthesis. These results suggest that EC secrete a PDGF-like protein that is biochemically distinct from the majority of EC-derived mitogenic activity.
Nucleotide sequence analysis of the chicken c-myc gene reveals homologous and unique coding regions by comparison with the transforming gene of avian myelocytomatosis virus MC29, delta gag-myc.
Authors: Watson D K , Reddy E P , Duesberg P H , Papas T S
Published: May 2006

Myelocytomatosis virus MC29 is a defective avian retrovirus with a hybrid transforming gene (delta gag-myc) consisting of a 1,358-base pair (bp) sequence from the retroviral gag gene and a 1,568-bp sequence (v-myc) shared with a cellular locus, termed c-myc. We have subjected to sequence analysis 2,735 bp of the cloned c-myc gene, which includes the v-myc-related region of 1,568 bp, an intervening sequence of 971 bp, and unique flanking sequences of 45 bp and 195 bp at the 5' and 3' ends, respectively. Analysis of the genetic information and alignment of the c-myc sequence with the known sequence of MC29 indicates that: (i) the two myc sequences share the same reading frame, including the translational termination signal; (ii) there are nine nucleotide changes between c-myc and v-myc that correspond to seven amino acid changes; (iii) the 971-bp intervening sequence of c-myc can be defined as an intron by consensus splice signals; (iv) the unique 5' sequence of c-myc could either extend its reading frame beyond the homology with v-myc or could be an intron because its junction with the myc region of the locus is a canonical 3' splice-acceptor site; (v) the v-myc contains 10 nucleotides at its 5' end not shared with the c-myc analyzed here and also not with known gag genes, probably derived from an upstream exon; and (vi) the c-myc locus can generate a mRNA whose termination signals have been identified to be located 83 bp and 119 bp from the point of divergence between the v-myc and c-myc. We conclude that the gene of the c-myc locus of the chicken and the onc gene of MC29 share homologous myc regions and differ in unique 5' coding regions and we speculate, on this basis, that their protein products may have different functions. The hybrid onc gene of MC29 must have been generated from the c-myc gene by deletion of the 5' cellular coding sequence, followed by substitution with the 5' region of the viral gag gene.
Molecular mimicry in virus infection: crossreaction of measles virus phosphoprotein or of herpes simplex virus protein with human intermediate filaments.
Authors: Fujinami R S , Oldstone M B , Wroblewska Z , Frankel M E , Koprowski H
Published: May 2006

Using monoclonal antibodies, we demonstrate that the phosphoprotein of measles virus and a protein of herpes simplex virus type 1 crossreact with an intermediate filament protein of human cells. This intermediate filament protein, probably vimentin, has a molecular weight of 52,000, whereas the molecular weights of the measles viral phosphoprotein and the herpes virus protein are 70,000 and 146,000, respectively. Crossreactivity was shown by immunofluorescent staining of infected and uninfected cells and by immunoblotting. The monoclonal antibody against measles virus phosphoprotein did not react with herpes simplex virus protein and vice versa, indicating that these monoclonal antibodies recognize different antigenic determinants on the intermediate filament molecule. The significance of these results in explaining the appearance of autoantibodies during virus infections in humans is discussed.
recF-dependent induction of recA synthesis by coumermycin, a specific inhibitor of the B subunit of DNA gyrase.
Authors: Smith C L
Published: May 2006

Genetic and biochemical studies on enzymes known to be involved in regulating DNA supercoiling yield a complex spectrum of effects on the Escherichia coli SOS system. Previous studies indicated that only inhibition of DNA gyrase by antibiotics that act on the DNA gyrase A subunit results in turning on the E. coli SOS system. Here we show that coumermycin, an antibiotic that acts on the DNA gyrase B subunit, can also induce. Like nalidixic acid induction, coumermycin induction is dependent on the recBC DNase. In both cases induction apparently results from a response of the cell to the DNA gyrase-inhibitor complex rather than just the loss of DNA gyrase activity. However, unlike induction by the DNA gyrase A-specific antibiotics, coumermycin induction also requires the recF gene product. This demonstrates a functional relationship between DNA gyrase and the recF gene product.
Localization of a family of MRNAS in a single cell type and its precursors in sea urchin embryos.
Authors: Lynn D A , Angerer L M , Bruskin A M , Klein W H , Angerer R C
Published: May 2006

Spec 1 mRNAs increase 100-fold in abundance per embryo during early sea urchin development. Previous studies indicated an enrichment of this mRNA in ectoderm fractions of gastrulae and plutei. We have determined the precise localization of this mRNA by in situ hybridization techniques. In pluteus larvae, the mRNA is highly restricted to a set of morphologically uniform ectoderm cells in the dorsal part of the embryo. The mRNA is not detectable in other regions of ectoderm or in endoderm and mesoderm. The pattern of localization is already established at the gastrula stage, before these cells are distinguishable by morphological criteria. This pattern of distribution of Spec 1 mRNA is distinct from that of bulk poly(A)+ mRNA. Measurements of the amount of Spec 1 mRNA per embryo and the number of cells containing this RNA indicate that there are about 500 Spec 1 mRNA molecules per cell at the pluteus stage and probably twice as many at the gastrula stage. These results indicate that the sensitivity of the in situ hybridization method allows detection of sequences that comprise approximately equal to 0.05% of the embryo mRNA nucleotides.
Base substitution mutations induced by metabolically activated aflatoxin B1.
Authors: Foster P L , Eisenstadt E , Miller J H
Published: May 2006

We have determined the base substitutions generated by metabolically activated aflatoxin B1 in the lacI gene of a uvrB- strain of Escherichia coli. By monitoring over 70 different nonsense mutation sites, we show that activated aflatoxin B1 specifically induced GxC leads to TxA transversions. One possible pathway leading to this base change involves depurination at guanine residues. We consider this mechanism of mutagenesis in the light of our other findings that the carcinogens benzo[a]pyrene diol epoxide and N-acetoxyacetylaminofluorene also specifically induce GxC leads to TxA transversions.
Unique spectrum of activity of 9-[(1,3-dihydroxy-2-propoxy)methyl]-guanine against herpesviruses in vitro and its mode of action against herpes simplex virus type 1.
Authors: Cheng Y C , Huang E S , Lin J C , Mar E C , Pagano J S , Dutschman G E , Grill S P
Published: May 2006

A guanosine analog, 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine (DHPG), was found to inhibit herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2, cytomegalovirus, and Epstein-Barr virus replication by greater than 50% at concentrations that do not inhibit cell growth in culture. The potency of the drug against all of these viruses is greater than that of 9-[(2-hydroxyethoxy)methyl]guanine (acyclovir). DHPG was active against HSV-1 growth during the early phase of virus replication and had no activity when added at a later time after infection. Its antiviral activity was irreversible. Thymidine partially neutralized its action. The anti-HSV-1 activity of DHPG was dependent on the induction and the properties of virus-induced thymidine kinase. Virus variants that induced altered virus thymidine kinase and became resistant to acyclovir were still as sensitive to DHPG as the parental virus. DHPG is active against five different HSV variants with induced altered DNA polymerase and resistance to acyclovir.
Calculation of the polarized fluorescence from a labeled muscle fiber.
Authors: Morales M F
Published: May 2006

Equations are derived that explicitly relate fluorescence polarization observables on a labeled muscle fiber to attitude of the cross-bridges and to attitude of the labels within the cross-bridges.
Radiation inactivation of oligomeric enzyme systems: theoretical considerations.
Authors: Verkman A S , Skorecki K , Ausiello D A
Published: May 2006

Radiation inactivation has been used as a tool to explore the size, structure, and function of soluble and membrane-bound enzymes. Enzyme systems consisting of a single functional unit yield a single exponential dependence of enzyme activity on radiation dose. Complex, nonexponential, inactivation curves suggest the presence of a multiunit enzyme system. A concave-upward inactivation curve suggests the presence of multiple independent functional units of distinct size and activity that do not interact with each other. An oligomeric enzyme, consisting of n identical subunits in equilibrium with monomers, can give simple exponential decay curves or more complex inactivation curves with various degrees of upward or downward concavity, depending upon the extent of oligomer-monomer equilibration among subunits after radiation, oligomer-to-monomer size and activity ratios, and multihit requirements for oligomer inactivation. For each of these possibilities, equations for the inactivation curves are derived, calculated numerically, and discussed in qualitative terms. A systematic approach to the evaluation of complex radiation inactivation curves is proposed and limitations of the radiation inactivation method are reviewed.
Properties of a human immunoglobulin epsilon-chain fragment synthesized in Escherichia coli.
Authors: Kenten J , Helm B , Ishizaka T , Cattini P , Gould H
Published: May 2006

A fragment of the cloned gene for the human myeloma ND epsilon chain, coding for the second, third, and fourth domains of the immunoglobulin, has been coupled to the tryptophan control region of an expression plasmid and subcloned in Escherichia coli. Induction of gene expression results in the synthesis of the expected, antigenically active polypeptide of Mr 40,000, which constitutes 18% of total bacterial protein and yields 55 mg/liter of culture. The immunoglobulin, which is aggregated and packed into large inclusion bodies within the bacterial cell, can be dissolved by denaturing solvents and purified by affinity chromatography using anti-IgE Sepharose. Reduced monomeric chains assemble spontaneously into dimers. On assay to measure the inhibition of binding of 125I-labeled human E myeloma protein to Fc epsilon receptors on cultured human basophils, the cloned gene product exhibited 20% of the activity of the native protein.
Development of a delayed outward-rectifying K+ conductance in cultured mouse peritoneal macrophages.
Authors: Ypey D L , Clapham D E
Published: May 2006

Patch clamp techniques were used to study ionic currents in cultured mouse peritoneal macrophages. Whole-cell voltage clamp studies of cells 1-5 hr after isolation showed only a high-resistance linear membrane. After 1 day in culture, 82 of 85 cells studied had developed a voltage- and time-dependent potassium (K+) conductance similar to the delayed outward rectifier in nerve and muscle cells. The current activated when the membrane was depolarized above -50 mV. The sigmoidally rising current rose to a peak at a rate that increased with depolarization. Inactivation proceeded exponentially with a time constant of approximately equal to 450 ms. Recovery from inactivation was slow (tau = 12 s). The reversal potentials for varying extracellular K+ concentrations followed the Nernst predictions for a K+ -specific channel. The conductance was blocked by extracellular 4-aminopyridine and by intracellular tetraethylammonium chloride, barium, and cesium. Single-channel K+ currents comprising this net current had a conductance of 16 pS, exhibited bursting behavior, and inactivated with time. No inward currents were ever detected in macrophages cultivated for up to 4 days. Short-term exposure to chemoattractant and transmitter agents failed to activate an inward current. Macrophages may change their membrane electrophysiological properties depending on their state of functional activation. We postulate that the K+ conductance develops prior to depolarizing conductances involved in the macrophage's immunological functions.
Developmental studies of phospholipid-sensitive Ca2+-dependent protein kinase and its substrates and of phosphoprotein phosphatases in rat brain.
Authors: Turner R S , Raynor R L , Mazzei G J , Girard P R , Kuo J F
Published: May 2006

Ontogenetic changes in the protein phosphorylation/dephosphorylation systems in rat brain were investigated. It was found that the activity level of phospholipid-sensitive Ca2+-dependent protein kinase (PL-Ca-PK) in the particulate fraction of grey and white matter and the soluble fraction of grey matter increased rapidly and markedly after birth, reached the highest level at day 30, and declined slightly or remained unchanged thereafter. The enzyme level in the soluble fraction of white matter, in contrast, remained constant throughout the development and maturation of brain. Various ontogenetic changes in the substrate proteins for PL-Ca-PK were also noted. The levels of myelin basic protein and other substrates (notably the Mr 87,000, 58,000, 54,000, and 50,000 protein in grey matter) progressively increased during development, reaching the highest level at adulthood. The level of the Mr 66,000 protein from the particulate fraction of white and grey matter, on the other hand, increased rapidly after birth, reached a peak at day 18, and then declined to the initial neonatal level at the adult stage. The time scale for the increases in the levels of PL-Ca-PK and its many substrates paralleled that of brain development and maturation (synaptogenesis and myelinogenesis). The activity levels of phosphoprotein phosphatases (assayed using 32P-labeled myelin basic protein, histone, and protamine sulfate) were found to only slightly (up to 60%) increase or decrease in certain fractions from different brain regions during development, suggesting that phosphorylation, compared to dephosphorylation, may be more important in determining the phosphorylation state of cellular proteins.
Chromosomal assignment of the gene for human elongation factor 2.
Authors: Kaneda Y , Yoshida M C , Kohno K , Uchida T , Okada Y
Published: May 2006

Elongation factor 2 (EF-2), polypeptidyl -tRNA translocase, is an essential factor for protein synthesis in eukaryotic cells and Archebacteria . We isolated diphtheria toxin-resistant human primary embryo cells that contain EF-2 that cannot be ADP-ribosylated by diphtheria toxin and Pseudomonas exotoxin A (PA toxin). Somatic cell hybrids were constructed from mouse L cells and toxin-resistant human embryo cell mutants. Forty-one hybrid clones were isolated, of which 15 clones were resistant to PA toxin. Karyotypic analysis and isozyme studies revealed that there was an absolute correlation between human chromosome 19 and resistance to PA toxin in the hybrids. On subcloning of PA toxin-resistant hybrid cells, we obtained one PA toxin-resistant hybrid subclone containing human chromosome 19 as the only human chromosome. Furthermore, the resistance to PA toxin of hybrid cell strains was lost after infection with poliovirus, for which sensitivity is conferred by human chromosome 19. It was confirmed by using two-dimensional gel electrophoresis that PA toxin resistance in hybrid cells was caused by the presence of EF-2 resistant to ADP-ribosylation by fragment A of diphtheria toxin. These facts suggest that the gene encoding EF-2 is located on human chromosome 19.
Bis[cyclo(histidylhistidine)]copper(II) complex that mimicks the active center of superoxide dismutase has its catalytic activity.
Authors: Kubota S , Yang J T
Published: May 2006

The formation of copper complexes with bis[cyclo( histidylhistidine )] copper(II) was determined potentiometrically; the maximum coordination number was four deprotonated histidine residues per Cu(II) ion. This complex mimicks the active center of Cu/Zn superoxide dismutase (superoxide:superoxide oxidoreductase, EC 1.15.1.1). Its absorption spectrum showed a broad band above 600 nm as compared with the 670- to 680-nm band of the enzyme. Its CD spectrum had a negative 600- to 605-nm band and a more intense positive band near 780 nm. The band positions were identical with, and the intensities were higher than, those of the enzyme, but the corresponding bands had opposite signs. This can be attributed to the difference in the distorted planar structure between the complex and enzyme. The complex catalyzed the dismutation of superoxide anion; the activity was 1/10th that of the enzyme on a molar basis, but about three times that of the enzyme on a weight basis.
Brain fodrin: substrate for calpain I, an endogenous calcium-activated protease.
Authors: Siman R , Baudry M , Lynch G
Published: May 2006

The calcium-activated thiol-protease calpain I, which is present in cytosolic and membrane preparations from rat brain, was tested for its capacity to degrade the neuronal spectrin-like protein fodrin. In the presence of micromolar calcium concentrations purified calpain I degraded both purified fodrin and the fodrin present in hippocampal and cerebellar membranes. Fodrin was identified as a high molecular weight protein present in brain membranes by the following criteria: (i) comigration on NaDodSO4/polyacrylamide gels with purified fodrin, (ii) reactivity with antibodies to purified fodrin, and (iii) a proteolytic map following calpain activation comparable to that found after calpain-mediated degradation of purified fodrin. The fodrin breakdown was selective in that calpain I did not affect at least 15 other membrane-associated polypeptides. Fodrin degradation by the protease was rapid and was accompanied by the appearance of a lower molecular weight breakdown product. Calpain I had a high affinity for fodrin, with a Km for degradation of about 50 nM. Purified calpain I also degraded purified spectrin and the spectrin present in erythrocyte membranes. Calpain I-mediated degradation of spectrin-like proteins could provide a mechanism by which brief increases in intracellular free calcium levels modify the structure of the submembraneous cytoskeleton and the distribution of cell surface receptors and alter cell shape.
Titin is an extraordinarily long, flexible, and slender myofibrillar protein.
Authors: Wang K , Ramirez-Mitchell R , Palter D
Published: May 2006

" Titin " is a term used to describe a pair of closely related megadalton polypeptides that together are the third most abundant myofibrillar protein in a wide range of striated muscles. It has been proposed that titin and another giant protein, nebulin , are the major components of an elastic cytoskeletal lattice within the sarcomere. We have now purified the leading band, titin -2 (T2), of the titin doublet in native forms by extraction with Guba -Straub solution followed by chromatography. Electron microscopy of low-angle-shadowed and negatively stained specimens revealed that T2 chains self-assembled into extremely long (from 0.1 micron to over 1.0 micron), flexible, and extensible slender strands (4-5 nm in diameter) with axial periodicity. Furthermore, these strands tended to associate to form filamentous bundles and meshworks. Thus, titin appears to be ideally suited as a component of an elastic lattice that serves as an organizing scaffold or template for thick and thin filaments.
Differentiation of Friend erythroleukemia cells induced by benzodiazepines.
Authors: Wang J K , Morgan J I , Spector S
Published: May 2006

Many benzodiazepines of the type whose receptors are found in peripheral tissues cause the differentiation of cultured Friend erythroleukemia cells. The maximal level of induction of hemoglobin synthesis is 10-80% of the cells, depending on the compound tested. The induction is concentration and time dependent, requiring micromolar amounts of the drugs and about 5 days of treatment for full expression. The time course is very similar to that observed for a well-studied inducer, dimethyl sulfoxide. The affinities of the agents for the peripheral-type benzodiazepine binding site are not correlated with their capacity to induce differentiation. Also, the biological effect is stereospecific since the (3S) stereoisomer Ro11 -6896 is at least an order of magnitude more potent than its (3R) enantiomer, Ro11 -6893. The benzodiazepine effect exhibits definite structure-activity relationships. A 1-methyl group is an absolute requirement, although this is not sufficient in itself. Hydroxyl and methoxyl groups at the 4' position enhance the biological activity, but 4'-chloro decreases it. Substitutions at the 2', 6', and 4 positions also decrease the biological activity, as does the lack of a 2-carbonyl group. These data suggest that the benzodiazepines act in a specific manner to induce the differentiation of Friend erythroleukemia cells.
Modification of low density lipoprotein by endothelial cells involves lipid peroxidation and degradation of low density lipoprotein phospholipids.
Authors: Steinbrecher U P , Parthasarathy S , Leake D S , Witztum J L , Steinberg D
Published: May 2006

Low density lipoprotein (LDL) incubated with cultured endothelial cells from rabbit aorta or human umbilical vein is altered in several ways (EC-modified): (i) It is degraded by macrophages much faster than LDL similarly incubated in the absence of cells or incubated with fibroblasts. (ii) Its electrophoretic mobility is increased. (iii) Its density is increased. We report here that antioxidants completely prevent these changes. We also report that these changes do not take place if transition metals in the medium are chelated with EDTA. During EC-modification as much as 40% of the LDL phosphatidylcholine is degraded to lysophosphatidylcholine by a phospholipase A2-like activity. When incubation conditions in the absence of cells were selected to favor oxidation--for example, by extending the time of incubation of LDL at low concentrations, or by increasing the Cu2+ concentration--LDL underwent changes very similar to those occurring in the presence of cells, including degradation of phosphatidylcholine. Hence, some phospholipase activity appears to be associated with the isolated LDL used in these studies. The results suggest a complex process in which endothelial cells modify LDL by mechanisms involving generation of free radicals and action of phospholipase (s).
L and ε functions for GSp(4) × GL(2)
Authors: Piatetski-Shapiro Ilya I. , Soudry David
Published: May 2006

The construction of L functions for automorphic cuspidal representations of GSp(4, A) × GL(2, A) with a Whittaker model has been given by Novodvorsky [Novodvorsky, M. (1979)
Proc. Symp. Pure Math.
33 (2), 87-95]. In this paper, we prove that this L function has nontrivial poles if and only if the representation of GSp(4, A) is a lifting from split O
4
. We also introduce a different construction of L functions for GSp(4) × GL(2) that is applicable to representations that do not have a Whittaker model—for instance, those that correspond to holomorphic modular forms. This construction is based on the lifting of these automorphic forms to Sp(4). Lifted forms will have a Whittaker model. This allows us to write integral expressions yielding these L functions.