bluuebunny/crossref_metadata_2025 huggingface dataset:
Inborn errors of molybdenum metabolism: combined deficiencies of sulfite oxidase and xanthine dehydrogenase in a patient lacking the molybdenum cofactor.
Authors: Johnson J L , Waud W R , Rajagopalan K V , Duran M , Beemer F A , Wadman S K
Published: May 2006

A patient suffering from a combined deficiency of sulfite oxidase (sulfite dehydrogenase; sulfite:ferricytochrome c oxidoreductase, EC 1.8.2.1) and xanthine dehydrogenase (xanthine:NAD+ oxidoreductase, EC 1.2.1.37) is described. The patient displays severe neurological abnormalities, dislocated ocular lenses, and mental retardation. Urinary excretion of sulfite, thiosulfate, S-sulfocysteine, taurine, hypoxanthine, and xanthine is increased in this individual, while sulfate and urate levels are drastically reduced. The metabolic defect responsible for loss of both enzyme activities appears to be at the level of the molybdenum cofactor common to the two enzymes. Immunological examination of a biopsy sample of liver tissue revealed the presence of the xanthine dehydrogenase protein in near normal amounts. Sulfite oxidase apoprotein was not detected by a variety of immunological techniques. The plasma molybdenum concentration was normal; however, hepatic content of molybdenum and the storage pool of active molybdenum cofactor present in normal livers were below the limits of detection. Fibroblasts cultured from this patient failed to express sulfite oxidase protein or activity, whereas those from the parents and healthy brother of the patient expressed normal levels of this enzyme.
Predicted structure of two adenovirus tumor antigens.
Authors: Perricaudet M , Le Moullec J M , Pettersson U
Published: May 2006

Early adenovirus type 2(Ad2) mRNA sequences have been cloned by using the pBR322 plasmid as a vector. Two clones that include sequences from region E1B were identified and their DNAs were characterized by hybridization, restriction enzyme cleavage, and DNA sequence analysis. The results showed that the clones were derived from two different spliced mRNAs. By combining our results with the established DNA sequence for region E1B of the closely related adenovirus type 5[Maat, J., van Beveren, C.P. & van Ormondt, H. (1980) Gene, in press] it was possible to deduce the structure of a 13S and a 22S mRNA. The two mRNAs differ from each other by the size of their intervening sequences. If translation starts at the first AUG following the cap, the 22S mRNA encodes a Mr 67,000 polypeptide that is terminated by a UGA stop codon located immediately before the splice, whereas the 13S mRNA encodes a Mr 20,000 polypeptide that is translated in different reading frames before and after the splice. The Mr 20,000 and 67,000 polypeptides correspond in molecular weight to two proteins that invariably are precipitated from infected cell extracts by antisera from animals carrying adenovirus-induced tumors.
Interaction between beta-adrenergic receptors and guanine nucleotide sites in turkey erythrocyte membranes.
Authors: Vauquelin G , Bottari S , Andre C , Jacobsson B , Strosberg A D
Published: May 2006

beta 1-Adrenergic receptors from turkey erythrocyte membranes have been identified by specific binding of the radiolabeled antagonist (-)-[3H]dihydroalprenolol. These receptors are inactivated by the alkylating agent N-ethylmaleimide when occupied by beta-adrenergic agonists but not when occupied by antagonists or when unoccupied. A time-dependent decrease of the number of receptor sites is observed. Inactivation affects 45-60% of the sites, regardless of the agonist or N-ethylmaleimide concentration. The guanine nucleotides GTP and 5'-guanylyl imidodiphosphate effectively protect the receptors against agonist-mediated inactivation by N-ethylmaleimide. Protection by ATP necessitates a 100-fold higher concentration; 10 mM NaF is ineffective. The guanine nucleotide effect is reversible and occurs via interaction with N-ethylmaleimide-insensitive sites. These observations establish that guanine nucleotide sites interact with and caused structural modification of the agonist-occupied beta-adrenergic receptors in turkey erythrocyte membranes.
Calmodulin activates prokaryotic adenylate cyclase.
Authors: Wolff J , Cook G H , Goldhammer A R , Berkowitz S A
Published: May 2006

The adenylate cyclase of Bordetella pertussis is stimulated 100- to 1000-fold in a dose-dependent manner by calf brain calmodulin. The system has the following properties. (i) The activation is prevented by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and restored by Ca2+. (ii) Oxidation of the methionine residues of calmodulin abolishes the ability to activate the cyclase. (iii) Trifluoperazine inhibits calmodulin-activated cyclase. (iv) A troponin C preparation stimulates the B. pertussis cyclase with < 0.01 the potency of calmodulin. Although calmodulin has not been demonstrated in prokaryotes, this is an example of a (eukaryotic) calmodulin effect in a prokaryote.
Carboxypeptidase A mechanisms.
Authors: Lipscomb W N
Published: May 2006

The mode of binding of a ketonic substrate, which is an analogue of esters in which the O of the scissile bond is replaced by CH2, to carboxypeptidase A is similar to that of Gly-Tyr. The site is S'1, with the side chain in the pocket of the enzyme, the carboxylate salt-linked to Arg-145, and the carbonyl group bound to Zn. Thus, esters are probably cleaved at the peptide cleavage site, although not necessarily with the same rate-controlling step or by the same detailed mechanism. The large differences found between the behavior of the enzyme in solution and in one crystalline phase do not apply to a different crystalline phase.
Vesicular stomatitis virus glycoprotein is anchored in the viral membrane by a hydrophobic domain near the COOH terminus
Authors: Rose J. K. , Welch W. J. , Sefton B. M. , Esch F. S. , Ling N. C.
Published: May 2006

We have determined the COOH-terminal and NH
2
-terminal amino acid sequences of the vesicular stomatitis virus (VSV) glycoprotein (G). A sequence of 122 COOH-terminal amino acids was deduced from the complete sequence of a cloned DNA insert carrying 470 nucleotides derived from the 3′ end of the G mRNA. Evidence presented indicates that this portion of the polypeptide includes the domains of G that reside inside the virion and span the lipid bilayer of the virion. This seems clear because a partial amino acid sequence of a fragment of G that remains associated with the membrane of the virion after exhaustive proteolytic digestions can be located unambiguously in the predicted sequence. This predicted sequence contains an uninterrupted hydrophobic domain beginning 49 amino acids and ending 30 amino acids from the COOH terminus. This region presumably spans the lipid bilayer. The COOH-terminal portion of 29 amino acids contains a high proportion of basic residues and resides inside the virion. The COOH-terminal portion of the VSV G protein therefore resembles in structure that of glycophorin, an erythrocyte membrane protein well characterized previously. The configuration of G in the viral membrane demonstrated here is probably similar for other viral glycoproteins, although this has not been tested as directly in any other case. From the sequence of a DNA primer extended on the RNA genome from the adjacent M protein gene into the G protein gene, we have deduced an NH
2
-terminal G protein sequence of 53 amino acids, including the leader sequence of 16 amino acids. Our sequence confirms, extends, and corrects two partial amino acid sequences reported for this region previously.
Hepatic uptake and degradation of unilamellar sphingomyelin/cholesterol liposomes: a kinetic study.
Authors: Hwang K J , Luk K F , Beaumier P L
Published: May 2006

The kinetics of hepatic uptake and degradation of sphingomyelin/cholesterol (2:1, M/M) small unilamellar liposomes were investigated in a BALB/c mouse. The tissue distribution of liposomes was determined by scintillation spectrometry. The percentage of intact liposomes in tissues was estimated by the technique of gamma-ray perturbed angular correlation. A kinetic model was developed to analyze the above data. A remarkable agreement was noted between the experimental data and the corresponding theoretical values. Our results indicate that the sphingomyelin/cholesterol unilamellar liposomes had an unusually long half-life of 16.5 hr in the circulation after intravenous administration to mice. The hepatic degradation of the liposomes in vitro at 37 degrees C followed first-order kinetics, with a half-life of 3.5 +/- 0.2 (SEM) hr. Furthermore, the rate of the in vivo degradation of liposomes in the liver was found to be quite similar to that in vitro, with a half-life of 3.6 +/- 0.4 hr. The rate of release of the liposome-encapsulated agent, indium-111, in the liver was not constant, and reached a maximum at about 8 hr after the administration of liposomes. The approach developed in the present study is general and can be applied to the investigation of factors that may control the release of pharmacologically active agents in any tissue.
Stability of DNA in nucleosomes.
Authors: Bina M , Sturtevant J M , Stein A
Published: May 2006

Heats of thermal denaturation of chromatin core particles and core particles with covalently crosslinked histones were measured by differential scanning calorimetry. The additional stabilization of the nucleoprotein complex by crosslinking is not reflected in the transition enthalpy. The contribution of protein denaturation to the total heat was estimated by comparison of core particles with core particle DNA in a high-salt solution. By taking into account the temperature dependence of the transition enthalpy of DNA, we conclude that the enthalpy change for denaturation of DNA in core particles is nearly the same as that for naked DNA in solution.
Apical membrane aminopeptidase appears at site of cell-cell contact in cultured kidney epithelial cells.
Authors: Louvard D
Published: May 2006

A dog kidney epithelial cell line (MDCK), grown in monolayer, displayed in vitro an asymmetric localization of surface proteins. Aminopeptidase [alpha-aminoacylpeptide hydrolase (microsomal), EC 3.4.11.2] was found only in the apical face whereas Na+, K+-ATPase (ATP phosphohydrolase, EC 3.6.1.3) was found in the basolateral faces. These two faces are delineated by the junctional complex at which close cell-cell contact occurs. alpha-Actinin, a protein associated with plasma membranes, was concentrated near the region of cell-cell contact. When membrane proteins in the apical surface were crosslinked and subsequently removed from the surface by endocytosis, crosslinked antigens reappeared in the apical face at the region of cell-cell contact. Antigens that were not crosslinked were also (re)inserted in the same region. This process was not affected by cycloheximide, presumably because a large pool of apical membrane proteins (observed in small cytoplasmic vesicles) was used to replace the endocytosed antigens. It is psoposed that the region containing the junctional complex is involved in guiding apical membrane proteins to their final location.
Coated pits act as molecular filters.
Authors: Bretscher M S , Thomson J N , Pearse B M
Published: May 2006

Two proteins--theta and H63 antigens--are found to be excluded from coated pits on fibroblast plasma membranes. Coated pits thus act as molecular filters, pinching off, into the cell, lipid vesicles containing a limited number of specific receptors but excluding other plasma membrane proteins. This constitutes part of a lipid flow cycle that was proposed earlier to explain the capping of crosslinked surface antigens.
Antibodies from myasthenic patients that compete with cholinergic agents for binding to nicotinic receptors.
Authors: Fulpius B W , Miskin R , Reich E
Published: May 2006

We have purified immunoglobulins from sera of myasthenic patients and have identified antibodies directed against the cholinergic ligand-binding site of the nicotinic acetylcholine receptor. In the serum of one patient analyzed in detail these antibodies belonged to the IgG3 class, and their effects were as follows: (i) In chicken embryo myogenic cultures, antibody binding was both competitive with 125I-labeled alpha-bungarotoxin and irreversible on a time scale of hours. (ii) 125I-Labeled alpha-bungarotoxin was not displaced by antibody from preformed complexes and, conversely, antibody was not displaced by toxin. (iii) Antibody binding was competitive with some, but not all, nicotinic agents. Thus, acetylcholine, carbamoylcholine, and dimethyltubocurarine competed effectively whereas decamethonium and hexamethonium did not, suggesting that the two classes of nicotinic ligands probably interact at different, nonoverlapping receptor subsites. (iv) There was no competitive binding between these antibodies and the muscarinic antagonist atropine. (v) Both this class of myasthenic immunoglobulins and rabbit antibodies raised against Torpedo acetylcholine receptors increased the rate of receptor degradation. However, synthesis and degradation remained coupled and there was a compensating increase in receptor synthesis. We propose that immunoglobulins directed against the ligand-binding site of acetylcholine receptors may account for the characteristic curare-like symptoms of early myasthenia and their response to cholinesterase inhibition, for the apparent decrease in receptors measurable by 125I-labeled alpha-bungarotoxin binding, and for initiating localized complement activation at the postsynaptic membrane.
Nerve growth factor: acceleration of the rate of wound healing in mice.
Authors: Li A K , Koroly M J , Schattenkerk M E , Malt R A , Young M
Published: May 2006

Earlier studies have shown that removal of the submandibular glands of mice retards the rate of contraction of experimentally induced wounds and that communal licking of wounds accelerates contraction in intact animals [Hutson, J. M., Niall, M., Evans, D. & Fowler, R. (1979) Nature (London) 279, 793-795]. In the light of the observation that nerve growth factor (NGF) is secreted in high concentrations in mouse saliva, we have studied the effect of topically applied high molecular weight nerve growth factor (HMW-NGF) upon the rate of wound contraction in sialoadenectomized animals. Results show that HMW-NGF significantly accelerates the rate of wound contraction and that this phenomenon is probably dependent upon the enzymic activity of the protein. Neither diisopropyl fluorophosphate-inactivated NGF nor 2.5S NGF [isolated according to Bocchini, V. & Angeletti, P. U. (1969) Proc. Natl. Acad. Sci. USA 64, 787-794] displays this biological activity. Thus, it may be that one of the physiological roles of NGF in saliva is to promote wound healing by the licking process.
Temperature and pressure dependence of viscosity of quantum liquid
4
He according to significant structure theory
Authors: Ryoo Ryong , John Mu Shik , Eyring Henry
Published: May 2006

Both solid-like and gas-like flow structures are considered in the calculation of viscosity of liquid
4
He using the significant structure theory of liquids. The predominance of quantum mechanical zero-point motion over that arising from thermal excitation is taken into account for the solid-like motion of molecules. The viscosity of liquid
4
He under its own vapor pressure calculated over its whole temperature range is in reasonably good agreement with experimental data. The pressure dependence of viscosity also was obtained. The calculation does not yield good results at high densities where the free jumping of molecules into the nearest neighboring vacancies through the kinetic zero-point motion is no longer plausible.
Observation of an exothermic process associated with the in vitro polymerization of brain tubulin.
Authors: Berkowitz S A , Velicelebi G , Sutherland J W , Sturtevant J M
Published: May 2006

The polymerization of tubulin has been studied with a high-sensitivity differential scanning microcalorimeter, with results which indicate that microtubule assembly can proceed via one or possibly two exothermic reactions. The amount of heat evolution has been found to be far in excess of GTP hydrolysis. The heat liberated has been observed to depend strongly upon the exact experimental conditions, varying from many hundreds of kilocalories per mole of tubulin dimer when dilute tubulin solutions are heated rapidly to a few kilocalories per mole of tubulin dimer when concentrated tubulin solutions are heated slowly. The results are tentatively interpreted in terms of the existence of at least two pathways for the formation of energetically distinct polymers. These findings indicate the importance of kinetic factors in studying tubulin polymerization.
On the process of cellular division in Escherichia coli: isolation and characterization of penicillin-binding proteins 1a, 1b, and 3.
Authors: Tamura T , Suzuki H , Nishimura Y , Mizoguchi J , Hirota Y
Published: May 2006

Multiple mutants of Escherichia coli defective in penicillin-binding proteins (PBPs) were constructed, and into these strains Co1E1 plasmids carrying the genes for PBP-1a, -1b, or -3 were introduced. From these plasmid-carrying strains, PBP-1a and -1b were purified by ampicillin-Sepharose affinity chromatography and PBP-3 by cephalexin-Sepharose affinity chromatography. Improved purification was achieved by differential elution with NH2OH. Purified PBP-1b synthesized murein when added to the membrane fraction of a PBP-1b-defective mutant, which by itself failed to support murein synthesis in vitro. The PBP-1b preparation was able to synthesize murein from the lipid intermediate extracted with chloroform/methanol but was unable to utilize UDP-linked precursors for murein synthesis. Murein synthesis was inhibited by vancomysin, ristocetin, moenomycin, and enduracidin, but not by beta-lactam antibiotics. The synthesized murein was shown to contain crosslinked muropeptides. Their crosslinking was abolished by action of beta-lactam antibiotics. The PBP-1a and -3 preparations showed substantially no activity for murein synthesis in the same reaction system. None of the three PBPs showed D-alanine carboxypeptidase activity with UDP-N-acetylmuramoyl-pentapeptide as substrate or endopeptidase activity with bis(disaccharide-peptide) as substrate.
Yeast fatty acid synthetase: Structure—function relationship and nature of the β-ketoacyl synthetase site
Authors: Stoops James K. , Wakil Salih J.
Published: May 2006

Yeast fatty acid synthetase consists of two multifunctional proteins, α and β, which are arranged in a complex of α
6
β
6
. Electron microscopic studies of this complex led to a model for the synthetase as an ovate structure consisting of an equatorial plate-like structure to which six arches are equally distributed on either side. The bifunctional reagent 1,3-dibromo-2-propanone inhibits the synthetase by reacting rapidly (
t
½
≈7 sec) with two juxtapositioned active sulfhydryl groups. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of the dibromopropanone-inhibited synthetase shows that the β subunit is intact and the α subunit nearly absent with a concomitant appearance of oligomers with an estimated molecular weight of 0.4-1.2 × 10
6
. These results indicate that the α subunits are crosslinked by this bifunctional reagent. Because the active centers of dibromopropanone are 5 Å apart, it is concluded that the α subunits are closely packed so that the reacting thiols of the adjacent α subunits are within 5 Å of each other. Furthermore, because the plate-like structures in our model are the only components that are arranged closely enough to satisfy this requirement, it is proposed that the α subunits are the “plates” and the β subunits therefore are the “arches.” Assay of the partial reactions shows that dibromopropanone inhibits the β-ketoacyl synthetase reaction but none of the six other partial reactions, indicating that the site of action of the bifunctional reagent is the condensing reaction. This conclusion was supported by the finding that pretreatment of the synthetase with acetyl-CoA or iodoacetamide prevented dibromopropanone from interacting at this site and obviated the formation of the crosslinked oligomer. These observations and other lead us to propose that a site of action of the dibromopropanone is the active cysteine-SH of the β-ketoacyl synthetase of one α subunit and the pantetheine-SH of the acyl carrier protein moiety of an adjacent α subunit. Thus, the enzymically active center of the β-ketoacyl synthetase consists of an acyl group attached to the cysteine-SH of one α subunit (plate) and a malonyl group attached to the pantetheine-SH of an adjacent α subunit. This arrangement appears to be necessary for the coupling of the acyl and β-carbon of the malonyl group to occur to yield CO
2
and the β-ketoacyl product.
Cell density determines epithelial migration in culture.
Authors: Rosen P , Misfeldt D S
Published: May 2006

The dog kidney epithelial cell line (MDCK) has been shown to exhibit a density-correlated inhibition of growth at approxmately 6.6 X 10(5) cells per cm2. When a confluent monolayer at its maximal density was wounded by removal of a wide swath of cells, migration of the cell sheet into the denuded area occurred. Precise measurements of the rate of migration for 5 day showed that the cells accelerated at a uniform rate of 0.24 micrometer . hr-2 and, by extrapolation, possessed an apparent initial velocity of 2.8 micrometer . hr-1 at the time of wounding. The apparent initial velocity was considered to be the result of a brief (< 10 hr) and rapid acceleration dependent on cell density. To verify this, wounds were made at different densities below the maximum. In these experiments, the cells did not migrate until a "threshold" density of 2.0 X 10(5) cells per cm2 was reached regardless of the density at the time of wounding. At the threshold density, the cell sheet began to accelerate at the previously measured rate (0.24 micrometer . hr-2). Any increase in density by cell division was balanced by cell migration, so that the same threshold density was maintained by the migrating cells. Each migrating cell sustained the movement of the cell sheet at a constant rate of acceleration. It is proposed that an acceleration is, in general, characteristic of the vectorial movement of an epithelial cell sheet.
Duplicated cytosolic malate dehydrogenase genes in
Zea mays
Authors: McMillin David E. , Scandalios John G.
Published: May 2006

Six inbred lines of
Zea mays
expressing different soluble (cytosolic) malate dehydrogenase (sMDH) zymogram phenotypes were analyzed genetically. sMDH was found to be coded for by unlinked duplicated loci in four of these inbred lines. The remaining two lines were found not to possess these duplicated loci. Furthermore, the duplicated loci,
sMdh1
and
sMdh2
, have been found to be located on different chromosomes:
sMdh1
on chromosome 1L linked to
Amp1
, and
sMdh2
on chromosome 5S linked to
Cat1
and
Amp3
. The importance of finding sMDH encoded by duplicated loci is discussed in relation to the role of chromosomal rearrangements, the relationship between the cytoplasmic and mitochondrial enzymes, and the evolution of
Z. mays
.
Structural organization of human genomic DNA encoding the pro-opiomelanocortin peptide.
Authors: Chang A C , Cochet M , Cohen S N
Published: May 2006

We have isolated a human genomic DNA segment encoding the corticotropin-beta-lipotropin precursor peptide from a fetal DNA library, using previously cloned bovine cDNA for this peptide as a probe. The human genomic DNA was studied by electron microscope heteroduplex analysis and gel blotting methods, and its nucleotide sequence was determined and compared with that of cDNA corresponding to bovine pro-opiomelanocortin mRNA. From this sequence, segments of interspecies conservation and divergence, punctuated by pairs of the basic amino acid residues lysine and arginine, were identified. No noncoding intervening sequence was observed over an 830-base-pair DNA segment extending from a position near the 5' end of the structural pro-opiomelanocortin gene through the 3' terminus of the cDNA and including sequences for the component peptide hormones corticotropin and beta-lipotropin.
Complete amino acid sequence of a mouse immunoglobulin alpha chain (MOPC 511).
Authors: Robinson E A , Appella E
Published: May 2006

The complete amino acid sequence of the 432-residue heavy (alpha) chain of mouse myeloma MOPC 511 has been determined. The variable region of the alpha chain of IgA 511, a phosphocholine-binding protein, is highly homologous to that of the other phosphocholine-binding immunoglobulins. Comparison of the 511 alpha chain constant region with that of other mouse and human heavy chains shows that sequence divain. The CH3 domain disulfide bridge of the 511 alpha chain, for example, consists of only 28 amino acid residues compared to 60 residues for other chains and domains. Sequence divergences are alsos apparent at the CH2/CH3 domain boundary, an area where a number of frameshift mutations have occurred. One mutant, mouse IgA 47A, lacks the entire CH3 domain. Comparison of the 511 alppha chain with the 47A alpha chain reveals two noncconservative amino acid changes at the COOH terminus of the 47A chain, Ser-Gln for VAl-Thr in the 511 chain. These changes and the deletion of the CH3 domain can be explained by a single genetic event--namely, a frameshift mutation followed by premature chain termination. The remainder of the 47A constant region, including the hinge region, is identical to the 511 alpha chain, except for two conservative changes in the CH1 domain: serine-126 and theonine-197 in the 511 alpha chain are both replaced by alanine in the 47A chain.
Enkephalin biosynthetic pathway: proteins of 8000 and 14,000 daltons in bovine adrenal medulla.
Authors: Lewis R V , Stern A S , Kimura S , Stein S , Udenfriend S
Published: May 2006

Two [Met]enkephalin-containing polypeptides have been purified from extracts of bovine and adrenal chromaffin granules. One is 8000 daltons in size and contains the enkephalin sequence at the carboxy terminus. The other (14,000 daltons) has two internal enkephalin sequences and a third sequence at the carboxy terminus. All of these sequences can be released by trypsin, indicating a possible enkephalin precursor role for the polypeptides.
Urotensin II: a somatostatin-like peptide in the caudal neurosecretory system of fishes.
Authors: Pearson D , Shively J E , Clark B R , Geschwind I I , Barkley M , Nishioka R S , Bern H A
Published: May 2006

Urotensin II, a peptide hormone from the caudal neurosecretory system of the teleost, Gillichthys mirabilis, was isolated by using classical chromatographic techniques and high-performance liquid chromatography (HPLC). Direct microtechniques for sequence determination were used to establish its structure. Urotensin II from Gillichthys is a 1363-dalton dodecapeptide with the amino acid sequence Ala-Gly-Thr-Ala-Asp-Cys-Phe-Trp-Lys-Tyr-Cys-Val. This sequence is homologous with somatostatin in positions 1 and 2 and 7-9. The sequence has been verified by the production of a bioactive synthetic urotensin II. The possible chemical and physiological significance of its homology to somatostatin is discussed.
Mechanism of negative cooperativity in glyceraldehyde-3-phosphate dehydrogenase deduced from ligand competition experiments.
Authors: Henis Y I , Levitzki A
Published: May 2006

It is shown that the modulation in the negative cooperativity of ligand binding by another, competing ligand that binds noncooperatively is accounted for exclusively by the ligand-induced sequential model. It is therefore suggested that whenever such a phenomenon is observed it argues strongly in favor of the sequential model. The advantages and limitations of this approach are evaluated. The binding of the coenzymes NAD+ and nicotinamide-1-N6-ethenoadenine dinucleotide to rabbit muscle apo-glyceraldehyde-3-phosphate dehydrogenase [D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating; EC 1.2.1.12] exhibits strong negative cooperativity, whereas acetylpyridine adenine dinucleotide, ATP, and ADP-ribose bind noncooperatively to the NAD+ sites. The strong abolished in the presence of acetylpyridine adenine dinucleotide and strongly weakened by ATP, ADP, and AMP, but was not affected by addition of ADP-ribose. These findings demonstrate that the negative cooperativity in coenzyme binding to this enzyme results from sequential conformational changes and exclude the pre-existent asymmetry model as a possible explanation. These results also support the view that the structure of the pyridine moiety of the coenzyme analogs plays a role in orienting the adenine moiety at the adenine subsite, therefore affecting the cooperativity in the binding of the coenzyme analog which is mediated through the adenine subsites.
Comparison of the structures of free and ribosome-bound tRNAPhe by using slow tritium exchange.
Authors: Farber N , Cantor C R
Published: May 2006

The rate of incorporation of tritium from the solvent into the C-8 position of purines in RNA is markedly sensitive to the microenvironment. This slow tritium exchange reaction has been used to study the structure and interactions of yeast tRNAPhe bound to poly(U)-programed tight-couple 70S ribosomes of Escherichia coli. The tritium incorporation into specific sites of the tRNA was determined by enzymatic digestion and measurement of the specific activity of each of the isolated radioactive fragments. Ribosome binding leads to marked suppression in the exchange rate of a number of fragments. This delineates extensive regions of tRNA-ribosome contact. No change in exchange rates is seen for fragments from the corner of the molecule, indicating that this region of bound tRNA is readily accessible to the solvent. Ribosome binding results in an enhanced exchange rate at the T loop. This appears to be the result of a conformational change that is most likely an unfolding of the T and D loops. Additional tritium exchange reactions suggest this conformational change is induced by ribosomes and not by messenger.
Cloning and analysis of a cDNA coding for bovine prothrombin.
Authors: MacGillivray R T , Degen S J , Chandra T , Woo S L , Davie E W
Published: May 2006

Poly(A)-RNA enriched for prothrombin was isolated by specific immunoprecipitation of bovine liver polysomes. Prothrombin consisted of about 8% of the cell-free translation products of this RNA. A double-stranded cDNA was synthesized by using reverse transcriptase (RNA-dependent DNA nucleotidyltransferase) and made blunt-ended with nuclease S1. After tailing with dCTP and terminal transferase, the double-stranded cDNA was annealed to pBR322 DNA that had been cleaved previously at the single Pst I site and similarly tailed with dGTP. The resulting plasmids were used to transform Escherichia coli strain RR1 under P3-EK1 conditions. Sixty-three tetracycline-resistant clones were obtained that hybridized to 32P-labeled cDNA synthesized from prothrombin-enriched mRNA. Recombinants containing cDNA to prothrombin mRNA sequences were screened by a solution hybridization assay with a [3H]cDNA synthesized from mRNA. This enriched mRNA was 50% prothrombin mRNA, as determined by a reticulocyte lysate translation assay. Three positive clones were identified by this assay; they contained bovine DNA inserts of 700, 500, and 400 base pairs. The DNA sequence of the 700-base-pair insert was then determined. This recombinant plasmid contained DNA coding for the carboxyl-terminal 160 residues of bovine prothrombin followed by a noncoding region of 119 base pairs and a poly(A) tail of 60 base pairs.
Cellular sequences are present in the presumptive avian myeloblastosis virus genome.
Authors: Souza L M , Strommer J N , Hillyard R L , Komaromy M C , Baluda M A
Published: May 2006

EcoRI restriction endonuclease fragments from a lambda proviral DNA hybrid containing the entire presumptive avian myeloblastosis virus (AMV) provirus, and from a lambda proviral hybrid containing a partial myeloblastosis-associated virus type 1 (MAV-1)-like provirus were compared by heteroduplex analysis. The cloned presumptive AMV provirus was also analyzed by electron microscopy, using R-loop formation with purified 35S RNA isolated from virions of the standard AMV complex. The results indicate that the putative AMV genome contains a segment absent in its MAV-1-like helper virus. This segment represents a substitution in the region of the genome that in MAV-1 virus is occupied by the envelope gene and is approximately 900 +/- 160 nucleotide pairs in length. Hybridization of specific probes from the presumptive AMV genome to Southern blots of EcoRI-digested cellular DNA has revealed that these substituted sequences are homologous to chicken and duck DNA that is not related to chicken endogenous proviral sequences.
Communication between catalytic subunits in hybrid aspartate transcarbamoylase molecules: effect of ligand binding to active chains on the conformation of unliganded, inactive chains.
Authors: Yang Y R , Schachman H K
Published: May 2006

In the regulatory enzyme aspartate transcarbamoylase (aspartate carbamoyltransferase; carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2) of Escherichia coli, the six catalytic polypeptide chains are arranged as two distinct catalytic trimers "crosslinked" by three regulatory dimers. Because in allosteric proteins it is assumed that the binding of a ligand to one site promotes a conformational change that affects the subsequent binding to other sites in the oligomeric protein, it was of interest to determine directly whether the effects of ligand binding to chains in one catalytic subunit are "communicated" to unliganded chains in the other subunit. Accordingly, hybrid enzyme molecules were constructed containing sensitive chromophores on the three inactive catalytic chains in one subunit along with an active catalytic subunit and three native regulatory subunits. The derivative exhibited the allosteric properties characteristic of the native enzyme. Communication between the two catalytic subunits was demonstrated by spectral measurements showing that the effects of ligand binding to the three active chains are propagated to the chromophores on the unliganded, inactive chains in the other subunit. Moreover, the change in the tertiary structure of the unliganded catalytic chains is tightly linked to the alteration in the quaternary structure.
Transforming growth factors produced by certain human tumor cells: polypeptides that interact with epidermal growth factor receptors.
Authors: Todaro G J , Fryling C , De Larco J E
Published: May 2006

Three different human tumor lines in culture, a rhabdomyosarcoma, a bronchogenic carcinoma and a metastatic melanoma, release proteins (transforming growth factors, TGFs) into the medium that confer the transformed phenotype on untransformed fibroblasts. These proteins are acid and heat-stable; produce profound morphologic changes in rat and human fibroblasts; and enable normal anchorage-dependent cells to grow in agar. Removal of the transforming protein results in a reversion of cell phenotype. The major activity interacts with epidermal growth factor (EGF) cell membrane receptors. The peptides from these tumor cells are similar in their action to the sarcoma growth factor (SGF) released by murine sarcoma virus-transformed rodent cells. The most anchorage-independent tumor cells released the most TGFs. EGF-related TGFs were not detectable in fluids from cultures of cells with high numbers of free EGF membrane receptors (normal human fibroblasts and human carcinomas).
Le interferon mRNA from human fibroblasts.
Authors: Pang R H , Hayes T G , Vilcek J
Published: May 2006

Human F and Le interferon can be clearly distinguished on the basis of different antigenic properties and host range. After inoculation with Newcastle disease virus (NDV), GM-258 fibroblasts produced Le as well as F interferon; in contrast, only F interferon was detectable after stimulation with poly(I) . poly(C). Polyadenylylated mRNA isolated from fibroblasts induced with poly(I) . poly(C) or NDV was injected into Xenopus laevis oocytes and the interferon activities thus produced were analyzed. Only F interferon production was demonstrable in oocytes injected with mRNA from cells induced with poly(I) . poly(C), whereas both F and Le interferons were made in oocytes injected with mRNA from NDV-induced cultures. The time course of accumulation of F and Le interferon mRNAs in NDV-induced cells corresponded to the kinetics of F and Le interferon synthesis in intact cells. The ratio of F and Le interferons made in oocytes was similar to that observed in intact GM-258 cells. F and Le interferon mRNA activities isolated from GM-258 cells could not be separated by sucrose density gradient centrifugation. However, the profile of F mRNA activity was more heterogeneous and its peak sedimented somewhat more slowly than that of Le interferon mRNA. These results suggest that the varying ratios of F and Le interferon synthesis in different cells after different modes of stimulation are determined at the level of mRNA. The induction mechanisms of F and Le interferon mRNA synthesis appear to be closely related but not identical.
Maintenance of an aminopeptidase allele frequency cline by natural selection.
Authors: Koehn R K , Newell R I , Immermann F
Published: May 2006

The product of the Lap locus in the marine bivalve Mytilus edulis is a neutral, membrane-associated aminopeptidase that is primarily localized on intestinal microvilli and in digestive cell lysosomes. Natural populations are genetically differentiated at the Lap locus between areas of differing salinity. A steep (0.55-0.15) allele frequency cline connects differentiated populations between the Atlantic Ocean and Long Island Sound. We demonstrate an annual gene flow/mortality cycle in cline populations whereby gene frequencies after mortality are correlated with salinity and enzyme activity. The cline is spatially and temporally unstable in immigrants, but stable in residents after mortality. Mortality is nonrandom with regard to the Lap locus; genotype-dependent properties of the aminopeptidase enzyme apparently led to a differential rate of the utilizaiton of nutrient reserves because selected genotypes exhibited an increased rate of tissue weight loss. Aminopeptidase genotypes are differentially adapted to different temperatures and salinities, which provides a mechanism for the relationship among biochemical, physiological, and population phenotypes.
Prolonged derangements of canine myocardial purine metabolism after a brief coronary artery occlusion not associated with anatomic evidence of necrosis
Authors: DeBoer Laurence W. V. , Ingwall Joanne S. , Kloner Robert A. , Braunwald Eugene
Published: May 2006

Changes in myocardial purine metabolism were studied after temporary coronary artery occlusion and subsequent reperfusion in the dog. Sequential myocardial biopsies were performed to allow for measurements of ATP, adenine nucleotide, nucleoside, and base concentrations after 15 min of ischemia, and after 90 min and 72 hr of reperfusion following this period of ischemia. Control, nonischemic sites were also sampled. After 15 min of coronary occlusion, subendocardial ATP concentrations (reported in nmol/mg of protein; mean ± SEM) were depressed in the ischemic zone at 19.9 ± 3.5 compared to 38.1 ± 2.8 in the nonischemic zone (
P
< 0.001). Subepicardial ATP concentrations also were depressed at 27.0 ± 2.2 in ischemic sites compared to subepicardial nonischemic sites (40.0 ± 4.0,
P
< 0.005). After 90 min of reperfusion ATP concentrations remained depressed in the previously ischemic subendocardium 26.8 ± 4.2 (
P
< 0.025 vs. nonischemic sites). After 72 hr of reperfusion, ATP was still depressed in the previously ischemic subendocardium at 29.2 ± 2.5 (
P
< 0.025 vs. nonischemic) and subepicardium (27.9 ± 3.3,
P
< 0.05 vs. nonischemic). Total purines were determined as the sum of ATP, ADP, AMP, adenosine, inosine, and hypoxanthine. After 15 min of occlusion, the total purine pool in the ischemic subendocardium tended towards being lower than in the nonischemic zone (42.0 ± 5.9 vs. 53.8 ± 5.2, not significant) but in the ischemic subepicardium the total purine pool was similar to that in the nonischemic zone. After 90 min of reperfusion the previously ischemic subendocardial purine pool was reduced compared to the nonischemic zone (39.0 ± 4.8,
P
< 0.025). Total purines were also depleted in both the subendocardium and subepicardium of previously ischemic zones after 72 hr of reperfusion (44.5 ± 2.9 and 40.0 ± 4.4, respectively,
P
< 0.05). Histologic analysis of the previously ischemic tissue revealed no evidence of necrosis. Therefore, brief temporary coronary artery occlusions not associated with anatomic evidence of necrosis may result in prolonged abnormalities of ATP concentration and significant depletion of the total purine pool.
Regulation of beta-galactoside phosphate accumulation in Streptococcus pyogenes by an expulsion mechanism.
Authors: Reizer J , Panos C
Published: May 2006

Streptococcus pyogenes pregrown on lactose took up glucose, lactose, or methyl beta-D-thiogalactopyranoside (MeSGal or TMG) by a phosphoenolpyruvate-dependent phosphotransferase system. MeSGal accumulated in the cell as MeSGal-phosphate (MeSGalP). Three effects were noted when various sugars were added to MeSGal preloaded cells: (i) no decrease in intracellular MeSGalP concentration after addition of fructose, sucrose, o-nitrophenyl-beta-D-galactoside, glycerol, 6-deoxyglucose, alpha-methyl D-glucoside, 2-deoxygalactose, glucose 1-phosphate, or glucose 6-phosphate; (ii) slow loss of preaccumulated MeSGalP evoked by lactose, 2-deoxy-D-glucose, or unlabeled MeSGal; and (iii) a short lag followed by extremely rapid expulsion of intracellular MeSGalP elicited by glucose or mannose and a slower expulsion elicited by glucosamine. The expelled compound was free MeSGal, indicating the involvement of dephosphorylation in the expulsion mechanism. Deoxyglucose inhibited the expulsion evoked by mannose, and prepoisoning of cells with fluoride or arsenate prevented the glucose-dependent expulsion. The expulsion is due to activation of an expulsion mechanism rather than to turnover of MeSGalP and leak of internal MeSGal with concomitant inhibition of MeSGal influx. The results suggest the need for phosphotransferase-dependent translocation of a preferential sugar or accumulation of the sugar catabolite for expulsion activation. The significance of the expulsion mechanism in synthesis regulation of enzymes involved in carbohydrate utilization is proposed.
Localization of cyclic GMP-dependent protein kinase and substrate in mammalian cerebellum.
Authors: Schlichter D J , Detre J A , Aswad D W , Chehrazi B , Greengard P
Published: May 2006

The regional and cellular distribution of guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase (ATP:protein phosphotransferase,EC 2.7.1.37) in mammalian brain was examined by use of the photoaffinity label 8-azidoinosine 3',5'-cyclic monophosphate. Of the regions examined, cerebellum had by far the highest concentration of this enzyme. The cellular localization of cGMP-dependent protein kinase within the cerebellum was determined by examination of mutant mice missing specific types of cerebellar neurons. Mutant mice lacking Purkinje cells had greatly reduced amounts of cGMP-dependent protein kinase, whereas the loss of another cell type, granule cells, did not reduce cGMP-dependent protein kinase levels. By using the same strains of mutant mice, a 23,000-dalton soluble cerebellar substrate for cGMP-dependent protein kinase was also shown to be enriched in Purkinje cells. In contrast, the concentration of type I 3',5'-cyclic AMP-dependent protein kinase in the cerebellum was unaffected by the absence of Purkinje cells and only slightly reduced by the absence of granule cells. The enrichment in Purkinje cells of the cGMP-dependent protein kinase and its substrate suggests an important role for cGMP and cGMP-dependent protein phosphorylation in the function of this type of neuronal cell.
Identification of a promoter component involved in positioning the 5' termini of simian virus 40 early mRNAs.
Authors: Ghosh P K , Lebowitz P , Frisque R J , Gluzman Y
Published: May 2006

The 5' termini of the principal early mRNAs produced in cells transformed by wild-type simian virus 40 lie 21-25 nucleotides downstream from an A-T-T-T-A-T sequence on the DNA template. The 5' termini of early mRNAs produced by five origin-defective mutants containing deletions downstream from the A-T-T-T-A-T sequence and one viable mutant dl892 with a deletion starting 15 nucleotides upstream from this sequence were determined by a method involving synthesis, separation, and determination of the sequences of DNAs complementary to 5' termini. Mutant dl892 produced early mRNAs with the same principal 5' termini as wild-type virus; the origin-defective mutants produced mRNAs with principal 5' termini shifted downstream by a distance equivalent to the length of the deleted DNA segment. These data suggest that a DNA sequence of 29 nucleotides, which includes the A-T-T-T-A-T sequence, contains a component(s) of a promoter for early transcription. This component functions in positioning the 5' ends of the principal early mRNAs 21-25 nucleotides downstream from the A-T-T-T-A-T sequence and acts independently of these downstream sequences.
Nucleotide sequence of cloned unintegrated avian sarcoma virus DNA: viral DNA contains direct and inverted repeats similar to those in transposable elements.
Authors: Swanstrom R , DeLorbe W J , Bishop J M , Varmus H E
Published: May 2006

We have determined the nucleotide sequence of portions of two circular avian sarcoma virus (ASV) DNA molecules cloned in a prokaryotic host--vector system. The region whose sequence was determined represents the circle junction site--i.e., the site at which the ends of the unintegrated linear DNA are fused to form circular DNA. The sequence from one cloned molecule, SRA-2, shows that the circle junction site is the center of a 330-base-pair (bp) tandem direct repeat, presumably representing the fusion of the long terminal repeat (LTR) units known to be present at the ends of the linear DNA. The circle junction site is also the center of a 15-bp imperfect inverted repeat, which thus appears at the boundaries of the LTR. The structure of ASV DNA--unique coding region flanked by a direct repeat that is, in turn, terminated with a short inverted repeat--is very similar to the structure of certain transposable elements. Several features of the sequence imply that circularization to form the SRA-2 molecule occurred without loss of information from the linear DNA precursor. Circularization of another cloned viral DNA molecule, SRA-1, probably occurred by a different mechanism. The circle junction site of the SRA-1 molecule has a 63-bp deletion, which may have arisen by a mechanism that is analogous to the integration of viral DNA into the host genome. Flanking one side of the tandem direct repeat is the binding site for tRNATrp, the previously described primer for synthesis of the first strand of viral DNA. The other side of the direct repeat is flanked by a polypurine tract, A-G-G-G-A-G-G-G-G-G-A, which may represent the position of the primer for synthesis of the second strand of viral DNA. An A+T-rich region, upstream from the RNA capping site, and the sequence A-A-T-A-A-A are present within the direct repeat sequence. These sequences may serve as a promoter site and poly(A) addition signal, respectively, as proposed for other eukaryotic transcription units.
On the importance of changes in the gravity field on seismic recording at ultralong periods
Authors: Okal Emile A.
Published: May 2006

The effect of changes in gravity induced by the Earth's deformation on seismic recording at ultralong periods is studied quantitatively for the low-order spheroidal modes of the Earth. Because this effect can either enhance or reduce the recorded amplitude of a mode, depending on its geometry, it may become nontrivial at the longest free-oscillation periods, and contribute to relative deviations of up to 17%.