bluuebunny/crossref_metadata_2025 huggingface dataset:
Stereochemical course of the reaction catalyzed by the pyridoxal phosphate-dependent enzyme 1-aminocyclopropane-1-carboxylate synthase.
Authors: Ramalingam K , Lee K M , Woodard R W , Bleecker A B , Kende H
Published: May 2006

(+/-)-S-adenosyl-DL-(3R*,4S*)-[3,4-2H2]-methionine [a 1:1 mixture of (3R,4S) and (3S,4R)] and (+/-)-S-adenosyl-DL-(3R*,4R*)-[3,4-2H2]methionine [a 1:1 mixture of (3R,4R) and (3S,4S)] were synthesized from (Z)- and (E)-[1,2-2H2]ethene, respectively. Key steps in the synthesis were the antiperiplanar addition of methanesulfenyl chloride to (Z)-[1,2-2H2]ethene, to give a 1:1 mixture of (R,R)- and (S,S)-1-chloro-2-(methylthio)[1,2-2H2)ethane, followed by alkylation with sodium acetamidomalonate and hydrolysis to give an equal mixture of four stereoisomers of [3,4-2H2]methionine [(2R,3R,4S), (2R,3S,4R), (2S,3R,4S), and (2S,3S,4R)]. The other four stereoisomers of [3,4-2H2]methionine were prepared from (E)-(1,2-2H2]ethene. The two sets of stereoisomers of [3,4-2H2]methionine were chemically converted to S-adenosylhomocysteine, methylated to give the corresponding (+/-)-S-adenosyl-DL-methionines, and then incubated with 1-aminocyclopropane-1-carboxylate synthase partially purified from tomato (Lycopersicon esculentum, L.) pericarp tissue. The stereochemistry of the resulting samples of 1-aminocyclopropane-1-carboxylic acid was determined by comparison with the 1H NMR of the chemically synthesized and regio- and stereo-specifically deuterated compound. The results indicate that the hydrogens at the beta carbon of the methionine portion of S-adenosylmethionine do not participate in the reaction and that the ring closure occurs with inversion of configuration at the gamma carbon of the methionine portion of S-adenosyl-methionine, probably through a direct SN2-type displacement of the 5'-methylthio-5'-deoxyadenosine moiety by a carbanion equivalent formed at the alpha carbon of the methionine portion of S-adenosylmethionine.
Human beta-hexosaminidase alpha chain: coding sequence and homology with the beta chain.
Authors: Myerowitz R , Piekarz R , Neufeld E F , Shows T B , Suzuki K
Published: May 2006

We have isolated a cDNA clone, p beta H alpha-5, from an adult human liver library that contains the entire coding sequence of the alpha chain of beta-hexosaminidase. The cDNA insert of p beta H alpha-5 is 1944 base pairs long and contains a 168-base-pair 5' untranslated region, a 186-base-pair 3' untranslated region, and an open reading frame of 1587 base pairs corresponding to 529 amino acids (Mr, 60,697). The first 17-22 amino acids satisfy the requirements of a signal sequence. A striking sequence homology with a published partial amino acid sequence for the beta chain [O'Dowd, B. F., Quan, F., Willard, H. F., Lamhonwah, A. M., Korneluk, R. G., Lowden, J. A., Gravel, R. A. & Mahuran, D. J. (1985) Proc. Natl. Acad. Sci. USA 82, 1184-1188] suggests that both chains may have evolved from a common ancestor. A shorter alpha-chain cDNA was found to hybridize to the long arm of chromosome 15, the known location for the alpha-chain gene. In addition, we isolated another alpha-chain cDNA clone, p beta H alpha-4, from a simian virus 40-transformed human fibroblast library that contained an extra 453-base-pair piece at its 3' end. A probe consisting of this additional sequence hybridized exclusively to a single mRNA species (2.6 kilobases) in mRNA preparations from cultured human fibroblasts. In contrast, p beta H alpha-5 hybridized to both a 2.1-kilobase major and a 2.6-kilobase minor mRNA species in these same mRNA preparations, indicating the presence of two distinct alpha-chain mRNA species differing at the 3' end. Fibroblasts from an Ashkenazi Jewish patient with classic Tay-Sachs disease were deficient in both species of mRNA, confirming their genetic relationship.
Confirmation of the assignment of the low-field proton resonance of serine proteases by using specifically nitrogen-15 labeled enzyme.
Authors: Bachovchin W W
Published: May 2006

Proton NMR spectra of serine proteases in 1H2O solutions typically show a single resonance at very low magnetic field--i.e., 14-18 ppm from dimethylsilylapentanesulfonate. This resonance has been assigned to the proton hydrogen bonded between aspartic acid-102 and histidine-57 (chymotrypsin numbering system) of the "charge-relay system" or catalytic triad of serine proteases [Robillard, G. & Shulman, R. G. (1972) J. Mol. Biol. 71, 507-511]. Since then, there have been a number of reports that have cast doubt on its correctness. In the present work we have tested this assignment using alpha-lytic protease (EC 3.4.21.12, Myxobacter alpha-lytic proteinase), a bacterial serine protease homologous to elastase, which is specifically labeled with nitrogen-15 at N delta 1 of its single histidine residue. The low-field region of the proton spectra of this labeled enzyme shows a single resonance having the properties reported [Robillard, G. & Shulman, R. G. (1974) J. Mol. Biol. 86, 519-540], which, in addition, exhibits spin-spin splitting to the nitrogen-15 label. The observation of this 15N delta 1-H coupling makes the assignment of this resonance to the charge-relay proton unequivocal.
Ion etching bacteriophage T4: support for a spiral-fold model of packaged DNA.
Authors: Black L W , Newcomb W W , Boring J W , Brown J C
Published: May 2006

Ion etching of bacteriophage T4 erodes virus components progressively from the outside to the inside while preserving the overall structure. The terminal portion of the T4 DNA molecule packaged can be specifically radiolabeled and was found to be eroded more rapidly than the remainder of the DNA. This strongly suggests that the first DNA to enter the prohead is condensed in the center of the capsid and is therefore shielded from the ion beam by the surrounding last packaged DNA. The results support a "spiral-fold" model for the arrangement of DNA within the icosahedral bacteriophage head. According to this model, phage T4 DNA strands run parallel to the long axis of the phage, with sharp (180 degrees) bends at the top and bottom of the capsid. The folds themselves are arranged radially about the long axis of the head in spirally organized shells.
Human monocyte-endothelial cell interaction in vitro.
Authors: Pawlowski N A , Abraham E L , Pontier S , Scott W A , Cohn Z A
Published: May 2006

We have examined the interaction of freshly isolated human blood monocytes with cultured human umbilical vein endothelial cells in vitro. Purified monocytes incubated with confluent primary or passaged endothelial cells (EC) for 90 min at 37 degrees C bound at maximal densities of 6.5-7.0 X 10(3)/mm2 (8 or 9 per EC) without causing disruption of the monolayer. Monocyte-EC binding proceeded in the presence of plasma proteins or optimal phagocytic doses of opsonized zymosan particles. The avidity of attachment was not diminished by alternative monocyte isolation techniques. Monocyte attachment to EC was dependent upon the presence of divalent cations (magnesium greater than calcium) and was inhibited at 4 degrees C. Monocytes selectively bound to EC when incubated with monolayers composed of smooth muscle cells and EC. Neither EC monolayer confluence nor a variety of EC culture conditions affected the high levels of monocyte binding. In contrast, human neutrophils (less than 1 per EC) and lymphocytes (less than 2-3.5 per EC) bound at lower maximal densities under the same conditions, while platelet reactivity remained minimal. The distinctively higher affinity of human blood monocytes relative to other circulating white cells for binding to cultured human EC may have relevance to their function in vivo.
Monosynaptic connections made by the sensory neurons of the gill- and siphon-withdrawal reflex in
Aplysia
participate in the storage of long-term memory for sensitization
Authors: Frost William N. , Castellucci Vincent F. , Hawkins Robert D. , Kandel Eric R.
Published: May 2006

We have found that in the gill- and siphon- withdrawal reflex of
Aplysia
, the memory for short-term sensitization grades smoothly into long-term memory with increased amounts of sensitization training. One cellular locus for the storage of the memory underlying short-term sensitization is the set of monosynaptic connections between the siphon sensory cells and the gill and siphon motor neurons. We have now also found that these same monosynaptic connections participate in the storage of the memory underlying long-term Sensitization. We examined the amplitudes of the direct synaptic connections produced by siphon sensory neurons on the gill motor neuron L7 in nervous systems removed from control and from long-term sensitized animals 1 day after the end of long-term sensitization training. The connections were significantly larger in long-term sensitized animals than in control animals. The finding that long-term memory occurs at the same synaptic locus as the short-term memory should facilitate study of the cellular and molecular mechanisms involved in the conversion of short-term to long-term memory.
Calculus of twisted vertex operators
Authors: Lepowsky J.
Published: May 2006

Starting from an arbitrary isometry of an arbitrary even lattice, twisted and shifted vertex operators are introduced. Under commutators, these operators provide realizations of twisted affine Lie algebras. This construction, generalizing a number of known ones, is based on a self-contained “calculus.”
Effects of phospholipids and ADP-ribosylation on GTP hydrolysis by Escherichia coli-synthesized Ha-ras-encoded p21.
Authors: Tsai S C , Adamik R , Moss J , Vaughan M , Manne V , Kung H F
Published: May 2006

The Ha-ras protooncogene product p21, which may be involved in control of cellular growth, is a membrane protein that binds guanine nucleotides and hydrolyzes GTP. p21 GTPase activity is stimulated by lysophosphatidylcholine; a delay in activation was observed unless p21 was incubated with the phospholipid prior to assay. Maximal activation by the phospholipid was observed over a narrow concentration range; the presence in the assay mixture of lysophosphatidylcholine at concentrations above this optimum markedly inhibited p21 GTPase. GTP hydrolysis was also stimulated, but to a lesser degree, by phosphatidylcholine. Phosphatidylinositol and phosphatidylserine did not significantly enhance GTPase activity. The stimulatory effect of phospholipid was mimicked, in part, by nonionic detergents. p21 may be related to other GTPases, the regulatory guanine nucleotide-binding G proteins of the hormone-sensitive adenylate cyclase complex and transducin of the retinal light-activated phosphodiesterase system. The G proteins and transducin are heterotrimers; the alpha subunits possess GTPase activity and the beta gamma subunit complex along with agonist-receptor complex or light-activated rhodopsin enhance GTP hydrolysis. p21 GTPase activity was slightly stimulated by rhodopsin, but, in contrast to the GTPase activity of transducin, stimulation was not light-dependent. GTP hydrolysis was enhanced somewhat by beta gamma subunit complex in the absence, but not in the presence, of rhodopsin. Like the G proteins and transducin, activity of p21 was altered by ADP-ribosylation. Modification of p21 catalyzed by an NAD: arginine ADP-ribosyltransferase purified from turkey erythrocytes decreased both GTPase activity and guanine nucleotide binding activity.
Purified lens junctional protein forms channels in planar lipid films.
Authors: Zampighi G A , Hall J E , Kreman M
Published: May 2006

Junctions isolated from bovine lenses were solubilized with the detergent octyl glucoside, and their protein(s) was reconstituted in unilamellar vesicles. The protein(s) appears as annular-shaped intramembrane particles approximately equal to 10 nm in diameter on the vesicles' fracture faces. The addition of the vesicle-containing junctional protein(s) to both sides of preformed lipid films induced voltage-dependent channels. The channels have a conductance of 200 pS in 0.1 M salt solutions and are thus large enough to account for the electrical coupling observed between intact lens fibers; they turn off when the magnitude of the voltage is increased and in the presence of octanol. Although the identity of the reconstituted channels as the communicating pathway between lens fibers remains to be proven, it is most likely that the reconstituted channels are formed by MIP-26, the major protein component of the isolated lens junctions.
Stabilized complexes of epidermal growth factor and its receptor on the cell surface stimulate RNA synthesis but not mitogenesis.
Authors: Wakshull E M , Wharton W
Published: May 2006

Treatment of mouse fibroblasts, prelabeled at 4 degrees C with 125I-labeled epidermal growth factor (EGF), with the lectin concanavalin A (Con A) stabilized the 125I-labeled EGF-receptor complex to dissociation and prevented receptor-mediated endocytosis; after 5 hr at 37 degrees C, approximately 50% of the 125I-labeled EGF initially bound at 4 degrees C remained cell associated, compared to less than 15% in control cells. The radioactivity lost from the Con A-treated cells was found as intact hormone in the medium, with almost no hormone degradation evident, whereas in control cells most of the medium radioactivity was in the form of low molecular weight degradation products. The trimolecular complex Con A-EGF-receptor was capable of stimulating RNA synthesis to levels greater than control (untreated) or EGF alone and maintained this stimulation for prolonged periods of time. However, there was no effect of Con A treatment on the stimulation of DNA synthesis induced by EGF prebound at 4 degrees C. Thus, maintaining the EGF-receptor complex at the cell surface allows enhanced stimulation of an acute biological response to EGF (RNA synthesis) but not stimulation of DNA synthesis. These data support the idea that processing subsequent to receptor binding is necessary to produce the mitogenic signal.
Type beta transforming growth factor controls the adipogenic differentiation of 3T3 fibroblasts.
Authors: Ignotz R A , Massagué J
Published: May 2006

Differentiating mouse 3T3-L1 preadipocytes have been used as a model system to study the ability of type beta transforming growth factor (TGF-beta) to modulate cell development. We find that TGF-beta inhibits potently (ID50 approximately equal to 25 pM) the adipogenic conversion of 3T3-L1 cells. Inhibition is observed only when cells are exposed to TGF-beta before they become committed to differentiation. Even a transient (4 hr) exposure to TGF-beta immediately before the commitment point is sufficient to prevent differentiation. This point coincides with the time point immediately preceding the onset of coordinate expression of differentiation-specific proteins in 3T3-L1 cells. TGF-beta interacts with cell surface receptors in 3T3-L1 cells that have structural and binding properties similar to TGF-beta receptors in other cell types in which TGF-beta acts as a growth activator or a growth inhibitor. However, TGF-beta does not markedly alter differentiation-related mitosis in 3T3-L1 cells. The action of TGF-beta on 3T3-L1 cells does not involve changes in cAMP or prostaglandin E levels. These results suggest that TGF-beta is a unique modulator of adipogenic differentiation of fibroblasts.
Molecular association between major histocompatibility complex class I antigens and insulin receptors in mouse liver membranes.
Authors: Fehlmann M , Peyron J F , Samson M , Van Obberghen E , Brandenburg D , Brossette N
Published: May 2006

Molecular association between major histocompatibility complex (MHC) antigens and cellular proteins are thought to be involved in various immunological and nonimmunological functions of MHC antigens, including hormone signaling. The existence of physical interactions between insulin receptors and MHC class I antigens was investigated in liver plasma membranes from congenic H-2k mice. Insulin receptors were specifically labeled with a 125I-labeled photoreactive insulin analogue, and cellular proteins were solubilized and incubated with various monoclonal antibodies. Immunoprecipitates were analyzed by polyacrylamide gel electrophoresis followed by autoradiography. Antibodies reacting with distinct epitopes on H-2k class I antigens were all able to precipitate up to 25% of the labeled insulin receptors in H-2k mouse liver membranes, whereas no insulin receptors were precipitated in H-2b mouse liver membranes. Sequential immunoprecipitations showed that insulin receptors and H-2 antigens were coprecipitated and that no cross-reactivity occurred. The specificity of the interaction between insulin receptors and H-2 antigens was demonstrated after double labeling of membrane proteins by photoreactive insulin and lactoperoxidase-catalyzed iodination. These results thus show that, in mouse liver membranes, insulin receptors are physically associated to class I antigens of the MHC.
Neutral endopeptidase 24.11 in human neutrophils: cleavage of chemotactic peptide.
Authors: Connelly J C , Skidgel R A , Schulz W W , Johnson A R , Erdös E G
Published: May 2006

Membrane metallo-endopeptidase (NEP; neutral endopeptidase, kidney-brush-border neutral proteinase, enkephalinase, EC 3.4.24.11) cleaves peptides at the amino side of hydrophobic amino acids. While the enzyme is known to be in organs such as kidney and brain, we found it in human neutrophils. These cells cleaved the NEP substrate glutaryl (Glut)-Ala-Ala-Phe-(4-methoxynaphthylamine) (Glut-Ala-Ala-Phe-MNA) at a rate of 9.5 nmol X hr-1 per 10(6) cells, and phosphoramidon (1 microM) inhibited the hydrolysis by 90%. Intact neutrophils from donors who smoked had NEP activities about twice that of nonsmokers. Subcellular fractionation and sucrose density gradient centrifugation of lysed neutrophils showed that most of the NEP activity was membrane bound. A washed membrane fraction from human neutrophils rapidly cleaved 0.5 mM Glut-Ala-Ala-Phe-MNA (96 nmol X min-1 X mg-1) and the hydrolysis was inhibited by phosphoramidon and by specific antiserum to human renal NEP. The washed membrane fraction also rapidly cleaved 0.1 mM bradykinin (34 nmol X min-1 mg-1) and 0.1 mM fMet-Leu-Phe (49 nmol X min-1 X mg-1). The membrane-bound enzyme cleaved the peptide substrates at the same site as the homogeneous human renal NEP, and phosphoramidon and thiorphan inhibited the hydrolysis. Kinetic studies with pure human renal NEP showed that the chemotactic peptide fMet-Leu-Phe was one of the best biologically active substrates (Km, 59 X 10(-6) M; kcat, 3654 min-1). Immunocytochemistry at the light microscopic level revealed a high concentration of NEP on the cell membrane of neutrophils. This was confirmed with electron microscopy using the immunogold technique on ultrathin cryosections. These studies indicate that NEP in neutrophils may have important functions in inflammation and chemotaxis.
Midgestational abnormalities associated with in vitro preimplantation N-methyl-N-nitrosourea exposure with subsequent transfer to surrogate mothers.
Authors: Bossert N L , Iannaccone P M
Published: May 2006

Mouse blastocyst functions have been shown to be disrupted by in vitro exposure to N-methyl-N-nitrosourea (MeNU). After exposure, the chemically treated blastocysts were transferred to the uteri of pseudopregnant surrogate mothers. Implantation rate and birth rate have been shown previously to decrease in a concentration-dependent manner. Because of the large progressive decrease in the 50% effective concentration (EC50) for cytotoxicity, implantation rate, and live birth rate, we have investigated the midgestational effects of preimplantation exposure to MeNU after the transfer of treated embryos to surrogate mothers. A concentration-dependent decrease in normal implantation and a concurrent concentration-dependent increase in resorption number was observed in surrogates sacrificed at gestational age day 12 or day 15. Gross malformations were significantly increased by preimplantation exposure, in vitro, to MeNU. Fetal body length did not differ between fetuses developed from solvent-treated blastocysts and those that developed from natural pregnancies (nontransferred control) at either gestational age examined. Fetal body length was significantly shorter in fetuses developed from MeNU-treated blastocysts.
Calcium-regulating hormones modulate carbonic anhydrase II in the human erythrocyte.
Authors: Arlot-Bonnemains Y , Fouchereau-Peron M , Moukhtar M S , Benson A A , Milhaud G
Published: May 2006

The effect of calcitonin (CT) and parathyroid hormone (PTH) on carbonic anhydrase (carbonate hydrolyase, EC 4.2.1.1.) activity was tested in human erythrocyte hemolysates and with purified carbonic anhydrases I and II. The most important effect was on carbonic anhydrase II: CT showed a 2-fold increase and PTH showed a 50% decrease of carbonic anhydrase activity. This effect was observed at low hormonal concentrations and suggests the importance of CT in regulating carbonic anhydrase activity in the two important sites of CO2 exchange, erythrocyte and lung.
Phosphorylation of lens fiber cell membrane proteins.
Authors: Garland D , Russell P
Published: May 2006

Two intrinsic membrane proteins of calf lens fiber cells can be phosphorylated by a soluble bovine lens cAMP-dependent protein kinase and rabbit muscle cAMP-dependent protein kinase. After electrophoresis of the phosphorylated membranes, 32P comigrates with the lens main intrinsic protein at 26-27 kDa and with a minor band of protein that migrates at 19-20 kDa. 32P is also found with proteins that, based on the molecular sizes, are likely multimers of the 19-kDa and 26-kDa proteins. Upon boiling in NaDodSO4, all the radioactivity is found at the top of the gel, suggesting that both phosphoproteins are intrinsic membrane proteins. Serine is the only phospho amino acid detected in both proteins regardless of the source of protein kinase. The phosphorylation sites of both proteins are lost upon cleavage with trypsin and chymotrypsin. The smaller phosphoprotein is likely not a crystallin, because antibodies directed against alpha-, beta-, or gamma-crystallins do not cross-react with the 19-kDa protein. The 19-kDa 32P-labeled protein does not migrate coincident with calf alpha-crystallin.
Antisera against a guanine nucleotide binding protein from retina cross-react with the beta subunit of the adenylyl cyclase-associated guanine nucleotide binding proteins, Ns and Ni.
Authors: Gierschik P , Codina J , Simons C , Birnbaumer L , Spiegel A
Published: May 2006

Antisera were produced in rabbits against a guanine nucleotide binding protein (N protein), transducin, purified from bovine retina. Antiserum AS/1, which recognized all three subunits (alpha, beta, and gamma) of the holoprotein, was tested for cross-reactivity with the subunits of the adenylyl cyclase [adenylate cyclase; ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1]-associated stimulatory (Ns) and inhibitory (Ni) N proteins purified from human erythrocytes. As/1 showed strong reactivity against the beta subunits of both Ns and Ni but failed to cross-react with either the alpha or gamma subunits of Ns and Ni. Seven additional antisera against transducin reacted with the beta subunits but not with the alpha or gamma subunits of Ns and Ni. A single antiserum against transducin reacted with the alpha subunit of Ni but not of Ns. Immunostaining of the beta subunits of Ns and Ni was proportional to the amount of beta subunit blotted and to the antiserum concentration. Immunostaining of either human erythrocyte or bovine cerebral cortical plasma membrane proteins with AS/1 showed a single band, comigrating with the beta subunit of transducin; this band was absent in bovine erythrocyte membranes. Estimation of the amount of beta subunit by immunoblotting with AS/1 showed that the beta subunit comprises approximately equal to 2% of bovine cerebral cortical plasma membrane protein, approximately equal to 100-fold more than in human erythrocyte membranes. These findings provide immunochemical evidence for similarities in the beta subunits and differences in the alpha and gamma subunits of this family of N proteins. Antisera against transducin react specifically with the beta subunits of Ns and Ni in crude plasma membranes and, thus, can serve as specific probes for the beta subunit.
A protein kinase from Xenopus eggs specific for ribosomal protein S6.
Authors: Erikson E , Maller J L
Published: May 2006

A protein kinase specific for ribosomal protein S6 has been purified from eggs of Xenopus laevis. As visualized on a silver-stained polyacrylamide gel, the major protein in the preparation migrated with a Mr of 90,000. Incubation of the enzyme preparation with [gamma-32P]ATP led to phosphorylation of this protein on serine residues. Upon glycerol gradient centrifugation, the S6 kinase activity and the Mr 90,000 protein both sedimented with a Mr of 50,000-55,000. Two-dimensional gel electrophoresis demonstrated that up to 4-5 phosphate groups per S6 molecule could be incorporated with this enzyme in vitro, and two-dimensional peptide mapping demonstrated that the phosphopeptides from S6 labeled in vitro with the enzyme comigrated with those from highly phosphorylated S6 labeled in vivo in response to progesterone treatment. The purified S6 protein kinase did not phosphorylate at a significant rate ribosomal protein S10, histone H1, histone H4, mixed histones, casein, or phosvitin, indicating a high degree of substrate specificity. These results indicate that activation of a single S6 protein kinase may be sufficient to account for increased S6 phosphorylation after a growth stimulus.
Heparin affinity of anionic and cationic capillary endothelial cell growth factors: analysis of hypothalamus-derived growth factors and fibroblast growth factors.
Authors: Klagsbrun M , Shing Y
Published: May 2006

Bovine hypothalamus-derived growth factors (HDGF), pituitary fibroblast growth factor (FGF), and brain FGF were analyzed by chromatography on immobilized heparin and tested for the ability to stimulate the proliferation of capillary endothelial (CE) cells. Two distinct CE cell growth factors were found in hypothalamus, one anionic (aHDGF; pI of about 5) and one cationic (cHDGF; pI of about 8). Both aHDGF and cHDGF adhered tightly to immobilized heparin. They were eluted with 0.9-1.1 M NaCl and 1.3-1.5 M NaCl, respectively. Pituitary and brain FGF were also found to bind to immobilized heparin and to stimulate CE cell proliferation. Pituitary FGF was eluted at 1.4-1.6 M NaCl. The elution profile of brain FGF showed that two peaks of CE cell growth factor activity were eluted from the heparin column, one at 1.0 M NaCl and a second at 1.4-1.6 M NaCl. The tight binding of all of these growth factors to heparin (particularly aHDGF, whose binding is unexpected because of its negative charge) is presented as evidence that CE cell growth factors all share an affinity for heparin.
23Na and 39K NMR studies of ion transport in human erythrocytes.
Authors: Ogino T , Shulman G I , Avison M J , Gullans S R , den Hollander J A , Shulman R G
Published: May 2006

Ion transport in human erythrocytes was studied by 23Na and 39K NMR with an anionic paramagnetic shift reagent, Dy(P3O10)2(7-). The intra- and extracellular 23Na and 39K NMR signals were well separated (over 10 ppm) at 5 mM concentration of the shift reagent. The NMR visibility of the intracellular Na+ and K+ was determined to be 100% in human and duck erythrocytes. The intracellular ion concentrations were 8.1 +/- 0.8 mM Na+ (n = 7) and 110 +/- 12 mM K+ (n = 4) for fresh human erythrocytes. The ouabain-sensitive net Na+ efflux was 1.75 +/- 0.08 mmol/hr per liter of cells at 37 degrees C (n = 3). The gramicidin-induced ion transport in human erythrocytes was also studied by 23Na and 39K NMR or by simultaneous measurements of 23Na NMR and a K+-selective electrode. The time courses of the Na+ and K+ transport induced by the ionophore were biphasic. The initial rapid fluxes were due to an exchange of Na+ for K+, which were found to occur with a 1:1 stoichiometry. The subsequent slow components were the net Na+ and K+ effluxes rate-limited by the Cl- permeability and accompanied by a reduction in cell volume. The Cl- permeability determined from the NMR measurements of these slow fluxes was 3.2 +/- 0.5 X 10(-8) cm/sec at 25 degrees C (n = 4).
Construction of the mycoplasma evolutionary tree from 5S rRNA sequence data.
Authors: Rogers M J , Simmons J , Walker R T , Weisburg W G , Woese C R , Tanner R S , Robinson I M , Stahl D A , Olsen G , Leach R H
Published: May 2006

The 5S rRNA sequences of eubacteria and mycoplasmas have been analyzed and a phylogenetic tree constructed. We determined the sequences of 5S rRNA from Clostridium innocuum, Acholeplasma laidlawii, Acholeplasma modicum, Anaeroplasma bactoclasticum, Anaeroplasma abactoclasticum, Ureaplasma urealyticum, Mycoplasma mycoides mycoides, Mycoplasma pneumoniae, and Mycoplasma gallisepticum. Analysis of these and published sequences shows that mycoplasmas form a coherent phylogenetic group that, with C. innocuum, arose as a branch of the low G+C Gram-positive tree, near the lactobacilli and streptococci. The initial event in mycoplasma phylogeny was formation of the Acholeplasma branch; hence, loss of cell wall probably occurred at the time of genome reduction to approximately to 1000 MDa. A subsequent branch produced the Spiroplasma. This branch appears to have been the origin of sterol-requiring mycoplasmas. During development of the Spiroplasma branch there were several independent genome reductions, each to approximately 500 MDa, resulting in Mycoplasma and Ureaplasma species. Mycoplasmas, particularly species with the smallest genomes, have high mutation rates, suggesting that they are in a state of rapid evolution.
Exclusive nuclear location of estrogen receptors in Squalus testis.
Authors: Callard G V , Mak P
Published: May 2006

An estrogen (E)-binding molecule having both occupied and unoccupied sites is restricted to nuclear subfractions in the testis of the spiny dogfish (Squalus acanthias). We investigated the hypothesis that a species characterized by high body-fluid osmolarity (1010 mosM) has an estrogen receptor (ER) that binds to chromatin with high affinity and consequently resists redistribution during tissue processing. Although the steroid binding and sedimentation properties of the Squalus nuclear ER conformed to those of classical ER, its elution maximum from DNA-cellulose was unusually high (0.55 M NaCl). A tendency to adhere tightly to cell nuclei was reflected in the high salt concentration (0.43 M KCl) required to extract 50% of the receptors from the nuclear compartment during homogenization and in the stability of the nuclear ER population in the presence of high concentrations of a nonionic solute (urea) or increased buffer volume. Mixing and redistribution experiments showed that nuclear ER could be quantitatively and qualitatively measured in cytosolic extracts, ruling out the possibility that soluble receptors were being masked. Although Squalus oviduct ER was similar to that of testis, ER in the testis and liver of a related elasmobranch (Potamotrygon) that maintains osmotic equilibrium at 300 mosM more closely resembled mammalian ER in its elution maximum from DNA-cellulose (0.22 M NaCl) and cytosolic/nuclear ratios in low-salt buffers. We conclude that Squalus testis has a single ER pool located exclusively in the nuclear compartment. These observations support a revised concept of steroid action and further indicate that the chromatin affinity of the hormone-ER complex is an important factor in determining subfractional distribution during tissue processing.
Similarity between the corticotropin (ACTH) receptor and a peptide encoded by an RNA that is complementary to ACTH mRNA.
Authors: Bost K L , Smith E M , Blalock J E
Published: May 2006

An interesting pattern in the genetic code was recently observed: Codons for hydrophilic and hydrophobic amino acids on one strand of nucleic acid are complemented by codons for hydrophobic and hydrophilic amino acids on the other strand, respectively. The average tendency of codons for "uncharged" (slightly hydrophilic) amino acids is to be complemented by codons for "uncharged" (slightly hydrophilic) amino acids. We have postulated that this pattern can result in the binding of peptides that are encoded by complementary RNA strands and we have presented supporting evidence. In this report we demonstrate the specific and high-affinity binding of naturally occurring peptides [corticotropin (ACTH) and gamma-endorphin] to synthetically derived counterparts that were specified by RNA sequences complementary to the mRNA for ACTH and gamma-endorphin, respectively. That this binding might result from one peptide being an "internal image" of the other was strongly suggested by the observation that antibody to the peptide that was encoded by the complementary RNA for ACTH recognized the adrenal cell ACTH receptor. Based on these findings, a theory on the evolution of peptides and their receptors is suggested.
On the origin of species of fibronectin.
Authors: Schwarzbauer J E , Paul J I , Hynes R O
Published: May 2006

Multiple different subunits of fibronectin are known to occur and their origin has been unclear. Recent results showing that a single fibronectin gene can give rise to several different mRNAs by alternative splicing suggested an explanation for some of this diversity of fibronectin subunits. Because the alternative splicing events occur within the coding region, the mRNAs differ in coding potential. We have prepared recombinant phage containing a rat fibronectin cDNA segment that is present in some fibronectin mRNAs and not in others. This segment was inserted in the beta-galatosidase gene of lambda gt11, and fusion protein produced by lysogens of the recombinant phage was purified and used as immunogen. The resulting antisera recognized some subunits of rat and hamster fibronectins but not others, indicating that inclusion or removal of this segment gives rise to mRNAs that encode different fibronectin subunits. In particular, presence or absence of a 95 amino acid segment appears to account for differences in size among the subunits of plasma fibronectin, whose origin is therefore explained by alternative patterns of RNA splicing.
Isolation and biochemical characterization of the mammalian reovirus type 3 cell-surface receptor.
Authors: Co M S , Gaulton G N , Fields B N , Greene M I
Published: May 2006

A cell-surface receptor for the mammalian reovirus type 3 hemagglutinin was isolated by using antiidiotypic anti-receptor antibodies. The receptor is a glycoprotein with a molecular mass of 67,000 daltons and a pI of 5.9. Evidence that the isolated structure represents the reovirus receptor was obtained by electrophoretic immunoblot studies, which demonstrated that the 67,000-dalton glycoprotein is the only cell-surface structure recognized by both reovirus type 3 and the anti-receptor immunoglobulin. Comparison of the reovirus receptor on murine thymoma (R1.1) and rat neuroblastoma (B104) cells indicated that similar structures on the cell surface are recognized by the reovirus type 3 and the anti-receptor antibodies as previously suggested from cellular and binding studies. This receptor was found on mouse, rat, monkey, and human cells. Furthermore, diverse tissue types, including lymphoid and neuronal cells, express the receptor structure. The receptor structure is discussed in terms of its role in mediating viral tropism and as an essential cell-surface protein.
Nerves in the spine of a sea urchin: a neglected division of the echinoderm nervous system.
Authors: Smith D S , Brink D , del Castillo J
Published: May 2006

Electrical stimulation of the primary spines (greater than 10 cm long) of the tropical sea urchin Diadema antillarum elicits graded compound action potentials that are conducted at a constant speed of approximately equal to 27 cm/sec. Ion substitution experiments suggest that these are due to the summation of calcium spikes. Structural studies have revealed the presence of up to 21 regularly disposed nerves within the spine shaft, each nerve bundle including greater than 1000 neurites in the basal region, narrowing to slender groups of processes near the spine tip. The neurites in each nerve range in diameter from less than 0.1- approximately equal to 2 micron. Most appear to be distal processes of presumed sensory perikarya situated at the level of the tissue cone surrounding the spine base, or more proximally, although some neurites may arise from perikarya near the spine tip. These nerve tracts are thought to correspond to the nerve fibers described by Hamann [(1887) Jena Z. Naturwiss. 21, 114-176] almost a century ago in spines of Centrostephanus longispinus and, thus, to represent a long-neglected region of the echinoid nervous system.
Mechanism of inhibition of mitochondrial adenosine triphosphatase by dicyclohexylcarbodiimide and oligomycin: relationship to ATP synthesis.
Authors: Penefsky H S
Published: May 2006

Measurement of the rate of [gamma-32P]ATP binding (k1) and release (k-1) from catalytic sites on submitochondrial particles permitted calculation of the affinity constant in catalytic sites (k1 = K1/k1-1) of 10(12) M-1. This value is the same as that determined previously for the solubilized ATPase (F1) from beef heart mitochondria. Treatment of submitochondrial particles with dicyclohexylcarbodiimide or oligomycin so as to cause about 90% inhibition of ATPase activity was accompanied by a decrease in the binding of [gamma-32P]ATP in high-affinity catalytic sites. Under the conditions of the experiment, it is expected that the inhibitors reacted not with the ATPase itself but with other proteins in the oligomycin-sensitive ATPase complex (F0-F1). It is proposed that dicyclohexylcarbodiimide and oligomycin inhibit ATPase activity by causing a conformational change in the F0 portion of the complex that is transmitted to F1, resulting in an impaired binding of substrate in catalytic sites. These observations of apparent conformational interactions between F0 and F1 on the mitochondrial membrane are relevant to the mechanism of the coupling device that links the energy store to ATP formation in oxidative phosphorylation. It is proposed that a change in the state of ionization of one or more charged amino acid residues in F0 results in a conformational change in F0 which, transmitted to F1, reversibly alters the catalytic sites and facilitates the release of product ATP.
Complete sequence of a gene encoding a human type I keratin: sequences homologous to enhancer elements in the regulatory region of the gene.
Authors: Marchuk D , McCrohon S , Fuchs E
Published: May 2006

We report here the complete nucleotide sequence of a gene encoding the 50-kDa keratin expressed in abundance in human epidermal cells. According to its sequence, this gene has a single transcriptional initiation site and a single polyadenylylation signal. Nuclease S1 mapping of this gene with total human epidermal mRNA confirmed the presence of a single initiation site for the 50-kDa keratin gene. When the regulatory sequences 5' upstream from this gene were examined, three sequences that share significant homology with viral and immunoglobulin enhancer elements were found. In comparison, the sequence of the regulatory region of vimentin, a structurally similar intermediate filament gene, was highly divergent [Quax, W., Egberts, W. V., Hendriks, W., Quax-Jeuken, Y. & Bloemendal, H. (1983) Cell 35, 215-223]. This finding may provide a clue to understanding the molecular mechanisms underlying the widely varying levels of expression of different intermediate filament genes in different tissues.
Complete sequence of the chicken glyceraldehyde-3-phosphate dehydrogenase gene.
Authors: Stone E M , Rothblum K N , Alevy M C , Kuo T M , Schwartz R J
Published: May 2006

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an evolutionarily conserved glycolytic enzyme, is constitutively expressed in most cell types yet is induced to high levels during the development of fast twitch muscle fibers. To analyze the organization and regulation of the chicken GAPDH gene, we first constructed a nearly full-length GAPDH cDNA clone (pGAD-28). pGAD-28 was used in the current study to screen a genomic library, and several overlapping clones were selected. The GAPDH coding region was detected within a 4.65-kilobase Xho I/EcoRI genomic fragment that was completely sequenced by using the M13 cloning vector system. A small portion of pGAD-28 was used as a primer to extend a 33-nucleotide sequence from the 5' end of GAPDH mRNA. The canonical promoter "TATA" region was found 22 base pairs from the 5' end of the mRNA. The 5' end of the GAPDH gene is extraordinarily G+C-rich (80%) and contains two inverted sequences with a 9-base-pair homology found at -58 (G-G-G-G-C-G-G-G-C) and -93 (G-C-C-C-G-C-C-C-C) nucleotides from the transcription start site. Sequencing also revealed the location of 11 introns within the transcribed portion of the GAPDH gene. The placement of at least 3 of the introns corresponds to the boundaries of protein domains within prokaryotic and eukaryotic GAPDHs that were previously detected by x-ray crystallography. This concordance suggests that introns may have participated in the construction of the earliest GAPDH gene.
Isolation and point of action of a factor from Escherichia coli required to reconstruct translation.
Authors: Ganoza M C , Cunningham C , Green R M
Published: May 2006

To study the mechanism of translation we have attempted to reconstruct the process from purified components. Protein synthesis was programmed by the RNAs of wild-type or amber mutants of bacteriophages f2 or MS2. Translation programmed by MS2 or f2am3 RNA does not occur using ribosomes, precharged aminoacyl-tRNAs, and the sum of the purified proteins involved in initiation (initiation factors; IF-1, IF-2, and IF-3), propagation (elongation factors; EF-Tu, EF-Ts, and EF-G) and termination (release factors; RF-1 or RF-2) of protein synthesis. The requirement for a protein called W was demonstrated. Protein W was purified free of all translation factors, activating enzymes, and other proteins such as the RR, "rescue," and EF-P implicated in translation. The stimulation of propagation by W depended on the position of the amino acid residue to be added in the synthesis of the NH2-terminal hexapeptide of the coat protein. In the reconstructed system, with the sum of all translation factors but in the absence of W, only dipeptides and smaller quantities of tripeptides were synthesized under the direction of f2am3 RNA. W stimulated the synthesis of the hexapeptide, fMet-Ala-Ser-AspNH2-Phe-Thr directed by this RNA. In addition, W stimulated ejection of non-cognate tRNAs that bind to ribosomal particles.
Cell lines and clinical isolates derived from Ph1-positive chronic myelogenous leukemia patients express c-abl proteins with a common structural alteration.
Authors: Konopka J B , Watanabe S M , Singer J W , Collins S J , Witte O N
Published: May 2006

The Philadelphia chromosome (Ph1), observed in greater than 90% of chronic myelogenous leukemia (CML) patients, results from a specific chromosomal translocation involving the c-abl gene. The translocation breakpoint occurs near c-abl and correlates with the production of an altered c-abl mRNA. In the CML-derived cell line K562, Ph1 is accompanied by a structurally altered c-abl protein (P210c-abl) with in vitro tyrosine kinase activity not detected with the normal c-abl protein (P145c-abl). We have examined c-abl proteins in other Ph1-positive CML cell lines and found that they all express P210c-abl. P210c-abl was also detected in bone marrow cells from CML patients with Ph1 in the accelerated and blast crisis phases of the disease. Comparison of the [35S]methionine-labeled tryptic peptides generated from the normal P145c-abl and P210c-abl showed that they have closely related structures, but additional polypeptide sequences are present in P210c-abl. Based on these results we propose that translocation of c-abl in Ph1-positive CML results in the creation of a chimeric gene leading to the production of a structurally altered c-abl protein with activated tyrosine kinase activity. The altered P210 c-abl protein is strongly implicated in the pathogenesis of CML.
Optical measurement of voltage-dependent Ca2+ influx in frog heart.
Authors: Pizarro G , Cleemann L , Morad M
Published: May 2006

Sarcolemmal Ca2+ movements in frog ventricular strips were measured by monitoring Ca2+ depletion from the extracellular space with an impermeant Ca indicator dye, antipyrylazo III. Ca2+ depletion was measured as a weighted average of light signals recorded simultaneously at three different wavelengths. This weighting procedure was designed to reduce the motion-induced light scattering and to enhance the Ca2+-related optical signals. Comparison of the time course of Ca2+ depletion signal with that of contraction showed that the rate of Ca2+ depletion was maximal immediately after the upstroke of the action potential but prior to the onset of tension. Peak Ca2+ depletion was reached toward the end of the action potential and amounted to a 10-50 microM decrease in the total extracellular Ca2+ concentration. The reaccumulation of extracellular Ca2+ seen after the action potential was 2-5 sec slower than the relaxation of tension. The rate of Ca2+ depletion had a bell-shaped voltage dependence and was enhanced by epinephrine, suggesting that Ca2+ influx occurred primarily through a slowly inactivating ionic channel. Ca2+ transport through the Na+-Ca2+ exchange system was not significantly altered in the presence of strophanthidin or with decrease of extracellular K+ concentration despite marked potentiation of tension by these agents. Ca2+ depletion measured at the end of a 1-sec clamp pulse had a voltage dependence noticeably different from that of the developed tension. This finding may suggest that a fraction of activator Ca2+ is released from membrane-bound Ca2+ pools in a voltage-dependent manner. Our results show that Ca2+ indicator dyes can be used not only to measure rapid changes in the extracellular Ca2+ concentration during contraction, but also to quantify the contribution of various sarcolemmal Ca2+ transport systems to the generation of tension in cardiac muscle.
Alternative RNA processing events in human calcitonin/calcitonin gene-related peptide gene expression.
Authors: Jonas V , Lin C R , Kawashima E , Semon D , Swanson L W , Mermod J J , Evans R M , Rosenfeld M G
Published: May 2006

Two mRNAs generated as a consequence of alternative RNA processing events in expression of the human calcitonin gene encode the protein precursors of either calcitonin or calcitonin gene-related peptide (CGRP). Both calcitonin and CGRP RNAs and their encoded peptide products are expressed in the human pituitary and in medullary thyroid tumors. On the basis of sequence comparison, it is suggested that both the calcitonin and CGRP exons arose from a common primordial sequence, suggesting that duplication and rearrangement events are responsible for the generation of this complex transcription unit.