bluuebunny/crossref_metadata_2025 huggingface dataset:
Prostaglandin-stimulated GTP hydrolysis associated with activation of adenylate cyclase in human platelet membranes.
Authors: Lester H A , Steer M L , Levitzki A
Published: May 2006

In membranes purified from human blood platelets, basal guanosine triphosphate (GTP) hydrolysis is reduced by a factor of approximately 6 by exposure to N-ethylmaleimide (10 mM). This decreased background enables the detection of an additional GTP hydrolysis in the presence of prostaglandin E1 (PGE1). The PGE1-stimulated GTPase has several properties correlated with PGE1-stimulated adenylate cyclase in this preparation. The two enzymes have similar dose-response relationships (half-maximal stimulation at 0.1 microM PGE1). Exposure to cholera toxin blocks the PGE1-stimulated GTPase and activates adenylate cyclase. Both enzymes are activated by submicromolar concentrations of GTP, although the Km for the GTPase is about 10 times greater than that for the adenylate cyclase. The data are discussed in relation to the hypothesis that hormone-stimulated adenylate cyclase (i) is activated as a regulatory component binds a molecular of GTP and (ii) is deactivated as this molecule is hydrolyzed.
Evolutionary conservation of DNA polymerase beta structure.
Authors: Chang L M , Plevani P , Bollum F J
Published: May 2006

An immunological procedure that uses antiserum against homogeneous calf thymus DNA polymerase beta to detect immunoreactive peptides on NaDodSO4/polyacrylamide gel electrophoresis demonstrates a high degree of conservation of protein sequence and molecular weight for this enzyme, from parastic protozoans to man. By renaturation of DNA polymerase activity in situ after electrophoresis, the enzymatically active peptides are shown to correspond to the immunoreactive peptides. The persistence of sequence and molecular weight for the catalytic peptide of DNA polymerase beta through eons of evolutionary time suggests an essential role for this enzyme in DNA metabolism of complex cells.
Reconstitution in planar lipid bilayers of a Ca2+-dependent K+ channel from transverse tubule membranes isolated from rabbit skeletal muscle.
Authors: Latorre R , Vergara C , Hidalgo C
Published: May 2006

Addition of membrane vesicles prepared from transverse tubule (T-tubule) membranes of rabbit skeletal muscle to the aqueous phase of a planar lipid bilayer induces a stepwise increase in conductance. This conductance is both voltage and Ca2+ dependent. At 1 mM Ca2+, the steady-state conductance is maximal at voltages higher than +20 mV and decreases for more negative voltages. (Voltages refer to the side to which the vesicles are added, cis) Decreasing the Ca2+ concentration reversibly shifts the conductance-voltage curve toward the right along the voltage axis. Furthermore, Ca2+ can activate the conductance only if added to the cis compartment. Neither Mg2+, Ba2+, nor Cd2+ can activate the conductance induced by T-tubule vesicles. Addition of 5 mM tetraethylammonium ion to the trans, but not the cis, side abolishes the T-tubule-induced conductance. The Ca2+-dependent conductance appears as a consequence of ionic channel formation. Single-channel activity appears in bursts followed by periods of time in which the channel remains "silent". The conductance of the open channel averages 226 pS in 0.1 M KC1 and is voltage and Ca2+ independent. However, the fraction of time that the channel remains in the open state is voltage and Ca2+ dependent in a manner that parallels the voltage and Ca2+ dependence of the multichannel membrane. The channel is 6.6 times more permeable to K+ than to Na+ and is impermeable to C1-.
Presence of somatostatin-28-(1-12) in hypothalamus and pancreas.
Authors: Benoit R , Böhlen P , Ling N , Briskin A , Esch F , Brazeau P , Ying S Y , Guillemin R
Published: May 2006

Acid extracts from rat pancreas and hypothalamus were analyzed for the presence of the antigenic determinant corresponding to the NH2 terminus of somatostatin-28 (SS28), using an antiserum directed against amino acids 1 to less than or equal to 11 of the SS28 molecule. On gel permeation chromatography the majority of the immunoreactive material from each tissue extract eluted in one zone compatible with a peptide of 1250 daltons. Purification of this immunoreactive material by reverse-phase HPLC and cation-exchange chromatography yielded two immunoreactive peptides from each tissue extract. The amino acid compositions of both peptides in pancreas and hypothalamus correspond to the fragment 1-12 of SS28. The more hydrophobic peptide from each tissue coeluted with synthetic SS28-(1-12) on HPLC, while the other one coeluted with synthetic SS28-(1-12)-amide. We conclude that the prosomatostatin fragment Ser-Ala-Asn-Ser-Asn-Pro-Ala-Met-Ala-Pro-Arg-Glu-OH is present in both rat hypothalamus and rat pancreas.
Mössbauer studies of beef heart aconitase: evidence for facile interconversions of iron-sulfur clusters.
Authors: Kent T A , Dreyer J L , Kennedy M C , Huynh B H , Emptage M H , Beinert H , Münck E
Published: May 2006

Beef heart aconitase, isolated under aerobic conditions, has been studied with Mössbauer and EPR spectroscopy. In the oxidized state, the enzyme exhibits an EPR signal at g = 2.01. The Mössbauer data show that this signal is associated with a 3Fe cluster. In dithionite-reduced aconitase, the 3Fe cluster, probably of the [3Fe-3S] type, is in a paramagnetic state of interger electronic spin (S = 2); the Mössbauer spectra exhibit al the unique features reported for proteins with 3Fe clusters. On activation of aconitase with ferrous ion, the paramagnetic 3Fe cluster of dithionite-reduced enzyme is converted into a diamagnetic (S = 0) form. Activation studies with iron enriched in either 27 Fe or 56 Fe suggest that activation transforms the 3Fe cluster into a center that has a [4Fe-4S] core. This conclusion is supported by the observation that EPR signals characteristic of reduced [4Fe-4S] clusters can be elicited under appropriate conditions. It has frequently been assumed that the activation of aconitase with Fe2+ produces an active site containing a single ferrous ion. The data reported here suggest that a ferrous ion is used to rebuild a [4Fe-4S] cluster.
Two-dimensional rhodopsin crystals from disk membranes of frog retinal rod outer segments.
Authors: Corless J M , McCaslin D R , Scott B L
Published: May 2006

Two-dimensional crystals of rhodopsin have been prepared from purified frog disk membranes by using the detergent Tween 80. The space group of the orthorhombic crystals is p22121; the unit cell dimensions are 47 X 151 A. Projection maps of negatively stained preparations have been calculated to a resolution of approximately 22 A. The rhodopsin molecules are associated as dimers that appear to be slightly sigmoidal and are 20-25 A in width and 70-80 A in length.
Regulation of sulfate uptake in carrot cells: Properties of a hypercontrolled variant
Authors: Furner Ian J. , Sung Z. R.
Published: May 2006

Sulfate uptake in haploid carrot cultures can be experimentally controlled by the sulfur source provided for growth. The rate of sulfate uptake is low in cells grown on cystine or sulfate and high in sulfur-starved cells. A selenate-resistant variant cell line has been isolated from a haploid carrot line. The variant shows hypersuppression of sulfate uptake by cystine and essentially normal control by the other treatments. While both lines efflux intracellular sulfate in the presence of external sulfate, the rate of efflux from the variant is 4-6 times higher at comparable levels of initial intracellular sulfate. Further, properties of the efflux and uptake in both lines suggest that they are mediated by the same system. We propose that the variant possesses an altered uptake-efflux system that is more readily reversed and more subject to control by some metabolite derived from cystine.
Hydroxyl radical scavengers inhibit lymphocyte mitogenesis.
Authors: Novogrodsky A , Ravid A , Rubin A L , Stenzel K H
Published: May 2006

Agents that are known to be scavengers of hydroxyl radicals inhibit lymphocyte mitogenesis induced by phorbol myristate acetate (PMA) to a greater extent than they inhibit mitogenesis induced by concanavalin A or phytohemagglutinin. These agents include dimethyl sulfoxide, benzoate, thiourea, dimethylurea, tetramethylurea, L-tryptophan, mannitol, and several other alcohols. Their inhibitory effect is not associated with cytotoxicity. The hydroxyl radical scavengers do not inhibit PMA-dependent amino acid transport in T cells or PMA-induced superoxide production by monocytes. Thus, they do not inhibit the primary interaction of PMA with responding cells. Treatment of peripheral blood mononuclear cells with PMA increased cellular guanylate cyclase in most experiments, and dimethyl sulfoxide tended to inhibit this increase. In addition to inhibition of PMA-induced mitogenesis, hydroxyl radical scavengers markedly inhibited the activity of lymphocyte activating factor (interleukin 1). The differential inhibition of lymphocyte mitogenesis induced by different mitogens appears to be related to the differential macrophage requirements of the mitogens. The data suggest that hydroxyl radicals may be involved in mediating the triggering signal for lymphocyte activation. Some of the hydroxyl radical scavengers are inducers of cellular differentiation,. nd it is possible that their differentiating activity is related to their ability to scavenge free radicals.
Unique glycoprotein-proteoglycan complex defined by monoclonal antibody on human melanoma cells.
Authors: Bumol T F , Reisfeld R A
Published: May 2006

A monoclonal antibody, 9.2.27, with a high specificity for human melanoma cell surfaces has been utilized for biosynthetic studies in M21 human melanoma cells to define a unique antigenic complex consisting of a 250-kilodalton N-linked glycoprotein and a high molecular weight proteoglycan component larger than 400 kilodaltons. The 250-kilodalton glycoprotein has endoglycosidase H-sensitive precursors and shows a lower apparent molecular weight after treatment with neuraminidase. The biosynthesis of the proteoglycan component is inhibited by exposure of M21 cells to the monovalent ionophore monensin, this component can be labeled biosynthetically with 35SO4, is sensitive to beta-elimination in dilute base, and is degraded by both chondroitinase AC and ABC lyases, suggesting that it is a chondroitin sulfate proteoglycan. These data demonstrate that the antigenic determinant recognized by monoclonal antibody 9.2.27 is located on a glycoprotein-proteoglycan complex which may have unique implications for the interaction of glycoconjugates at the human melanoma tumor cell surface.
Replication and phenotypic expression of control and scleroderma human fibroblasts: responses to growth factors.
Authors: LeRoy E C , Mercurio S , Sherer G K
Published: May 2006

To explore the mechanism of increased collagen synthesis by scleroderma skin fibroblasts in vitro, control and scleroderma fibroblasts were compared in confluent monolayer cultures growth-arrested by serum deprivation; responses to optimal mitogenic doses of platelet-derived growth factor, fibroblast growth factor, epidermal growth factor and nerve growth factor were compared. Platelet-derived growth factor had a selective mitogenic effect on control skin fibroblasts not observed with scleroderma skin fibroblasts. None of the factors studied had a selective effect on collagen synthesis independent of cell replication; scleroderma and control fibroblasts responded similarly. Therefore, the growth factors studied may not be involved in generating the activated scleroderma fibroblast directly; platelet-derived growth factor may play an indirect role in fibroblast replication in human fibrotic disorders.U
Nicotinic acetylcholine receptor from chick optic lobe.
Authors: Norman R I , Mehraban F , Barnard E A , Dolly J O
Published: May 2006

An alpha-bungarotoxin-sensitive nicotinic cholinergic receptor from chick optic lobe has been completely purified. Its standard sedimentation coefficient is 9.1 S. The value near 12 S reported for the related component from other brain regions can be reproduced when the initial extraction is by Triton X-100 (rather than Lubrol PX), but other protein is then complexed with it. A single subunit of apparent molecular weight 54,000 is detected, and this subunit is specifically labeled by bromo-[3H]acetylcholine, but only after disulfide reduction. The same size subunit likewise is labeled in the protein (purified similarly) from the rest of the chick brain which can also bind alpha-bungarotoxin and nicotinic ligands. Immunological crossreactivity is demonstrated between both of these proteins with an antiserum to pure acetylcholine receptor from skeletal muscle. The acetylcholine receptor from chick optic lobe and the alpha-bungarotoxin-binding protein from the rest of the brain appear similar or identical by a series of criteria and are related to (but with differences from) peripheral acetylcholine receptors.
Tryptic digestion as a probe of myosin S-1 conformation.
Authors: Muhlrad A , Hozumi T
Published: May 2006

One of the products of the limited tryptic hydrolysis of chymotryptic myosin subfragment 1 is the 27,000-dalton NH2-terminal fragment. This fragment is generated by two parallel routes from either the 75,000- or 95,000-dalton peptide of the heavy chain: (i) through a 20,500-dalton precursor or (ii) directly without participation of a precursor. Lowering of pH and temperature and increasing of ionic strength inhibited route i digestion in comparison to route ii. MgATP and its derivatives in millimolar concentration substantially suppressed route i digestion. Suppression of route i digestion depended on the concentration of MgATP. It occurred after a lag phase when the ratio of MgATP to subfragment 1 concentrations was greater than 0.5. In contrast, the MgATP-induced increase in tryptophan fluorescence of myosin subfragment 1 appeared without a lag phase. The generation of the 27,000-dalton fragment by either route was not affected by F-actin however, the suppression of route i digestion induced by MgADP was abolished when myosin subfragment 1 was in ternary complex with actin and MgADP. We conclude that the 27,000/50,000-dalton hinge region is a flexible domain of the myosin head and that conformation of this region is sensitive to the presence of nucleotides and actin and to variations in ambient factors.
Acceleration of the adipocyte adenylate cyclase turn-off reaction by inhibitory hormonal factors.
Authors: Jakobs K H , Aktories K , Shultz G
Published: May 2006

beta-Adrenergic agonists such as isoproterenol stimulate hamster adipocyte adenylate cyclase by a GTP-dependent process, whereas prostaglandin E1, alpha-adrenergic agonists, and nicotinic acid inhibit the enzyme by a process also dependent on GTP and amplified by sodium ions. We have determined the first-order rate constant describing the decay of isoproterenol plus GTP-stimulated adenylate cyclase and the modulation of this off rate constant by sodium and inhibitory hormonal factors. With 1 microM GTP and 0.2 mM isoproterenol, the off rate constant was 5.0 min-1 at 25 degrees C. Addition of NaCl (100 mM), which increased basal and isoproterenol-stimulated cyclase activities, decreased the rate constant of the hormone-stimulated enzyme to 1.4 min-1. Prostaglandin E1 (10 microM) and nicotinic acid (30 microM), which decreased basal and hormonally stimulated enzyme activities, increased the NaCl-suppressed off rate constant to 6.1 and 6.0 min-1, respectively. Similar data were obtained with 1 mM isoproterenol with MnATP and MgATP as the adenylate cyclase substrate. On the other hand, the turn-on reaction of adipocyte adenylate cyclase, measured with the stable GTP analogue 5'-guanylyl imidodiphosphate (30 microM), was accelerated by isoproterenol (1 mM) and NaCl (100 mM). Under all of these conditions, inhibitory hormonal agents did not cause any delay in the turn-on reaction. These data indicate that, in hamster adipocyte membranes, inhibitory hormonal factors inhibit adenylate cyclase by increasing the enzyme's turn-off reaction.
Probable involvement of a glycoconjugate in IMR-32 DNA synthesis: decrease of DNA polymerase alpha 2 activity after tunicamycin treatment.
Authors: Bhattacharya P , Basu S
Published: May 2006

Multiple forms of DNA polymerase alpha activity (alpha 1, alpha 2, and alpha 3) from human neuroblastoma IMR-32 cells untreated or treated with tunicamycin (3 microgram/ml) were separated by DEAE-cellulose column chromatography. Loss of 40--60% of DNA polymerase alpha 2 activity was observed in tunicamycin-treated cells. Ricin 1B, a subunit of intact ricin (Mr, 64,000), was found to be a specific inhibitor of DNA polymerase alpha 2 isolated from control IMR-32 cells. However, DNA polymerase alpha 2 isolated from tunicamycin-treated cells was insensitive to ricin 1B. Heat treatment studies at 50 degrees C showed two completely different inactivation profiles for the DNA polymerase alpha 2 enzymes isolated from the tunicamycin-treated and untreated cells. A probable involvement of a beta-linked galactose-containing carbohydrate chain in the catalytic subunit of DNA polymerase alpha 2 is suggested.
Thyrotropin stimulation of the ADP-ribosyltransferase activity of bovine thyroid membranes.
Authors: Vitti P , De Wolf M J , Acquaviva A M , Epstein M , Kohn L D
Published: May 2006

Thyrotropin increases the ADP-ribosylation activity of bovine thyroid membranes. Rapid ADP-ribosylation of membrane components is followed by increasing ADP-ribosylation of components in the supernatant of the reaction mixture. One of the major membrane proteins ADP-ribosylated in the thyrotropin-stimulated reaction has an approximate molecular weight of 40,000; this same protein is also a major ADP-ribosylated product of the A promoter of cholera toxin and appears to be related to the G regulatory subunit of the adenylate cyclase complex. The ADP-ribosylated products appearing in the supernatant solution comigrate with thyrotropin and preparations of 125I-labeled alpha subunit of thyrotropin; the alpha subunit, but not the beta subunit, of thyrotropin can be ADP-ribosylated by the membrane ADP-ribosyltransferase activity. NAD can be shown to enhance the ability of thyrotropin to stimulate the adenylate cyclase activity of bovine thyroid membrane preparations and of membrane preparations of a rat thyroid tumor whose adenylate cyclase activity is otherwise unresponsive to thyrotropin. The beta subunit of thyrotropin inhibits thyrotropin stimulation of both the ADP-ribosylation and adenylate cyclase activities of the thyroid membrane.
Inhibition of kidney lysosomal phospholipases A and C by aminoglycoside antibiotics: possible mechanism of aminoglycoside toxicity.
Authors: Hostetler K Y , Hall L B
Published: May 2006

Nephrotoxicity is an important side effect of aminoglycoside antibiotics, which are used to control infections caused by Gram-negative bacteria. Accumulation of aminoglycosides and phospholipids in the lysosomes is a prominent and early feature of aminoglycoside nephrotoxicity and is characterized histologically by the presence of numerous multilamellar bodies in kidney proximal tubule cells. Previous studies have shown that the drug-induced phospholipid fatty liver in man and animals is due to concentration of certain cationic amphiphilic drugs in lysosomes with inhibition of lysosomal phospholipases. It seemed possible that this mechanism might also explain the elevated levels of phospholipid and increased numbers of multilamellar bodies reported in the kidney cortex in aminoglycoside nephrotoxicity. In this study, subcellular localization of acid phospholipases A and C has been shown to be lysosomal in rat kidney cortex. A soluble lysosomal protein fraction was isolated and found to contain both phospholipase A and phospholipase C activity. Streptomycin did not inhibit the release of fatty acids from [3H]dioleoylphosphatidylcholine. However, amikacin, dibekacin, gentamicin, and tobramycin inhibited both phospholipase A and phospholipase C. Our results suggest that the accumulation of phospholipids in lysosomes of kidney cortex, an early and pervasive feature of acute aminoglycoside nephrotoxicity, is due to inhibition of lysosomal phospholipases.
Compartmentalization of corticotropin-dependent steroidogenic factors in adrenal cortex: evidence for a post-translational cascade in stimulation of the cholesterol side-chain split.
Authors: Neher R , Milani A , Solano A R , Podestà E J
Published: May 2006

A sensitive cell-free assay was developed for the analysis of corticotropin-dependent factors that stimulate the rate-limiting step of adrenal steroidogenesis. In this assay adrenal post-mitochondrial supernates from corticotropin-stimulated rats caused a 10- to 100-fold increase in the de novo synthesis of pregnenolone and progesterone. A similar stimulation was observed by corresponding fractions from Leydig cells and mouse Y-1 adrenal tumor cells, but not from rat liver. Subcellular fractionation of rat adrenal tissue showed several steroidogenic factors to be present in various compartments. Recombination of them produced highly synergistic effects. The activation of some components could also be demonstrated in vitro, suggesting a cascade of events possibly linking the cAMP-dependent phosphorylation pathway with the rate-limiting step. Cycloheximide prevented the production of these steroidogenic factors in vivo upon stimulation but had no effect in vitro, suggesting a post-translational cascade involved in the activation of the cholesterol side-chain split.
Substrate binding affinity changes in mitochondrial energy-linked reactions.
Authors: Hatefi Y , Yagi T , Phelps D C , Wong S Y , Vik S B , Galante Y M
Published: May 2006

The effects of uncouplers and valinomycin plus nigericin (in the presence of K+) were studied on the apparent Km for substrates and apparent Vmax of the following energy-linked reactions catalyzed by submitochondrial particles: oxidative phosphorylation, NTP-33Pi exchange, ATP-driven electron transfer from succinate to NAD, and respiration-driven transhydrogenation from NADH to 3-acetylpyridine adenine dinucleotide phosphate. In all cases, partially uncoupling (up to 90%) concentrations of uncouplers of valinomycin plus nigericin were found to decrease apparent Vmax and to increase apparent Km. Results plotted as ln (Vmax/Km) versus the concentration of uncouplers or ionophores showed a linear decrease of the former as a function of increasing perturbant concentration (i.e., decreasing free energy). Because Vmax/Km may be considered as a measure of the apparent first-order rate constant for enzyme-substrate interaction and reflects the affinity between enzyme and substrate to form a complex, the results are consistent with the interpretation that membrane energization leads to a change in enzyme conformation with the resultant increase in enzyme-substrate affinity and facilitation of the reaction rate under consideration. The significance of these findings with respect to the mechanism of action of the energy-transducing systems studied is discussed.
Amplification of the uvrA gene product of Escherichia coli to 7% of cellular protein by linkage to the pL promoter of pKC30.
Authors: Yoakum G H , Yeung A T , Mattes W B , Grossman L
Published: May 2006

We have constructed a hybrid pKC30-uvrA plasmid (pGHY5003) in which transcription of the uvrA gene can be induced under pL control to amplify the uvrA gene product to 7% of cellular protein. To construct pGHY5003, we developed a genetic selection using the basal level of expression (30 degrees C) from pL in thermosensitive cI857 lysogens to isolate appropriately tailored repair genes inserted at the Hpa I site of pKC30 from recombinant DNA mixtures with a variety of products. In addition, a post-UV-irradiation radiolabeling method was adapted to screen inserts for temperature-inducible polypeptide synthesis directed by transcription under pL control rapidly. This should prove generally useful for isolating genes inserted at the Hpa I site of plasmid pKC30 with the following characteristics: (i) genetically functional hybrid plasmids selected from a large population of exonucleolytically tailored fragments ligated into Hpa I of pKC30 and (ii) production of high-level amplification for the gene product of interest by screening for post-UV-irradiation temperature inducibility of polypeptides synthesized from hybrid plasmids. The level of amplification obtained for the uvrA gene product from pGHY5003 is approximately 10,000-fold higher than estimates of the level of uvrA protein in logarithmic phase Escherichia coli.
Priming of DNA synthesis by diadenosine 5',5"'-P1,P4-tetraphosphate with a double-stranded octadecamer as a template and DNA polymerase alpha.
Authors: Zamecnik P C , Rapaport E , Baril E F
Published: May 2006

Diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) primes DNA synthesis in an in vitro system containing purified HeLa cell DNA polymerase alpha, deoxyadenosine triphosphate, and the double-stranded synthetic octadecamer template 5'-d-(G-G-A-G-G-C-T-T-T-T-T-T-G-G-A-G-G-C) (C-C-T-C-C-G-A-A-A-A-A-A-C-C-T-C-C-G)-d-5'; this octadecamer sequence is part of the origin region of DNA synthesis in simian virus 40. Ap4A is shown to be covalently linked to the first residue of the short deoxynucleotide chain synthesized under these experimental conditions. This template-primer system can initiate the new deoxynucleotide chain but cannot extend it beyond the A . T region.
Hemin-independent control of globin synthesis in Friend erythroleukemia cells induced to differentiate.
Authors: Stringer E A , Friend C
Published: May 2006

A hemin-independent translational inhibitor that prevents synthesis of rabbit globin when uninduced Friend leukemia (FL) cell and rabbit reticulocyte lysates are mixed [Cimadevilla, J. M. & Hardesty, B. (1975) Biochem. Biophys. Res. Commun. 63, 931-937] cannot be detected in FL cells induced to differentiate. Mixing of lysates of FL cells induced with hexamethylene bisacetamide or aminonucleoside of puromycin and rabbit reticulocytes does not cause inhibition of rabbit globin synthesis. Induction also results in the cells acquiring sensitivity to the inhibitor from uninduced FL cells. A reduction in total protein synthesis is observed when uninduced and induced FL cell lysates are mixed. Inhibition does not result from competition by an excess of uninduced FL cell mRNA species for the translational machinery because uninduced FL cell lysates retain their inhibitory activity after treatment with micrococcal nuclease. Rabbit globin mRNA recovered from rabbit reticulocyte lysates that have been incubated with lysates of uninduced FL cells can still be translated effectively, indicating that inhibition does not result from modification of other species of mRNA by uninduced FL cell lysates. A switch to hemin-dependent translational control does not follow induction of differentiation. The rate of amino acid incorporation in induced FL cell lysates--like that in uninduced FL cell lysates--is unaffected by omission of exogenous hemin from the system. Its presence is not required to prevent activation of heme-regulated inhibitor. From these data, it is clear that the control of protein synthesis in FL cells--whether or not they are induced--is different from that regulated by hemin in normal erythroid cells.
cDNA clones for the heavy chain of HLA-DR antigens obtained after immunopurification of polysomes by monoclonal antibody.
Authors: Korman A J , Knudsen P J , Kaufman J F , Strominger J L
Published: May 2006

A monoclonal antibody (HC 2.1) directed against the separated heavy chain of HLA-DR has been prepared. By binding HC 2.1 to polysomes from human B lymphoblastoid cells followed by the use of a protein A-Sepharose column as an immunoadsorbent, we have purified the mRNA coding for the HLA-DR heavy chain nearly to homogeneity. The immunopurified mRNA has been used to prepare labeled cDNA with which to probe cDNA libraries. Double-stranded cDNA was also made from the immunopurified mRNA and cloned directly into pBR322. Two clones, one from each of the above procedures, positively selected DR heavy chain message as assayed by cell-free translation and immunoprecipitation. One clone, pDRH-2 [500 base pairs plus 75 base pairs of poly(A)] contains the entire 3' untranslated region as well as coding information for the carboxy-terminal hydrophilic intracellular domain and part of the hydrophobic transmembrane region. Results of carboxypeptidase digestion of the heavy chains from detergent-solubilized (p34) and papain-treated (p33) HLA-DR antigen were consistent with the predicted protein sequence. Specific immunopurification of polysomes by defined monoclonal antibodies followed by direct cloning of cDNA to the highly purified mRNA is a powerful method for obtaining identified cDNA clones.
Probing the energetics of proteins through structural perturbation: sites of regulatory energy in human hemoglobin.
Authors: Pettigrew D W , Romeo P H , Tsapis A , Thillet J , Smith M L , Turner B W , Ackers G K
Published: May 2006

The sites of energy transduction within the human hemoglobin molecule for the regulation of oxygen affinity have been determined by an extensive study of the molecule's energetic response to structural alteration at individual amino acid residues. For 22 mutant and chemically modified hemoglobins we have determined the total free energy used by the tetrameric molecule for alteration of oxygen affinity at the four binding steps. The results imply that the regulation of oxygen binding affinity is due to energy changes which are mostly localized at the alpha 1 beta 2 interface. They also indicate a high degree of "internal cooperativity" within this contact region--i.e., the structural perturbations at individual residue sites are energetically coupled. Cooperativity in ligand binding is thus a reflection of cooperativity at a deeper level--that of the protein-protein interactions within the alpha 1 beta 2 interfacial domain.
Structure of the insulin-like growth factor receptor in chicken embryo fibroblasts.
Authors: Kasuga M , Van Obberghen E , Nissley S P , Rechler M M
Published: May 2006

The insulin-like growth factors (IGFs) and insulin stimulate DNA synthesis and cell multiplication in chicken embryo fibroblasts in culture. This response appears to be mediated by interaction with a single type of IGF receptor. The present study examines the subunit structure of this receptor by covalently crosslinking two 125I-labeled IGFs, IGF-I and multiplication-stimulating activity (MSA), to chicken embryo fibroblasts by using disuccinimidyl suberate. After solubilization, NaDodSO4/polyacrylamide gel electrophoresis, and autoradiography, IGF receptor complexes of appropriate specificity were identified; they had Mr approximately 130,000 (major band) and approximately 260,000 (minor band) under reducing conditions and Mr greater than 300,000 without disulfide reduction. The proportion of the Mr 260,000 component increased with increasing concentration of crosslinking agent, suggesting that it was formed from smaller proteins during the crosslinking procedure. The IGF receptor in chicken embryo fibroblasts resembles the insulin receptor in size and structure but can be distinguished by a higher affinity for IGF-I and MSA than for insulin. Although IGF receptors with different structure and specificity have been recognized in other tissues, the function of these binding sites is unknown. The present study demonstrates that the IGF receptor of chicken embryo fibroblasts that appears to mediate the growth-promoting effects of the IGFs contains a Mr approximately 130,000 binding subunit and exists as a native receptor complex of Mr greater than 300,000.
Relationship between the two components of the split promoter of eukaryotic tRNA genes.
Authors: Ciliberto G , Traboni C , Cortese R
Published: May 2006

Plasmids containing eukaryotic tRNA genes are faithfully transcribed in the nucleus of Xenopus laevis oocytes [Cortese, R., Melton, D. A., Tranquilla, T. & Smith, J. D. (1978) Nucleic Acids Res. 5, 4593-4611]. It has been established that two separated regions within the coding sequence of a tRNA gene are essential and sufficient for promotion of transcription [Hofstetter, H., Kressmann, A. & Birnstiel, M. L. (1981) Cell 24, 573-585; Ciliberto, G., Castagnoli, L., Melton, D. A. & Cortese, R. (1982) Proc. Natl. Acad. Sci. USA 79, 1195-1199]. We have constructed a hybrid tRNA gene containing one essential region from tDNALeu and the other from tDNAPro, both from Caenorhabditis elegans. This hybrid gene is efficiently transcribed, thus showing that the essential regions are independent transcriptional signals regardless of the overall regularities of the structure of tRNA genes. We have also constructed mutants of the tRNAPro gene in which the distance between the two essential regions is changed; optimal transcription occurs when this distance is about 40-50 nucleotides.
Carcinogenic epoxides of benzo[a]pyrene and cyclopenta[cd]pyrene induce base substitutions via specific transversions.
Authors: Eisenstadt E , Warren A J , Porter J , Atkins D , Miller J H
Published: May 2006

We have determined the spectrum of base-pair substitution mutations induced in the lacI gene of a uvrB- strain of Escherichia coli by two polycyclic aromatic hydrocarbons--(+/-)7 alpha,8 beta-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10 tetrahydrobenzo[a]pyrene (BPDE), and 3,4-epoxycylopenta[cd]pyrene (CPPE). Approximately 10% of all lacI mutations induced by either BPDE or CPPE are nonsense mutations, suggesting that base-pair substitutions are a large fraction of the mutational events induced by these agents in the uvrB- bacteria. Both carcinogens specifically induced the G . C leads to T . A and, to a lesser extent, the A . T leads to T . A transversions. One possible mechanism for transversion induction at G . C sites by BPDE might involve carcinogen binding to the exocyclic amino group of guanine in the template strand followed by a rotation of the modified base around its glycosylic bond from the anti to the syn conformation. This could allow specific pairing of modified bases with an imino tautomer of adenine.
Structure and expression of a mouse major histocompatibility antigen gene, H-2Ld.
Authors: Evans G A , Margulies D H , Camerini-Otero R D , Ozato K , Seidman J G
Published: May 2006

A genomic clone encoding H-2Ld, a mouse major transplantation antigen, has been identified and the structure of the H-2Ld gene has been partially determined. We isolated 35 genomic clones from a BALB/c (H-2d) genomic library by hybridization to mouse or human probes. One of these clones encodes H-2Ld as determined by two criteria. First, the gene encodes a protein that is identical at the 76 known amino acid positions for H-2Ld. Second, when introduced into L cells by DNA-mediated gene transfer, a new H-2 antigen is expressed that is recognized by anti-H-2Ld monoclonal antibodies. The sequence of the H-2Ld protein predicted by the DNA sequences shows more than 80% homology to known H-2 antigens. H-2L-like sequences are found in mutant H-2Kb molecules, suggesting that gene conversion or reciprocal recombination may play a role in the development of H-2 polymorphism.
Role of endothelial cells in relaxation of isolated arteries by bradykinin.
Authors: Cherry P D , Furchgott R F , Zawadzki J V , Jothianandan D
Published: May 2006

Bradykinin elicits relaxation of isolated transverse rings of canine coronary, celiac, superior mesenteric, renal, splenic, pulmonary, gastric, and femoral arteries. After endothelial cells of the vessel wall are removed by rubbing of the intimal surface, canine arteries fail to relax upon addition of bradykinin. The endothelium-dependent relaxation of canine arteries remains intact after treatment with cyclooxygenase inhibitors (indomethacin and flurbiprofen), and this argues against mediation by prostaglandins. When they are stimulated with bradykinin, endothelial cells of canine arteries appear to release a substance mediating vascular smooth muscle relaxation. In contrast, preparations of arteries of cats (superior mesenteric) and rabbits (superior mesenteric and celiac) may be rubbed on the intimal surface without a consistent loss of sensitivity to the relaxing effects of bradykinin. In addition, relaxation of the cat and rabbit arteries is completely blocked by cyclooxygenase inhibitors. Preliminary studies indicate that bradykinin relaxes human arteries in an endothelium-dependent manner and that this effect is not mediated by prostaglandins. We have previously reported that arteries of all species tested require the presence of endothelial cells for relaxation in response to acetylcholine and we have also demonstrated, using the rabbit aorta, that this effect is mediated by the release of an uncharacterized substance from these cells that relaxes vascular smooth muscle. We conclude that bradykinin relaxes canine and human arteries via a similar mechanism but that it relaxes cat and rabbit arteries by stimulating release of prostaglandins from as yet undefined cell types.
Use of monoclonal antibodies to subunits of human chorionic gonadotropin to examine the orientation of the hormone in its complex with receptor.
Authors: Moyle W R , Ehrlich P H , Canfield R E
Published: May 2006

Monoclonal antibodies were prepared against the alpha and beta subunits of human chorionic gonadotropin (hCG). Although all were selected on the basis of their ability to bind the intact hormone, each also bound one of the two subunits but not both. Using a solid phase double antibody system to measure the relative binding to sites on the surface of hCG, we observed that four of the five antibodies bound to different sites on the molecule. This information was correlated with the ability of each antibody to inhibit the biological activity of hCG. Of the five antibodies tested for their ability to inhibit hCG-induced stimulation of rat testes steroidogenesis in vitro, two proved to be potent inhibitors, whereas the other three had almost no effect. This inhibition of steroidogenesis was highly correlated with the ability of the antibodies to inhibit hCG binding to testes homogenates. Thus, we have begun to derive a scheme that describes the relative binding positions of individual monoclonal antibodies and receptor on hCG. The purified monoclonal antibodies were iodinated and employed to evaluate which antigenic sites on hCG remained free in hCG-receptor complexes. The data indicated that portions of the beta subunit in hCG-receptor complexes were buried (i.e., failed to bind radiolabeled antibody), whereas other portions remained exposed (i.e., they bound radiolabeled antibody). Those antibodies that interacted with portions of hCG that became inaccessible in the receptor complex also blocked the biological actions of hCG, whereas those that interacted with exposed sites had little or no effect on activity. Although we did not find antibodies to the alpha subunit that would bind to the hormone-receptor complex, we found that one of the two antibodies specific to alpha subunit epitopes blocked the actions of the hormone. Both antigenic determinants on the alpha subunit appeared to be lost after the hCG-receptor complex had formed. These studies suggest that each hCG subunit participates in the hormone-receptor complex and that portions of the beta subunit project from the surface of the receptor.
Mutant of Chinese hamster ovary cells with altered mannose 6-phosphate receptor activity is unable to synthesize mannosylphosphoryldolichol.
Authors: Stoll J , Robbins A R , Krag S S
Published: May 2006

B4-2-1 is a Chinese hamster ovary cell mutant previously isolated and characterized as deficient in mannose 6-phosphate receptor activity. We show here that B4-2-1 is a pleiotropic mutant, defective in biosynthesis of asparagine-linked oligosaccharides, B4-2-1 is unable to synthesize mannosylphosphoryldolichol; the consequences of this defect on glycosylation are (i) biosynthesis of one major lipid-linked oligosaccharide, characterized by its resistance to endoglycosidase H and decreased size; this oligosaccharide is similar to a minor species of lipid-liked oligosaccharide found in parental cells; (ii) transfer of this oligosaccharide to newly synthesized proteins; and (iii) absence of normal "high-mannose" oligosaccharides on mature glycoproteins isolated from B4-2-1; glycoproteins from the mutant contain complex oligosaccharides as well as endoglycosidase H-resistant, alpha-mannosidase-sensitive species. While the glycosylation defect may alter adversely the function of several glycoproteins in the mutant, including that of the mannose 6-phosphate receptor, it appears to have no effect on the formation or function of the mannose 6-phosphate recognition marker on acid hydrolases of B4-2-1.
Human lung small-cell carcinoma contains bombesin.
Authors: Erisman M D , Linnoila R I , Hernandez O , DiAugustine R P , Lazarus L H
Published: May 2006

The presence of immunoreactive bombesin in a human lung small-cell carcinoma grown in nude mice was established by several criteria: (i) Radioimmunoassay of tissue extracts for bombesin revealed approximately 6.5 pmol/g of tissue; (ii) bombesin was found in 12-14% of the tumor cells by immunohistochemical localization; (iii) gel filtration of small-cell carcinoma extract on Sephadex G-75 and Bio-Gel P-4 gave only a single peak of immunoreactivity, which occurred at the elution volume of bombesin; and (iv) reverse-phase HPLC of acid-solubilized extracts separated the immunoreactive material into three discrete peaks, one of which eluted with a retention time identical to that of synthetic bombesin. The presence of bombesin may represent the ectopic expression of this peptide in small-cell carcinoma, because immunoreactive bombesin was found in human fetal and neonatal lung but apparently not in adult lung tissue [Wharton, J., Polak, J. M., Bloom, S. R., Ghatei, M. A., Solcia, E., Brown, M. R. & Pearse, A. G. E. (1978) Nature (London) 273, 769-770]. The immunoreactive bombesin previously found in mammalian tissues is considerably larger than amphibian bombesin; these data substantiate the presence of a mammalian form of bombesin in a human tumor that may have a structure similar to that of the amphibian peptide.
Transmission by presynaptic spike-like depolarization in the squid giant synapse.
Authors: Llinás R , Sugimori M , Simon S M
Published: May 2006

Synaptic transmission produced by artificially generated presynaptic depolarizations, having the same waveform as normal presynaptic action potentials, was studied in the squid giant synapse. These stimulated spikes produced synaptic transmission virtually indistinguishable from that obtained with the original presynaptic spike activation. The technique allows a direct determination of the onset and amplitude of the Ca current, ICa, triggered by the presynaptic action potential and the relationship between ICa and postsynaptic response. The results closely resemble theoretical predictions by a previously published model for synaptic transmission in this synapse and demonstrate that ICa occurs at the falling phase of the presynaptic spike.
A salt bridge stabilizes the helix formed by isolated C-peptide of RNase A.
Authors: Bierzynski A , Kim P S , Baldwin R L
Published: May 2006

C-peptide, which contains the 13 NH2-terminal residues of RNase A, shows partial helix formation in water at low temperature (1 degree C, pH 5, 0.1 M NaCl), as judged by CD spectra; the helix is formed intramolecularly [Brown, J. E. & Klee, W. A. (1971) Biochemistry 10, 470-476]. We find that helix stability depends strongly on pH: both a protonated histidine (residue 12) and a deprotonated glutamate (residue 9 or 2 or both) are required for optimal stability. This information, together with model building, suggests that the salt bridge Glu-9- ... His-12+ stabilizes the helix. Formation of the helix is enthalpy driven [van't Hoff delta H, - 16Kcal/mol (1 cal = 4.18 J)] and the helix is not observed above 30 degrees C. Proton NMR data indicate that several side chains adopt specific conformations as the helix is formed. These results have two implications for the mechanism of protein folding. First, they indicate that short alpha-helices, stabilized by specific side-chain interactions within the helix, can be stable enough in water to function as folding intermediates. Second, they suggest that similar experiments with peptides of controlled amino acid sequence could be used to catalogue the intrahelix interactions that stabilize or destabilize alpha-helices in aqueous solution. These data might provide the code relating amino acid sequence to the locations of alpha-helices in proteins.
Serum enhances the cycling and survival of HeLa cells treated with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole.
Authors: Tamm I , Kikuchi T , Murphy J S
Published: May 2006

We have defined the growth kinetics of HeLa cell populations by determining the frequencies of mitoses and deaths and the lengths of intermitotic intervals. This was done by time-lapse cinemicrography. Untreated control cells proliferated at closely similar rates in medium enriched with 5% or 15% fetal calf serum, with an average of 4% dividing and less than 0.1% dying per hr. The mean intermitotic interval was 16 hr during exponential growth of the control populations. In contrast, in cultures treated with 40 or 60 microM 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), a selective inhibitor of heterogeneous nuclear RNA synthesis, the frequency of mitoses was markedly and directly dependent on serum concentration, whereas the frequency of deaths was inversely dependent. DRB prolonged the intermitotic interval in cells cycling in the presence of the drug, but the effect was less in 15% than in 5% serum. After prolonged treatment of HeLa cells with DRB, the inhibition of heterogeneous nuclear RNA synthesis by DRB appeared to be reduced, which was not due to inactivation of DRB in the culture medium.
Genetic heterogeneity in ataxia-telangiectasia studied by cell fusion.
Authors: Jaspers N G , Bootsma D
Published: May 2006

The effect of x-rays on the rate of semiconservative DNA replication was investigated by autoradiography in single cells obtained from normal individuals and from patients having ataxia-telangiectasia (AT). In the five AT cell strains studied, the rate of DNA synthesis was inhibited to a lesser extent that in two normal cell strains. By using this abnormal regulation of DNA replication in AT cells as a marker, an experimental procedure was developed that allowed genetic complementation analysis of AT. After Sendai virus-induced fusion of AT cells, the grains were counted over binucleate cells with both nuclei in S phase. In some cases, the inhibition of DNA synthesis caused by x-rays in the heterodikaryons was more pronounced than that in the parental homodikaryons and was comparable to that in normal binucleate cells, indicating complementation. By using this approach, the five AT cell strains that were investigated could be assigned to three complementation groups. The data suggest that extensive genetic heterogeneity exists in AT.
Absence of pork-like insulin in guinea pig tissues.
Authors: Eng J , Yalow R S
Published: May 2006

By using a technique for concentrating insulin 100-fold from tissue extracts with 75-95% recoveries, we earlier failed to detect pork-like insulin in guinea pig tissues and thus were unable to confirm reports from the National Institutes of Health that these tissues contain a pork-like insulin at concentrations averaging 1 ng/g. This difference could have been due to differences in strains of guinea pigs studied or in the species specificities of the antisera used for radioimmunoassay. In the current study, tissue extracts from both NIH and Hartley guinea pigs were assayed with three antisera routinely used in our laboratory and one antiserum that had been used in the National Institutes of Health laboratory. We observed that pork-like insulin in tissues from both strains of guinea pigs as determined with the four antisera is less than 0.02 ng/g. We therefore conclude that is is unlikely that nonpancreatic guinea pig tissues contain or synthesize a peptide resembling pork or other non-guinea pig mammalian insulin.