bluuebunny/crossref_metadata_2025 huggingface dataset:
Relationship between structure of the 5' noncoding region of viral mRNA and efficiency in the initiation step of protein synthesis in a eukaryotic system.
Authors: Yamaguchi K , Hidaka S , Miura K
Published: May 2006

To determine whether the rate of protein synthesis is controlled by the structure of mRNA near its 5' terminus, protein-synthesizing ability, especially in its initial stage, was compared among RNAs of plant viruses. Those viruses used here contain several definite pieces of single-stranded RNA. Each of these RNAs acts as a messenger. Cucumber mosaic virus (CMV) RNA 5 synthesizes a small amount of a protein, Mr 7000, in an in vitro protein-synthesizing system from wheat germ or reticulocyte. Brome mosaic virus (BMV) RNA 4 synthesizes a large amount of a coat protein under the same conditions. Both RNAs have the same 5'-cap structure and a short noncoding region (10 nucleotides in CMV RNA 5 and 9 in BMV RNA 4) between the 5' terminus and the initiation codon AUG. A sequence complementary to the 3' terminal of 18S ribosomal RNA is contained in BMV RNA 4 but is not apparent in CMV RNA 5. Formation of the initiation complex for protein synthesis by the 5'-terminal-labeled mRNA of cytoplasmic polyhedrosis virus was inhibited by the addition of unlabeled BMV RNA 4 whereas it was only slightly inhibited by unlabeled CMV RNA 5. BMV RNA 4, which has a sequence complementary to rRNA, can form the initiation complex more easily than CMV RNA5. It is concluded that an apparent complementary sequence in the 3' terminal of 18S rRNA in the 5' noncoding region of eukaryotic mRNA and the 5'-cap structure enhance the rate of initiation complex formation in protein synthesis.
Complete nucleotide sequence of a soybean actin gene
Authors: Shah Dilip M. , Hightower Robin C. , Meagher Richard B.
Published: May 2006

Soybean contains a small multigene family of actin-related sequences. We have determined the complete nucleotide sequence of a soybean actin gene carried on the recombinant plasmid pSAc3. As deduced from the nucleotide sequence, this soybean actin is composed of 376 amino acids. Compared to other eukaryotic actins, pSAc3 actin has a deletion of one amino acid between residues 118 and 122. The initiator methionine is followed by alanine, which is not found at this position in other eukaryotic actins. pSAc3 actin differs, in primary sequence, more from fungal and animal actins than any of the known nonplant actins differ from each other. pSAc3 actin appears to be related to both cytoplasmic and muscle specific actins in the location of specific NH
2
-terminal amino acids. The coding sequence is interrupted by three small introns, each less than 90 base pairs long. The splice junctions are similar to those found in other eukaryotic genes, suggesting the presence of a similar splicing apparatus in higher plants. Introns 1 and 3 interrupt the reading frame after codons 20 and 355, respectively. Intron 2 splits a glycine codon at position 151. None of these intron positions is conserved relative to the positions of introns in other actin genes examined.
In vitro formation of multimeric DNA structures mediated by purified simian virus 40 chromatin.
Authors: Waldeck W , Zentgraf H , Sauer G
Published: May 2006

Simian virus 40 chromatin was incubated after purification by sucrose density-gradient centrifugation with various circular double-stranded DNA substrates. Monomeric rings were converted in the presence of Mg2+ to structures possessing a higher degree of complexity. Dimeric catenanes, as well as multimeric linear structures and concatemers, were generated, indicating that recombination events had occurred in vitro involving covalent linkage between different DNA molecules. Furthermore, apparently fused dimeric rings were observed. Their structures suggest that they may be recombination intermediates such as those described in a prokaryotic system [Potter, H. & Dressler, D. (1976) Proc. Natl. Acad. Sci. USA 73, 3000-3004]. Recombination did not take place between heterologous DNA substrates, as exclusively homologous multimeric DNA structures were observed.
Beta-endorphin: replacement of tyrosine in position 27 by tryptophan increases analgesic potency--preparation and properties of the 2-nitrophenylsulfenyl derivative.
Authors: Li C H , Yamashiro D , Nicolas P
Published: May 2006

An analog of human beta-endorphin with tryptophan in position 27 has been synthetized by the solid-phase method. Bioassay of the analog showed that it had almost 4 times the analgesic potency of the parent hormone but only 68% of the opiate receptor-binding activity. The peptide is the most potent analgesic among the known synthetic analogs of beta-endorphin. The 2-nitrophenylsulfenyl derivative of the analog has been prepared and shown to have a lower analgesic potency and a higher opiate receptor-binding activity than the parent compound.
Monoclonal antibodies against polyoma virus tumor antigens.
Authors: Dilworth S M , Griffin B E
Published: May 2006

A method is described for the production of hybridoma cell lines secreting antibodies directed against the polyoma virus tumor antigens (T-Ags). Ten cloned lines have been established by this procedure and designated alpha Py Cl and C4, with activities against all three T-Ags; alpha Py LT1, -4 and -7, with activities against large T-Ag only; and alpha Py MT5, -7, -10, -13, and -16, with activities against middle T-Ag only. Some of the antibodies appear to react also with specific cellular protein. Data from studies on alpha Py MT7 point to the possible existence of more than one antigenic form of the viral middle T-Ag and, similarly, from studies on alpha Py LT7, to more than one form of large T-Ag. The approximate positions on the polyoma virus genome of the sequences encoding antigenic determinants have been mapped by using defined polyoma virus deletion mutants. The availability of these monoclonal antibodies allows the isolation of large amounts of viral T-Ags.
Relationship between the genetically determined acetylator phenotype and DNA damage induced by hydralazine and 2-aminofluorene in cultured rabbit hepatocytes.
Authors: McQueen C A , Maslansky C J , Glowinski I B , Crescenzi S B , Weber W W , Williams G M
Published: May 2006

The relationship between acetylation rates of rabbit hepatocytes and their susceptibility to genotoxicity by DNA-damaging chemicals that undergo N-acetylation was studied in primary cultures of hepatocytes from New Zealand White rabbits that have a genetically determined difference in acetylation rates. Hepatocytes from rapid and slow acetylator rabbits maintained in culture the difference in acetylation rates that existed in vivo, DNA repair, an index of DNA damage, was produced by hydralazine in hepatocytes from slow acetylator rabbits but not in those from rapid acetylators. In contrast to these results, hepatocytes from rapid acetylators were more sensitive than those from slow acetylators to toxicity from the carcinogen 2-aminofluorene and displayed greater amounts of DNA repair. The amount of DNA repair measured with either chemical was dose dependent. These phenotype-dependent differences in the genotoxicity of two DNA-damaging chemicals provide evidence for the role of the acetylation polymorphism as a factor in determining susceptibility to toxicity, and perhaps carcinogenicity, of these chemicals.
Immunohistochemical localization of guanylate cyclase within neurons of rat brain.
Authors: Ariano M A , Lewicki J A , Brandwein H J , Murad F
Published: May 2006

The immunohistochemical localization of guanylate cyclase [GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2] has been examined in rat neocortex, caudate-putamen, and cerebellum by using specific monoclonal antibodies. Immunofluorescence could be seen within somata and proximal dendrites of neurons in the these regions. A nuclear immunofluorescence reaction to guanylate cyclase was characteristically absent. The staining pattern for guanylate cyclase was coincident with previously described localizations of cyclic GMP immunofluorescence within medium spiny neurons of the caudate-putamen and pyramidal cells of the neocortex. Cerebellar guanylate cyclase immunoreactivity was primarily confined to Purkinje cells and their primary dendrites, similar to the pattern reported for cyclic GMP-dependent protein kinase localization. Guanylate cyclase immunofluorescence was abolished when the monoclonal antibodies were exposed to purified enzyme prior to incubation of the tissue slices or when control antibody was substituted for the primary antibody. Immunohistochemical localization of cyclic AMP in these same tissues was readily distinguished from that of guanylate cyclase or cyclic GMP, showing uniform fluorescence throughout the cell bodies of neurons and glial elements.
Isopeptidase: a novel eukaryotic enzyme that cleaves isopeptide bonds.
Authors: Matsui S , Sandberg A A , Negoro S , Seon B K , Goldstein G
Published: May 2006

In an attempt to clarify the regulatory mechanism that accounts for the shift of protein A24 in the mitotic cycle, we demonstrated the existence of an enzyme, provisionally termed isopeptidase, that cleaves A24 stoichiometrically into histone H2A and ubiquitin. Properties of this enzyme are (i) most eukaryotes, including mammals, amphibia, chicken, and yeast, contain isopeptidase in the cytoplasm; (ii) a significant increase in enzyme binding to chromatin occurs when cells enter mitosis; (iii) Escherichia coli does not contain isopeptidase; (iv) isopeptidase has a molecular weight of 38,000; (v) at an ionic strength that induces globular conformation of H2A, isopeptidase activity is repressed; (vi) a SH group is an essential cofactor; and (vii) most divalent cations (except Mg2+ and Ca2+) are inhibitory. In view of the stoichiometric conversion of A24 into H2A and ubiquitin by isopeptidase in vitro, A24 probably contains a Gly-Gly dipeptide in isopeptide linkage but no other intervening polypeptides. Since ubiquitin in various eukaryotes binds to protein other than H2A, and is proteolytically released, isopeptidase probably acts on isopeptide bonds in general and not uniquely on those of A24. Inasmuch as isopeptidase is present throughout the cell cycle, the level of A24 in chromatin appears to be controlled by a balance between isopeptidase and an as yet unestablished H2A-ubiquitin ligase.
Regulation of the colony-stimulating activity produced by a murine marrow-derived cell line (H-1).
Authors: Garnett H M , Cronkite E P , Harigaya K
Published: May 2006

The production of molecular species that stimulate growth of granulocyte or macrophage colonies (GM-CSF) by the fibroblastoid H-1 cell line is unaffected by either native or iron-saturated lactoferrin, although some inhibition is detected with 10 microM prostaglandin E1. The H-1 GM-CSF is able to support the formation of macrophage, neutrophil, and mixed colonies. Feeder layers of H-1 cells are also able to support the development of colony-forming units stimulated by GM-CSF (GM-CFUc) although the number of colonies produced when the optimal H-1 cell concentration is plated (2.5 x 10(3) cells) is only 30% of the number with conditioned medium alone. This inhibitory effect is observed irrespective of the presence of an additional agar layer between the feeder cells and plated bone marrow cells, implying that diffusable substances are involved. Addition of indomethacin (10 microM) to feeder layers derived from 2.5 x 10(3) H-1 cells increases the number of GM-CFUc detected to 50% of that seen with conditioned medium alone. This result suggests that released prostaglandin may be responsible for some, but not all, of the observed inhibition of colony formation. In the presence of the H-1 feeder layers, only macrophage colonies are detected and hence it appears that the H-1 cells produce, in addition to prostaglandin, a diffusible inhibitory substance that preferentially inhibits granulopoiesis.
Isolation and characterization of a monoclonal antibody against the saxitoxin-binding component from the electric organ of the eel Electrophorus electricus.
Authors: Moore H P , Fritz L C , Raftery M A , Brockes J P
Published: May 2006

A monoclonal hybridoma cell line secreting antibody against the saxitoxin-binding component from the eel Electrophorus electricus has been isolated. The specificity of this monoclonal antibody was established by (i) its ability to immunoprecipitate bound [3H]saxitoxin from a detergent extract of electroplax membranes in a dose-dependent manner, (ii) the inability of unrelated monoclonal antibodies to immunoprecipitate the toxin-binding activity in a similar assay, and (iii) the ability of excess unlabeled tetrodotoxin to displace [3H]saxitoxin from the immunoprecipitated component. The antibody is of the subclass IgG1 and binds specifically to a polypeptide component of Mr approximately 250,000 on NaDodSO4/polyacrylamide gels. The antigenic determinant is associated with the same polypeptide component throughout the purification procedure, indicating that this component is not a result of artifactual aggregation or degradation during isolation. We conclude that the 250,000-dalton polypeptide is part of the saxitoxin binding/sodium channel protein in the native electroplax membrane.
Animal cells dependent on exogenous phosphatidylcholine for membrane biogenesis.
Authors: Esko J D , Nishijima M , Raetz C R
Published: May 2006

A Chinese hamster ovary cell (CHO) mutant (strain 58), defective in CDP-choline synthetase (cholinephosphate cytidylyltransferase; CTP:cholinephosphate cytidylyltransferase, EC 2.7.7.15), is temperature sensitive for growth and contains less than half of the normal amount of phosphatidylcholine under nonpermissive conditions [Esko, J. D. & Raetz, C. R. H. (1980) Proc. Natl. Acad. Sci. USA 77, 5192-5196]. We now report that the addition of 40 microM egg phosphatidylcholine or lysophosphatidylcholine to the medium suppresses the temperature sensitivity of mutant 58 and permits the growth of colonies at the restrictive temperature. Phospholipids with different polar headgroups, lipoprotein-bound phospholipids, sphingomyelin, and glycerophosphocholine do not support prolonged growth at 40 degrees C, whereas phosphatidylcholine analogs such as phosphatidyldimethylethanolamine, D-phosphatidylcholine, and beta-phosphatidylcholine are quite effective. A broad range of saturated phosphatidylcholines, especially those with fatty acids 12-18 carbons in length, suppresses the phenotype. Phospholipids containing ether-linked hydrocarbons are ineffective, whereas polyunsaturated phosphatidylcholines are toxic. Residual endogenous synthesis of phosphatidylcholine by the mutant is not stimulated under conditions of phenotypic bypass, but the uptake of exogenous lipid is enhanced considerably compared to the wild type. Our findings demonstrate that exogenous phospholipid can provide at least 50% of the phosphatidylcholine required for membrane biogenesis in animal cells and that uptake of exogenous phospholipids may be regulated.
The fate of 8-methoxypsoralen photoinduced crosslinks in nuclear and mitochondrial yeast DNA: comparison of wild-type and repair-deficient strains.
Authors: Magaña-Schwencke N , Henriques J A , Chanet R , Moustacchi E
Published: May 2006

In Saccharomyces cerevisiae, after 8-methoxypsoralen [8-(OMe)Ps] photoaddition, more crosslinks are induced per unit dose in mitochondrial DNA than in nuclear DNA. In wild-type cells treated in the exponential phase of growth, single- and double-strand breaks are produced during crosslink removal and then are rejoined upon postexposure incubation. The incision step is almost blocked in the rad 3-2 mutant, which is also defective in excision-repair of UV-induced (254 nm) pyrimidine dimers. The cutting of crosslinks from nuclear DNA is depressed in wild-type stationary-phase cells. This is correlated with a higher sensitivity of such cells to 8-(OMe)Ps photoinduced cell killing. The incision of crosslinks is dramatically reduced in mitochondrial DNA. The rejoining of single- and double-strand breaks is not only dependent on the product of the RAD51 gene (as shown by others) but also of the PSO2 gene. A correlation was found between the ability to recombine and strand rejoining. Therefore, as in bacteria, both the excision and the recombinational repair systems are involved in crosslink repair in yeast. However, double-strand breaks in yeast constitute repair intermediates which are not detected in Escherichia coli. The LD37 (dose necessary to induce a mean of one lethal hit per cell) corresponds to about 120 crosslinks per genome in exponential-phase cells of the wild type and to 1-2 crosslinks in the pso2-1 mutant.
[half-Cys4,half-Cys10]-alpha-Melanocyte-stimulating hormone: a cyclic alpha-melanotropin exhibiting superagonist biological activity.
Authors: Sawyer T K , Hruby V J , Darman P S , Hadley M E
Published: May 2006

alpha-Melanocyte-stimulating hormone (alpha-melanotropin; alpha-MSH) is a linear tridecapeptide (Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2) that reversibly darkens amphibian skins by stimulating melanomsome (pigment granule) dispersion within melanophores. By using a number of in vitro melanocyte assays, we have examined the conformational requirements for alpha-MSH activity. Synthesis of [half-Cys4,half-Cys10]-alpha-MSH, a cyclic, conformationally restricted, "isosteric" analogue of alpha-MSH, provided a melanotropin with a potency greater than 10,000 times that of the native hormone in stimulating frog (Rana pipiens) skin darkening. The cyclic analogue also showed substantially prolonged activity relative to the native hormone. [half-Cys4,half-Cys10]-alpha-MSH was approximately 30 times more potent than alpha-MSH in stimulating lizard (Anolis carolinensis) skin melanophores in vitro. By using a cell-free Cloudman S-91 mouse melanoma plasma membrane preparation, we found the cyclic analogue to be approximately 3 times as potent as the native hormone in stimulating adenylate cyclase activity. These results provide insight into the conformational requirements for biological activity of alpha-MSH, and the comparative conformational requirements of alpha-MSH at a number of pigment cell receptors.
Effect of the B--Z transition in poly(dG-m5dC) . poly(dG-m5dC) on nucleosome formation.
Authors: Nickol J , Behe M , Felsenfeld G
Published: May 2006

We have studied the properties of complexes formed between histones and the methylated synthetic polydeoxynucleotide poly(dG-m5dC). poly(dG-m5dC). This polymer undergoes the transition from B DNA to left-handed Z DNA at moderate ionic strength. When the polymer is in the Z form it will bind histones, but nucleosomes are not detected. When the polymer in the B form is combined with equimolar quantities of the four core histones and digested with micrococcal nuclease, particles are formed which behave in all respects as normal nucleosome cores. When these core particles are placed in solvents that would result in conversion of the protein-free polymer to the Z form, no transition is observed. The formation of a nucleosome core particle thus stabilizes the B form, whereas the presence of the Z form prevents nucleosome formation. The results suggest that if Z DNA is present in eukaryotic nuclei, it will serve to disrupt the normal chromatin structure.
Tumor promoters increase the synthesis of a 32,000-dalton protein in BALB/c 3T3 cells.
Authors: Hiwasa T , Fujimura S , Sakiyama S
Published: May 2006

The effect of 12-O-tetradecanoylphorbol 13-acetate (TPA), a potent tumor promoter, on the synthesis of proteins in BALB/c 3T3 mouse fibroblasts has been studied. When [35S]methionine-labeled proteins synthesized after the addition of TPA were analyzed by one- or two-dimensional polyacrylamide gel electrophoresis, the increased synthesis of a 32,000-dalton protein (designated p32) was noted as one of the earliest changes. The synthesis of p32 increased approximately 2-fold within 2 hr at a promoter concentration of 20 ng/ml. Phorbol 12,13-didecanoate, another potent tumor promoter, showed the same effect as TPA, whereas 4-O-methyl-TPA, which has little promoting activity, did not enhance the synthesis of p32 at the same concentration but did effect a slight increase at 1 microgram/ml. p32 differed immunologically from a major excreted protein discovered by Gottesman [Gottesman, M. M. (1978) Proc. Natl. Acad. Sci. USA 75, 2767-2771]. The increased rate of the synthesis of p32 was considered to be regulated at the transcriptional level, because the increase in synthesis of p32 was inhibited by actinomycin D, and TPA-treated cells were shown to contain a higher level of translatable mRNA coding for p32 than do control cells. A possible relationship between the increase in p32 synthesis and transformation is discussed.
Amplification of expression of hepatitis B surface antigen in 3T3 cells cotransfected with a dominant-acting gene and cloned viral DNA.
Authors: Christman J K , Gerber M , Price P M , Flordellis C , Edelman J , Acs G
Published: May 2006

3T3 cells containing hepatitis B virus DNA sequences can be efficiently selected by exposure to methotrexate after cotransfection with cloned viral DNA and DNA coding for a methotrexate-resistant dihydrofolate reductase. More than 75% of methotrexate-resistant cells isolated after cotransfection with a head-to-tail tandem of the hepatitis B virus genome synthesized viral surface antigen. The antigen was released into the culture medium in the form of 22-nm particles with buoyant density of 1.20 g/ml. No other virally coded proteins were detected in the cells or the culture medium. Application of selective pressure by increasing the concentration of methotrexate resulted in an amplification of viral DNA sequences and a concomitant increase in the rate of synthesis and release of hepatitis B surface antigen. The ability to produce large amounts of surface antigen appears to be a stable trait and has been maintained in these cultures through more than 30 passages.
Clonal anergy: the universally anergic B lymphocyte.
Authors: Pike B L , Boyd A W , Nossal G J
Published: May 2006

The clonal anergy theory of induction of immunological tolerance states that differentiating B lymphocytes that encounter multivalent antigen at the pre-B to B cell transition stage can receive and store a negative signal, which renders them anergic to later triggering stimuli. The theory was tested by using an anti-mu chain monoclonal antibody, E4, as a model tolerogen. The fluorescence-activated cell sorter was used to select B cell-free cell populations from adult murine bone marrow or newborn spleen, and later, to analyze B cell neogenesis in vitro. The presence of E4 at greater than or equal to 1 microgram/ml was required to impede the development of normal numbers of B cells with full receptor status. The subsequent capacity of these B cells to respond in vitro to mitogens was assessed in a filter-cell free microculture system that allows single B cells to proliferate and differentiate. Concentrations of E4 far below those required to affect B cell neogenesis had profound inhibitory effects on the subsequent functional capacity of the B cells. In fact, 10(-3) micrograms/ml of E4 markedly impaired both proliferation and antibody formation, and 10(-1) micrograms/ml, which had no effect on Ig receptor development, abrogated functional capacity. Thus B cells formed in the presence of E4 at 10(-1) micrograms/ml, though possessing the receptor status typical of B cells, were functionally entirely anergic. Exposure to E4 appeared to accelerate the spontaneous death rate of newly formed B cells in vitro. Whether the anergic cell would also have a shortened life span in vivo is not known.
Selective networks capable of representative transformations, limited generalizations, and associative memory.
Authors: Edelman G M , Reeke G N
Published: May 2006

Two parallel sets of selective networks composed of intercommunicating neuron-like elements have been connected to produce a new kind of automaton capable of limited recognition of two-dimensional patterns. Salient features of this automaton are (i) preestablished unchanging connectivity, (ii) preassigned connection strengths that are selectively altered according to experience, (iii) local feature detection in one network with simultaneous global feature correlation in the other, and (iv) reentrant interactions between the two networks to generate a new function, associative memory. No forced learning, explicit semantic rules, or a priori instructions are used.
Model for primary charge separation in reaction centers of photosynthetic bacteria
Authors: Friesner Richard , Wertheimer Reiner
Published: May 2006

We present model calculations of the dynamics of primary electron transfer (ET) in reaction centers of photosynthetic bacteria. We obtain half times of [unk]1 ps and ≈5 ps for the first two ET processes, in excellent agreement with experimental observations. Our model is based on (
i
) a theoretical framework capable of describing ET in the presence of strong electronic interstate resonance coupling and (
ii
) energy parameters extracted from recent experimental data and molecular orbital calculations. Our analysis suggests that (
i
) strong electronic interstate mixing is crucial to the rapidity and efficiency of irreversible ET; (
ii
) possibly five rather than three electronic states participate in the transient ET prior to the reduction
in vivo
of the quinone complex; and (
iii
) conventional ET theories, which rely on weak electronic interstate mixing, are unfit for describing ET in reaction centers of photosynthetic bacteria.
Phosphorus-containing inhibitors of angiotensin-converting enzyme.
Authors: Thorsett E D , Harris E E , Peterson E R , Greenlee W J , Patchett A A , Ulm E H , Vassil T C
Published: May 2006

Several phosphonamides, phosphoramides, and phosphates having the general structure R-Y-P(O)(OH)-X-CH(CH3)-CO-Pro have been synthesized and tested for inhibition of angiotensin-converting enzyme (dipeptidyl carboxypeptidase; peptidyl-dipeptide hydrolase, EC 3.4.15.1). Inhibition was found to depend on the nature of R, Y, and X such that the maximal effect was observed when X = NH, Y = CH2, and R = phi CH2 (50% inhibition at 7 nM). Substitution of CH2 or O at X and O at Y produced significantly less potent inhibitors. Groups shorter or longer than R = phi CH2 led to less active inhibitors, presumably due to nonoptimal interaction of the side chain with the S1 subsite.
Solubilization and purification of the alpha 1-adrenergic receptor using a novel affinity resin.
Authors: Graham R M , Hess H J , Homcy C J
Published: May 2006

The highly selective alpha 1-adrenergic receptor antagonist prazosin was used to identify binding sites having alpha-adrenergic specificity in rat hepatic plasma membranes. Solubilization of the membrane-bound receptors was achieved by incubation with the nonionic detergent digitonin, and binding activity was assayed by using [3H]prazosin and a polyethylene glycol precipitation technique. Only 20-30% of the total receptor pool was released by the solubilization procedure. However, binding of [3H]prazosin was saturable [maximal value, 206 +/- 8 fmol/mg of protein (membrane) vs. 74 +/- 4 fmol/mg of protein (soluble)] and of high affinity [Kd, 0.6 +/- 0.2 nM (membrane) vs. 0.8 +/- 0.2 nM (soluble)]. To aid in purification of the receptors, an affinity resin was developed using an analog of prazosin, 2-(4-succinoylpiperazin-1-yl)-4-amino-6,7-dimethoxyquinazoline (CP 57,609; Kd 2.7 X 10(-7) M) immobilized via an amide linkage to agarose. The resulting resin demonstrated high affinity (Kd 3.2 X 10(-7) M) for the solubilized receptors, as determined by competitive inhibition assay. The degree of substitution to the resin was determined by a direct radioimmunoassay using antibodies against albumin-complexed CP 57,609 and found to be 0.1 to 0.2 mumol/ml of agarose. Affinity chromatography using the resin resulted in 513-fold purification in a single step. Moreover, the specificity of the purified binding sites was similar to that of membrane-bound receptors. This novel affinity resin should thus provide a powerful tool for isolating the receptor protein in quantities sufficient for detailed biochemical characterization.
Beta-endorphin: opiate receptor binding activities of six naturally occurring beta-endorphin homologs studied by using tritiated human hormone and naloxone as primary ligands--effects of sodium ion.
Authors: Nicolas P , Hammonds R G , Li C H
Published: May 2006

Six naturally occurring homologs of beta-endorphin were tested for their potency in inhibiting the binding of [3H]naloxone and [3H]-beta h-endorphin either in the presence or in the absence of Na+. Sodium ion reduces inhibitory potency of these homologs against [3H]-beta h-endorphin and [3H]naloxone to about the same extent. The ratio of the IC50 obtained with Na+ to that obtained without Na+ is not highly correlated with analgesic potency. Very high (greater than 300) sodium ratios are observed with some homologs. A significant rank correlation was found between the ratio of the relative potency in displacing [3H]beta h-endorphin to the relative potency in analgesia and potency in inhibiting the binding of the pure antagonist naloxone.
Partial resialylation of human asialotransferrin type 3 in the rat.
Authors: Regoeczi E , Chindemi P A , Debanne M T , Charlwood P A
Published: May 2006

After the injection of a small dose (1 micrograms/100 g of body weight) of 125I-labeled human asialotransferrin type 3 in rats, the radioactivity became rapidly associated with the liver. However, during the ensuing 12 hr a significant fraction of the dose returned to the circulation as protein-bound 125I. The protein released by the liver was indistinguishable by gel filtration from the original preparation and was precipitable by an antiserum to human transferrin. Nevertheless, it no longer bound to the immobilized Gal/GalN-specific lectin from rabbit liver. However, binding could be restored to a large extent by treatment with neuraminidase, indicating that the loss of binding was due to resialylation. Changes in the electrophoretic mobility of asialotransferrin released by the liver showed that resialylation was partial--i.e., it involved the attachment of two or three sialyl residues. From analysis by deconvolution of the plasma curve of partially resialylated asialotransferrin it was calculated that the liver "repaired" this way approximately one asialotransferrin molecule out of four. Plasma clearance of partially resialylated asialotransferrin was similar to that of nondesialylated transferrin.
Resistance to phosphatase of thiophosphorylated epidermal growth factor receptor in A431 membranes.
Authors: Cassel D , Glaser L
Published: May 2006

Epidermal growth factor (EGF) increases the phosphorylation of its receptor and other membrane proteins, and these proteins can be rapidly dephosphorylated by membrane-bound protein phosphatase [Carpenter, G., King L., Jr., & Cohen, S. (1979) J. Biol. Chem. 254, 4884]. We report that [35S]-adenosine 5'-[gamma-thio]triphosphate is as effective as [gamma-32P]ATP as substrate for the EGF receptor-associated protein kinase in A431 membranes. Both the kinetics and the extent of the EGF-dependent thiophosphorylation at 0 degrees C are similar to those obtained with [gamma-32P]ATP, provided that ATP hydrolysis by the membrane preparation is inhibited by addition of adenosine 5'-[beta, gamma-imino]-triphosphate. The thiophosphorylation reaction requires Mn2+ but differs from the phosphorylation reaction in the inability of Mg2+ to serve as cofactor. Both EGF-dependent phosphorylated and thiophosphorylated membrane proteins yield the same two major bands of Mr 145,000-160,000 in autoradiograms of NaDodSO4/polyacrylamide gel electrophorograms. The rate of dephosphorylation of membrane proteins that have been thiophosphorylated in the presence of EGF is dramatically slower (factors of 1/20 to 1/40) than that of the phosphorylated proteins at both 0 degrees C and 32 degrees C. This increased metabolic stability of the thiophosphorylated proteins will be useful for investigation of the role of phosphorylation in the biological effects of EGF.
Triphenylmethylphosphonium is an ion channel ligand of the nicotinic acetylcholine receptor.
Authors: Lauffer L , Hucho F
Published: May 2006

The lipophilic cation triphenylmethylphosphonium (Ph3MeP+), which is widely used as a sensor for membrane potential with cells, organelles, and membrane vesicles, is shown also to accumulate in membranes rich in nicotinic acetylcholine receptor in a voltage-independent way. Evidence is presented that Ph3MeP+ in this system is bound to a cation-binding site of the ion channel that is part of the acetylcholine receptor complex. Binding is stimulated by cholinergic effectors (Kd = 13 microM in the absence of carbamoylcholine; Kd = 1.5 microM in the presence of 10 microM carbamoylcholine), and this stimulation is blocked by alpha-bungarotoxin. Ph3MeP+ blocks efflux of 22Na from receptor-rich microsacs and appears to compete with the channel ligand phencyclidine for a common binding site. In contrast to the binding of other proven channel ligands, Ph3MeP+-binding is not affected by desensitization.
Sequence of galR gene indicates a common evolutionary origin of lac and gal repressor in Escherichia coli.
Authors: von Wilcken-Bergmann B , Müller-Hill B
Published: May 2006

The nucleotide sequence of the galR gene of Escherichia coli, which codes for galactose repressor, has been determined. The subunits of gal repressor are predicted to consist of 343 residues, including the NH2-terminal methionine. Twenty-six of the predicted NH2-terminal 55 residues of gal repressor are identical to the NH2-terminal residues of lac repressor. Additional homologies appear between residues 165 and 200, between residues 235 and 255, and around residue 325.
Methylation at D-aspartyl residues in erythrocytes: possible step in the repair of aged membrane proteins.
Authors: McFadden P N , Clarke S
Published: May 2006

Reversibly methylated aspartyl residues in human erythrocyte membrane proteins are shown to be in the "unnatural" D configuration. This is demonstrated by treatment of proteolytically derived aspartic acid beta-[3H]methyl ester with L- and D-amino-acid oxidases and by the resolution of diastereomeric L-leucyl dipeptides containing either L- or D-aspartic acid beta-methyl ester by ion-exchange chromatography. Based on this observation, we propose a novel role for eukaryotic protein carboxyl methyltransferases (protein O-methyltransferase; S-adenosyl-L-methionine:protein O-methyltransferase, EC 2.1.1.24). We suggest that these widely distributed enzymes function to recognize aspartyl residues that have racemized spontaneously for a subsequent repair reaction. This repair function is postulated to couple ester hydrolysis with the restoration of the original L configuration of the aspartyl residue. It is possible that similar types of racemization repair processes may occur by reversible covalent modifications at other residues. Other possible functions for D-aspartic acid beta-methyl ester residues in proteins are considered.
Some electrochemical and chemical properties of methoxatin and analogous quinoquinones.
Authors: Eckert T S , Bruice T C , Gainor J A , Weinreb S M
Published: May 2006

The present study establishes relationships between structure and reactivity for the pyrroloquinoline and phenanthroline quinones. The electrochemical reductions of 1,7- and 1,10-phenanthroline-5,6-quinones, like other quinones, are reversible and occur by 2e- transfer in a single step in aqueous solution and by two 1e(-)-transfer steps in aprotic media. The electron-withdrawing pyridine moieties both increase their potentials and stabilize their aprotic semiquinones. The electrochemistry of the cofactor methoxatin and its trimethylester derivative is similar to the phenanthroline quinones in aqueous solution. However, the electrochemical reductions of methoxatin and its triester in aprotic solutions are characterized by at least three potentials, each accounting for less than 1e-. This has been explained by the proposal of semiquinone complexing with itself and with quinone. Despite an electron-donating pyrrole moiety, methoxatin and its trimethylester have relatively high potentials in aprotic solution. This is presumably due to stabilization of radical anions by the aforementioned complexing or by delocalization with carboxylic acid and ester groups. The reduction potential of methoxatin, in both aqueous and aprotic solvent, suggests that oxidation of methanol should be a thermodynamically favorable process. No evidence for an electrochemically reduced state lower than the quinol was found for any of the compounds. Chemical reactivity is influenced by the orientation of the pyridine nitrogen. The two quinones with a pyridine nitrogen peri to a quinone carbonyl add and oxidize nucleophiles most readily.
Distribution of cytochemically detectable cholesterol in the electric organ of Torpedo marmorata.
Authors: Perrelet A , Garcia-Segura L M , Singh A , Orgi L
Published: May 2006

A cytochemical probe for cholesterol, the polyene antibiotic filipin, was applied to aldehyde-fixed samples of the electric organ of Torpedo marmorata to identify filipin-binding sites in the various membrane components of the organ and, hence, the probable cholesterol content at these levels. In both thin-sectioned and freeze-fractured samples, filipin-cholesterol complexes appeared numerous and homogeneously distributed on the Schwann cell plasma membrane. On the presynaptic membrane, filipin-cholesterol complexes occurred in patches alternating with unlabeled membrane segments. The postsynaptic, acetylcholine receptor-rich plasma membrane of the electroplax showed no or few filipin-cholesterol complexes in the flat region and upper part of the invaginations (both areas characterized by a lattice of small intramembrane particles); however, the membrane of the bottom part of the postsynaptic invaginations contained several complexes. The ventral, noninnervated plasma membrane of the electroplax showed a moderate, homogeneous filipin labeling. These data suggest that the distribution of cholesterol among membranes of the electroplax is not homogeneous and that the acetylcholine receptor-rich region of the postsynaptic membrane (as characterized by the lattice of small intramembrane particles) may contain little cholesterol.
Nucleotide sequences of gene segments encoding membrane domains of immunoglobulin gamma chains.
Authors: Yamawaki-Kataoka Y , Nakai S , Miyata T , Honjo T
Published: May 2006

The nucleotide sequences of the exons encoding membrane-bound IgG1, IgG2a, and IgG2b of mouse were determined and compared with the sequence of membrane-bound IgM. The sequences indicate that membrane-bound gamma chains bear an additional 71 residues at the COOH termini, including 17-residue acidic, 26-residue hydrophobic, and 28 residue hydrophilic portions. The hydrophobic portion, which seems to be anchored in the lipid bilayer of the membrane, is highly conserved among membrane-bound mu and gamma chains. We propose the presence of a membrane protein that recognizes the conserved hydrophobic segment and anchors the membrane-bound immunoglobulin. Comparison of the nucleotide sequences revealed another example of intervening sequence-mediated domain exchange around one of the membrane exons.
Synapsis-dependent allelic complementation at the decapentaplegic gene complex in Drosophila melanogaster.
Authors: Gelbart W M
Published: May 2006

Allelic complementation at the decapentaplegic gene complex (dpp: 2-4-0, cytogenetic location: polytene chromosome bands 22F1-3) of Drosophila melanogaster frequently occurs between site mutations. Two specific instances of allelic complementation are shown to be dependent upon normal somatic chromosome synapsis of homologous dpp genes. Numerous strains have been identified that bear lesions that disrupt allelic complementation when heterozygous with structurally normal chromosomes; each of these 57 strains contains a gross chromosomal rearrangement with a break on chromosome 2. The properties of the rearrangements carried by 50 of these strains are consonant with the idea that their effects are due to a disruption of somatic chromosome synapsis in the dpp region of chromosome arm 2L. In double heterozygotes of simple two-break rearrangements, allelic complementation is restored (presumably through the restoration of structural homozygosity). The types of rearrangements that disrupt complementation have properties very similar to those of rearrangements that disrupt the transvection effect at bithorax [Lewis, E. B. (1954) Am. Nat. 88, 225-239]. The existence of synapsis-dependent allelic complementation is a demonstration of the physiological importance of nuclear organization in gene expression.
Acetylcholine, melatonin, and potassium depolarization stimulate release of luteinizing hormone-releasing hormone from rat hypothalamus in vitro.
Authors: Richardson S B , Prasad J A , Hollander C S
Published: May 2006

We have examined the release of radioimmunoassayable luteinizing hormone-releasing hormone (LH-RH) from fragments of rat medial basal hypothalamus. These fragments were cultured overnight in medium containing serum and then preincubated in groups of three for 10 min in medium resembling cerebrospinal fluid in its electrolyte constituents and containing bacitracin. This was followed by 30-min incubation periods during which some of the hypothalami were exposed to test substances. Potassium depolarization, effected by the addition of 56 mM potassium chloride to the incubation medium, caused a marked stimulation in LH-RH release, but only in the presence of calcium. Acetylcholine at 10 nM and the parasympathomimetic anticholinesterase agent neostigmine at 1 microM markedly stimulated LH-RH release. Hexamethonium, a nicotinic antagonist, at 1 microM abolished the acetylcholine-induced increment in LH-RH release. Melatonin, a pineal indolamine, caused significant stimulation of LH-RH release at a concentration as low as 10 nM. Bacitracin (21 microM) was employed in all these experiments. It had no effect on LH-RH release but did prevent the degradation of LH-RH in this system. We conclude that acetylcholine and melatonin are capable of inducing LH-RH release from the rat medial basal hypothalamus. These actions may account for some of the progonadotropic properties previously ascribed to these agents.
Autoradiographic demonstration of the sodium-dependent high-affinity choline uptake system.
Authors: Ruch G A , Koelle G B , Sanville U J
Published: May 2006

Mouse phrenic nerve-hemidiaphragm preparations were incubated for 10 min at 37 degrees C in an oxygenated medium containing all the constituents reported as essential for the sodium-dependent high-affinity choline uptake system, including [3H]choline chloride, 3.1 Ci/mmol, 2.5 microM. The phrenic nerve was stimulated by a suction electrode with square waves of 4-msec duration, 3 V, at 10 Hz; some preparations were not stimulated. Similar preparations in which 25 microM hemicholinium was included in the medium served as controls. After exposure of emulsion-coated coverslips apposed to the preparations for 53 days, development, and staining of the motor endplates for acetylcholinesterase, there was a marked accumulation of silver grains over the motor endplates of stimulated preparations. Unstimulated preparations showed considerably lighter deposits; accumulation was blocked by hemicholinium.
DNA-mediated gene transfer: recombination between cotransferred DNA sequences and recovery of recombinants in a plasmid.
Authors: Anderson R A , Krakauer T , Camerini-Otero R D
Published: May 2006

Ltk- aprt- mouse L cells were transformed to the tk+ phenotype with 10 ng of the herpes simplex virus-1 thymidine kinase (tk) gene and 20 micrograms of pBR322 or simian virus 40 (SV40) DNA. DNAs from five cloned cell lines show restriction endonuclease fragments that hybridize to both tk and pBR322 or SV40 DNA. In all of the cell lines some of these fragments also contain cellular DNA sequences. The use of carrier DNAs with defined sequences has enabled us to demonstrate that the joining of carrier and selectable gene sequences occurs in mouse cells. In one case we have been able to use the ampicillin resistance marker of pBR322 to "rescue" a recombinant plasmid. An analysis of the junction between pBR322 and tk in this plasmid suggests that a small area of homology (16 of 19 base pairs) might be involved in the recombination process.
beta-globin transcript found in induced murine erythroleukemia cells is homologous to the beta h0 and beta h1 genes.
Authors: Brown B A , Padgett R W , Hardies S C , Hutchison C A , Edgell M H
Published: May 2006

RNA transcripts complementary to at least one of the four beta-globin homologous genes (beta h) are found in high concentration in the murine erythroleukemic (MEL) cell line GM979 after butyric acid induction. Hybridization data indicate that the gene expressed is Hbb-beta h0 or Hbb-beta h1, or both. The level of beta h0/1 transcripts in the MEL cell is similar to the level of adult transcripts. The Hbb-beta h0/1 transcript is about 800 nucleotides in length. In addition, there is a larger beta h0/1 transcript of the same size and relative intensity as the adult beta-globin precursor. We also report significant levels of embryonic gene Hbb-y transcripts in induced GM979 cells. We have determined that the GM979 cell line has the [Hbb]s haplotype on the basis of an examination of its globin DNA restriction pattern. An additional MEL cell line of haplotype [Hbb]d (DBA/2 line 6A11A) was examined and found to contain no significant level of Hbb-beta h0, Hbb-beta h1, Hbb-beta h2, or Hbb-y gene transcripts.
The beta-adrenergic receptor: rapid purification and covalent labeling by photoaffinity crosslinking.
Authors: Shorr R G , Heald S L , Jeffs P W , Lavin T N , Strohsacker M W , Lefkowitz R J , Caron M G
Published: May 2006

New procedures for the rapid purification and covalent labeling of the beta-adrenergic receptors have been developed that should greatly accelerate progress in the study of these widely distributed adenylate cyclase-coupled receptors. Chromatography of solubilized receptor preparations on a Sepharose-alprenolol affinity gel followed by HPLC on steric exclusion columns lead to rapid (2 days) and high yield (approximately 30%) purification of the receptors from frog erythrocytes. The receptor obtained by these rapid procedures appears to be composed entirely of 58,000 Mr subunit(s) and to be identical to that previously purified by much lengthier procedures [Shorr, R. G. L., Lefkowitz, R. J. & Caron, M. G. (1981) J. Biol. Chem. 256, 5820-5826]. A novel, very high affinity, specific beta-adrenergic antagonist, p-aminobenzylcarazolol, has also been synthesized. It can be radioiodinated to theoretical specific radioactivity with 125I (2,200 Ci/mmol). This radioligand, which possesses an arylamine moiety, may then be covalently incorporated into the receptor binding subunit (58,000 Mr peptide) of the frog erythrocyte membranes by the use of the bifunctional photoactive crosslinker N-succinimidyl-6-(4'-azido-2'- nitrophenylamino)hexanoate (SANAH). Covalent incorporation is blocked by various drugs with a strict beta-adrenergic specificity. This suggests that the photoaffinity crosslinking approach may be useful for labeling a variety of small molecule and neurotransmitter receptors when appropriate ligands can be synthesized.