bluuebunny/crossref_metadata_2025 huggingface dataset:
The subsynaptic 43-kDa protein is concentrated at developing nerve-muscle synapses in vitro.
Authors: Burden S J
Published: May 2006

A 43-kDa peripheral membrane protein is known to copurify with acetylcholine receptor (AcChoR)-rich membranes isolated from the electric organ of Torpedo californica. Immunoelectron microscopy and crosslinking studies have demonstrated that this 43-kDa protein is closely associated with the cytoplasmic domain(s) of the AcChoR and suggest that the 43-kDa protein could regulate the distribution of the AcChoR in the postsynaptic membrane. This paper demonstrates that this postsynaptic protein appears at developing neuromuscular synapses in Xenopus nerve/muscle cocultures as early as AcChoRs become clustered at synaptic sites. Moreover, this protein is concentrated at AcChoR clusters that occur on noninnervated muscle cells. The close spatial and temporal relationship of this subsynaptic protein and AcChoR clusters is consistent with a role for the 43-kDa protein in the formation and/or stabilization of AcChoR clusters.
Extension of the statistical theory of resonating valence bonds to hyperelectronic metals
Authors: Kamb Barclay , Pauling Linus
Published: May 2006

The statistical treatment of resonating covalent bonds in metals, previously applied to hypoelectronic metals, is extended to hyperelectronic metals and to metals with two kinds of bonds. The theory leads to half-integral values of the valence for hyperelectronic metallic elements.
Bipolaroxin, a selective phytotoxin produced by Bipolaris cynodontis.
Authors: Sugawara F , Strobel G , Fisher L E , Van Duyne G D , Clardy J
Published: May 2006

Two sesquiterpenes have been isolated from the fungal pathogen of Bermuda grass Bipolaris cynodontis. Chemical, spectral, and x-ray diffraction studies have led to the characterization of these as bipolaroxin and dihydrobipolaroxin, highly oxygenated members of the eremophilane family. Bipolaroxin is phytotoxic to some but not all of the plants tested. To our knowledge, a phytotoxin with host selectivity isolated from a weed pathogen has not been reported previously.
Reconstitution of regulatory properties of adenylate cyclase in Escherichia coli extracts.
Authors: Reddy P , Meadow N , Roseman S , Peterkofsky A
Published: May 2006

The inhibition of adenylate cyclase activity of Escherichia coli by methyl alpha-glucoside has been demonstrated in intact or in permeable cells but not in cell-free extracts. In intact or permeable cells, this inhibition is demonstrable only in strains expressing the genes for proteins of the phosphoenolpyruvate:glycose phosphotransferase system (PTS); in permeable cells, the inhibition also requires potassium phosphate. Using homogeneous proteins of the PTS, we have reconstituted in cell-free extracts many of the features of the regulated form of adenylate cyclase: (i) In the absence of K2HPO4, permeable cells have lower adenylate cyclase activity than extracts; addition of homogeneous PTS proteins to the extracts brings adenylate cyclase activity close to the level observed in permeable cells. (ii) The low activity observed in permeable cells is stimulated by potassium phosphate; this stimulation is also observed in extracts supplemented with PTS proteins and phosphoenolpyruvate. (iii) In permeable cells, potassium phosphate-stimulated adenylate cyclase activity is inhibited by methyl alpha-glucoside or pyruvate; extracts behaved similarly when supplemented with PTS proteins, K2HPO4, and phosphoenolpyruvate. Thus, the regulated form of adenylate cyclase has been reconstituted in cell-free extracts by addition of homogeneous PTS proteins.
Human apolipoprotein B-100: cloning, analysis of liver mRNA, and assignment of the gene to chromosome 2.
Authors: Law S W , Lackner K J , Hospattankar A V , Anchors J M , Sakaguchi A Y , Naylor S L , Brewer H B
Published: May 2006

Human apolipoprotein B-100 (apo B-100) is the major apolipoprotein of low density lipoproteins and the principal ligand for interaction with the low density lipoprotein receptor. The human apo B-100 gene has been inserted into a lambda gt-11 expression vector, and the apo B-100 cDNA clones have been identified by screening with a monospecific apo B-100 antiserum, by screening with synthetic oligonucleotides based on the amino acid sequence of peptides isolated from apo B-100, and by immunoblot analysis of the expressed protein with a monoclonal antibody to apo B-100. The complete nucleotide and derived-amino acid sequence of a 1.7-kilobase cDNA clone of apo B-100 was determined. The 560-amino acid residues of apo B-100 contain no unique linear or repeating sequences of amino acids. The computer-predicted conformation of the apo B-100 protein contains segments of helical structure; however, a large portion of the protein is organized into beta-structure. The beta-structure may be important in lipid-apo B-100 interactions in low density lipoprotein and may contribute to the insolubility of delipidated apo B-100 in aqueous buffers. RNA blot hybridization analysis of liver mRNA utilizing a Nco I/HindIII apo B-100 cDNA probe revealed that the apo B-100 mRNA is 15-18 kilobases long, which is of sufficient size to code for a 250-387 kDa apolipoprotein, the proposed molecular size of delipidated plasma apo B-100. The gene for human apo B-100 has been localized to chromosome 2 by filter hybridization of human-mouse somatic cell hybrids utilizing a 400-base-pair Nco I/HindIII apo B-100 cDNA probe. This location is in contrast to the low density lipoprotein receptor that has been localized to chromosome 19. The cloning of human apo B-100 has provided new insights into the structure and physicochemical properties of apo B-100 and will facilitate studies on the factors modulating apo B-100 biosynthesis and the expression of the apo B-100 gene in patients with dyslipoproteinemias.
Epidermal growth factor regulates the expression of its own receptor.
Authors: Clark A J , Ishii S , Richert N , Merlino G T , Pastan I
Published: May 2006

The epidermal growth factor (EGF) receptor gene is the cellular homolog of the avian erythroblastosis virus erbB oncogene. Control of EGF receptor expression determines cellular responsiveness to EGF and might play an important role in neoplastic development. Using RNA blot hybridization, we have found that exposure of human KB carcinoma cells to EGF results in elevated levels of EGF receptor mRNA. The phorbol ester 4 beta-phorbol 12-myristate 13-acetate also stimulates EGF receptor RNA accumulation. Immunoprecipitation of metabolically labeled (30 min) EGF receptor protein revealed that synthesis of new EGF receptor follows the increase in receptor RNA. Addition of cycloheximide together with EGF further enhances EGF receptor RNA accumulation. Results of nuclear runoff-transcription experiments suggest that the stimulatory effects of EGF and cycloheximide are most likely due to a posttranscriptional control mechanism.
Complete nucleotide sequence of dog heart creatine kinase mRNA: conservation of amino acid sequence within and among species.
Authors: Roman D , Billadello J , Gordon J , Grace A , Sobel B , Strauss A
Published: May 2006

Creatine kinase (CK; EC 2.7.3.2) plays an important role in energy metabolism in brain and muscle. Expression of CK isoenzymes is regulated during development and is tissue specific. To define the structures of canine CK isoenzymes and to elucidate the mechanism of regulation in their expression, CK cDNA clones from dog myocardium were isolated. Myocardial CK mRNA is predicted to encode a protein of 381 amino acids. The nontranslated regions of the mRNA comprise at least 38 bases at the 5' end and exactly 345 bases before the poly(A) tail. Partial protein sequences of dog muscle (M) CK and brain (B) CK subunits were determined and compared with the derived amino acid sequence of the myocardial enzyme and of M CK subunits of other species. The M CK subunits from different species share a very high degree (83-96%) of sequence identity. Dog M and B subunits share extensive sequence identity (74%), a degree of similarity not previously suspected. Southern blot analysis suggests that a CK gene family exists. These observations imply that evolutionary changes in the M CK subunit structure are constrained by the need for preservation of functional properties other than the kinase activity. This conservation is consistent with the possibility that the M subunit plays a structural role in cardiac and skeletal muscle.
Isolation and characterization of leukotriene C4 synthetase of rat basophilic leukemia cells.
Authors: Yoshimoto T , Soberman R J , Lewis R A , Austen K F
Published: May 2006

When leukotriene (LT) A4 was incubated with subcellular fractions of sonicated rat basophilic leukemia (RBL) cells in the presence of glutathione, the enzyme producing LTC4, designated LTC4 synthetase, was found in the 105,000 X g pellet (microsomes) with a 3-fold enrichment in specific activity over that of the sonicate. The identification of the reaction product as LTC4 was confirmed by its identical retention time on reverse-phase HPLC to that of synthetic LTC4, the incorporation of [3H]glutathione into the product, its reactivity in a radioimmunoassay, and its UV absorption spectrum. In contrast, glutathione S-transferase activity, measured spectrophotometrically with 1-chloro-2,4-dinitrobenzene, was detected predominantly in the 105,000 X g supernatant (89%) and also in the microsomes (7%). The microsomal glutathione S-transferase and LTC4 synthetase were solubilized with 0.4% Triton X-102 and separated by DEAE-Sephacel chromatography; the former appeared in the effluent and the latter in the eluate after the addition of 0.16 M NaCl to the equilibration buffer. Solubilized, microsomal glutathione S-transferase was inhibited by S-hexylglutathione with an IC50 of 36 microM and was stable at 40 degrees C for 5 min, whereas LTC4 synthetase was only slightly inhibited (IC50, 2.3 mM) by S-hexylglutathione and retained no activity after incubation at 40 degrees C for 5 min. The partially purified LTC4 synthetase showed a specific activity of 1.34 +/- 0.51 nmol of LTC4 per 10 min per mg of protein (mean +/- SD, n = 9), representing a 10-fold purification from the sonicate and catalyzed the dose- and time-dependent production of LTC4 from LTA4 and glutathione. The apparent Km values for LTA4 and glutathione were estimated by Lineweaver-Burk plots to be 5-10 microM and 3-6 mM, respectively. These results indicate that the conjugation of LTA4 with glutathione to form LTC4 is catalyzed by a unique microsomal enzyme.
Isolation of renin-producing human cells by transfection with three simian virus 40 mutants.
Authors: Pinet F , Corvol M T , Dench F , Bourguignon J , Feunteun J , Menard J , Corvol P
Published: May 2006

A human juxtaglomerular cell (JGC) tumor was used for the immortalization of renin-secreting cells. The transfection of primary JGC with three different simian virus 40 (SV40) mutants resulted in the continuous production of renin-secreting cells. The most efficient renin-producing cells (producing about 400 pg of renin per 24 hr per ml of culture medium) were those transfected with the PAS SV40 mutant. The renin production was stable and the cell cultures have been maintained for greater than 1 year. Two types of cells were cultured together and could not be separated: round and birefringent cells, which exhibited features of mast cells, and elongated cells containing myofilaments and secretory granules. Immunocytochemical staining showed the presence of renin in this latter cell type. The renin produced by the transfected cells was not stored within the cells but was released rapidly into the medium. More than 95% of the renin produced was prorenin, which, after activation, had characteristics similar to those of pure human standard renin as to its enzymatic, immunologic, and biochemical properties, except that it was less glycosylated. These stable JGC tumoral cell lines provide a unique system for studying human renin biosynthesis and its regulation in vitro.
Molecular analysis of chromosome 11 deletions in aniridia-Wilms tumor syndrome.
Authors: van Heyningen V , Boyd P A , Seawright A , Fletcher J M , Fantes J A , Buckton K E , Spowart G , Porteous D J , Hill R E , Newton M S
Published: May 2006

We describe five individuals who have constitutional deletions of the short arm of one chromosome 11, including all or part of the band p13. All of these individuals suffer from aniridia; two have had a Wilms tumor removed. We have established lymphoblastoid cell lines from these and in three cases constructed somatic cell hybrids containing the deleted chromosome 11. Analysis of DNA from the cell lines and hybrids with a cloned cDNA probe has shown that the catalase gene is deleted in four of five patients. The catalase locus must be proximal to the Wilms and aniridia-related loci. We have not detected a deletion of the beta-globin or calcitonin genes in any of these individuals; we conclude these genes are likely to be outside the region 11p12-11p15.4. In addition, we have used monoclonal antibodies in fluorescence-activated cell sorting analysis to measure expression in the hybrids of two cell surface markers encoded by genes that map to the short arm of chromosome 11. The genes for both of these are deleted in two individuals but are present in the individual with the smallest deletion.
Biological effects of background radiation: mutagenicity of 40K.
Authors: Gevertz D , Friedman A M , Katz J J , Kubitschek H E
Published: May 2006

The naturally occurring radioactive isotope 40K is the single largest contributor to the internal background radiation dose in living organisms. We examined cell growth and mutation rate or frequency in several strains of Escherichia coli in (i) media containing the natural content of 40K, (ii) media containing potassium from which essentially all of the 40K had been removed by isotope separation, and (iii) media highly enriched in 40K. Growth rates (doubling times) were identical in the present or absence of 40K. In more than 40 chemostat experiments, we were unable to detect any significant differences in mutation rate to bacteriophage T5 resistance or in mutation frequency to valine resistance or tryptophan prototrophy attributable to 40K. We conclude that, in the bacterial systems we have studied, 40K does not make a significant contribution to spontaneous mutation.
Growth hormone and insulin-like growth factors I and II produce distinct alterations in glucose metabolism in 3T3-F442A adipocytes.
Authors: Schwartz J , Foster C M , Satin M S
Published: May 2006

In 3T3-F442A adipocytes, human growth hormone (hGH) stimulates glucose oxidation in 4 hr. A maximal increase is evident at hGH concentrations of 50-100 ng/ml and rarely exceeds 50% above control. The stimulation is transient; after 48 hr of incubation with GH, glucose oxidation is significantly suppressed to 35% below control values. In view of the concept that insulin-like growth factors (IGF) may mediate the effects of GH, we compared the effects of hGH (500 ng/ml) and several preparations of IGF on glucose metabolism in 3T3 adipocytes. After 4 hr of incubation, IGF-I from human plasma stimulated glucose oxidation in a dose-related manner, producing a 10-fold increase at 50 ng/ml. Methionyl-IGF-I produced by recombinant DNA techniques was 85-88% as effective as IGF-I. IGF-II stimulated glucose oxidation 3-fold at 50 ng/ml after 4 hr of incubation. In contrast to the suppression observed with hGH after 48 hr, all three of the IGF preparations stimulated glucose oxidation after 48 hr of incubation and were as effective as they were after 4 hr. When each of the IGF preparations was tested (at 5 ng/ml) in combination with hGH, both the stimulatory and suppressive effects of GH were superimposed on the stimulation by the IGFs. Thus, the stimulatory properties of IGF differed from those of GH in that the maximum extent to which IGF increased glucose oxidation, compared with hGH, was as much as 20-fold greater. Furthermore, all of the IGF preparations stimulated glucose oxidation after 48 hr under conditions in which hGH suppressed glucose metabolism. Thus, it is unlikely that extracellular IGFs mediate the effects of hGH on glucose metabolism in 3T3-F442A adipocytes.
Pertussis toxin is required for pertussis vaccine encephalopathy.
Authors: Steinman L , Weiss A , Adelman N , Lim M , Zuniga R , Oehlert J , Hewlett E , Falkow S
Published: May 2006

A mouse model for encephalopathy induced by pertussis immunization has been described; it has features that closely resemble some of the severe reactions, including seizures and a shock-like state leading to death, occasionally seen after administration of Bordetella pertussis (whooping cough) vaccine. Susceptibility to encephalopathy maps to genes of the major histocompatibility complex and correlates as well with the genetic regulation of the level of antibody response to bovine serum albumin. In this study we have investigated which bacterial determinant is responsible for the encephalopathy. Two lines of evidence implicate pertussis toxin as the active bacterial component. Single-site mutants of B. pertussis with single affected virulence factors were tested. A mutant that produces a defective pertussis toxin had greatly diminished capacity to induce encephalopathy, whereas a hemolysin- and adenylate-cyclase-deficient avirulent mutant had the same activity in the mouse model as a virulent strain. Purified pertussis toxin plus bovine serum albumin was tested and found to induce the lethal encephalopathy, demonstrating that the toxin was the critical constituent of B. pertussis responsible for encephalopathy.
External Ca-independent release of norepinephrine by sympathomimetics and its role in negative feedback modulation.
Authors: Vizi E S , Somogyi G T , Harsing L G , Zimanyi I
Published: May 2006

The release of [3H]norepinephrine from isolated mouse vas deferens has been measured. 1-Phenylephrine and 1-norepinephrine significantly enhanced the spontaneous release of radioactivity. As shown by a combination of HPLC and scintillation spectrometry, the release of total radioactivity in response to 1-phenylephrine or 1-norepinephrine consisted mainly of [3H]norepinephrine. Evidence has been obtained that the release of endogenous norepinephrine by exogenous norepinephrine and 1-phenylephrine is independent of the external Ca2+ concentration. The released endogenous norepinephrine in turn inhibits the release of norepinephrine in response to electrical field stimulation. In the presence of yohimbine, the enhancement of spontaneous release due to 1-phenylephrine (or to 1-norepinephrine) was not affected, whereas there was a significant superimposed release of [3H]norepinephrine in response to field stimulation, indicating that the inhibition of stimulation-induced norepinephrine release is an alpha 2-adrenoceptor-mediated process. An important consequence of these findings is to question previous interpretations that the effects of administration of 1-norepinephrine or 1-phenylephrine are due exclusively to their direct effects on the effector cells. The Ca-independent release of endogenous norepinephrine might partly initiate their pharmacological responses. It is concluded that this Ca-independent release is of functional importance, since norepinephrine may accumulate in a concentration sufficient to modulate the release of norepinephrine from varicosities in response to electrical stimulation.
Expression of human adenosine deaminase using a transmissable murine retrovirus vector system.
Authors: Friedman R L
Published: May 2006

Human adenosine deaminase (ADA; adenosine aminohydrolase, EC 3.5.4.4) was expressed at high levels in cultured mouse cells using a transmissable murine retrovirus vector system. A cDNA clone encoding ADA has been inserted into a plasmid vector containing retroviral transcription and packaging signals as well as a selectable gene for G418 resistance. The constructions were transfected into psi 2 cells, which package the recombinant retroviral genomes into replication-defective virus particles. Isoenzyme analysis for ADA in G418-selected psi 2 cells showed at least 20-fold more human ADA activity than endogenous mouse ADA activity. A mouse T-cell lymphoma line, BL/VL3, was cocultured with transformed psi 2 cells producing human ADA, and some of the cocultured cells were selected for resistance to G418. Both G418-selected and unselected cocultured cells expressed human ADA activity at 25%-50% the level of the endogenous enzyme. Thus, efficient retroviral transduction of ADA expression was obtained.
Transposable element IS1 intrinsically generates target duplications of variable length.
Authors: Iida S , Hiestand-Nauer R , Arber W
Published: May 2006

Target duplication during transposition is one of the characteristics of mobile genetic elements. IS1, a resident insertion element of Escherichia coli K-12, was known to generate a 9-base-pair target duplication, while an IS1 variant, characterized by a nucleotide substitution in one of its terminal inverted repeats, was reported to duplicate 8 base pairs of its target during cointegration. We have constructed a series of transposons flanked by copies of either the normal or the variant IS1. The analysis of their transposition products revealed that transposons with normal termini as well as those with variant termini can intrinsically generate either 9- or 8-base-pair target duplications. We also observed that a normal IS1 from the host chromosome generated an 8-base-pair repeat. The possible relevance of the observation for the understanding of transposition processes and models to explain the variable length of target duplications are discussed.
Identification of B cells in multilineage hematopoietic colonies derived from cells of patients with lymphocytic lymphoma.
Authors: Fauser A A , Kanz L , Löhr G W
Published: May 2006

Pluripotent stem cells from human bone marrow can be identified in culture by their ability to form multilineage hematopoietic colonies containing different myeloid lineages and T cells of different phenotypes. The observation of a common progenitor of myeloid and lymphoid cells in normal and disturbed hematopoiesis prompted the question of whether B cells are part of the differentiation program of stem cells. The availability of hybridomas of azaguanine-resistant T-cell lines secreting monoclonal growth factors for B cells and clinical conditions that are considered to originate from malignant B cells might facilitate this investigation. We were able to identify surface immunoglobulin and B-cell-associated antigen-positive cells within such colonies, indicating that B cells are generated from a myelolymphopoietic stem cell. This report describes the presence of B cells in these colonies derived from bone marrow cells of patients with non-Hodgkin lymphoma.
Infectious hepatitis B virus from cloned DNA of known nucleotide sequence.
Authors: Will H , Cattaneo R , Darai G , Deinhardt F , Schellekens H , Schaller H
Published: May 2006

The infectivity of cloned hepatitis B viral DNA (HBV) has been tested in chimpanzees to identify a fully functional HBV genome and to assess the risk associated with its handling. Only one of two HBV DNA sequence variants tested was shown to be infectious. "Clone purified" virus of predicted nucleotide sequence was produced from the infectious HBV DNA, and the cloned viral genome was identical in structure with naturally occurring HBV. Infection could be initiated independent of whether circular monomeric or plasmid integrated dimeric forms of the viral genome were inoculated, but the infectivity of the DNA depended on liver cell transfection or intrahepatic injection. Intravenous injection of high doses of infectious HBV DNA did not induce hepatitis, suggesting that there is virtually no risk associated with routine laboratory handling of cloned HBV DNA.
Effects of phorbol ester on catecholamine secretion and protein phosphorylation in adrenal medullary cell cultures.
Authors: Pocotte S L , Frye R A , Senter R A , TerBush D R , Lee S A , Holz R W
Published: May 2006

The effects of phorbol 12-myristate 13-acetate (PMA) on catecholamine secretion and protein phosphorylation from intact and digitonin-treated chromaffin cells were investigated. PMA (10-300 nM), an activator of protein kinase C, caused a slow Ca2+-dependent release of catecholamine from intact chromaffin cells that was potentiated by the Ca2+ ionophore ionomycin. PMA also enhanced secretion induced by Ba2+. In cells with plasma membranes rendered permeable by digitonin to Ca2+, ATP, and protein, PMA (100 nM) enhanced Ca2+-dependent secretion approximately 70% at 0.5 microM Ca2+ and 30% at 10 microM Ca2+. PMA enhanced the maximal response to Ca2+ approximately 25% and decreased the Ca2+ concentration required for half-maximal secretion approximately 30%. The effects of PMA on chromaffin cells were associated with a 2- to 3-fold increase in the phosphorylation of a 56-kDa protein that may be tyrosine hydroxylase. Other proteins were phosphorylated to a lesser extent. The experiments suggest that PMA increases protein kinase activity and secretion in chromaffin cells and raise the possibility that protein kinase C modulates catecholamine secretion in chromaffin cells.
Demonstration and affinity labeling of a stereoselective binding site for a benzomorphan opiate on acetylcholine receptor-rich membranes from Torpedo electroplaque.
Authors: Oswald R E , Pennow N N , McLaughlin J T
Published: May 2006

The interaction of an optically pure benzomorphan opiate, (-)-N-allyl-N-normetazocine [(-)-ANMC], with the nicotinic acetylcholine receptor from Torpedo electroplaque was studied by using radioligand binding and affinity labeling. The binding was complex with at least two specific components having equilibrium dissociation constants of 0.3 microM and 2 microM. The affinity of the higher affinity component was decreased by carbamoylcholine but not by alpha-bungarotoxin. The effect of carbamoylcholine was not blocked by alpha-bungarotoxin. In comparison, the affinity of [3H]phencyclidine, a well-characterized ligand for a high-affinity site for noncompetitive blockers on the acetylcholine receptor, is increased by carbamoylcholine and the increase is blocked by alpha-bungarotoxin. The binding of (-)-[3H]ANMC was inhibited by a number of other benzomorphans, with (-) isomers being 4- to 5-fold more potent than (+) isomers. Phencyclidine inhibits the binding of (-)-[3H]ANMC to its high-affinity site by a mechanism that is not competitive. UV-catalyzed affinity labeling indicated that the high-affinity-binding site for (-)-[3H]ANMC is at least partially associated with the delta subunit. Tryptic degradation of the Torpedo marmorata delta chain suggested that (-)-ANMC labeled a 16,000-dalton COOH-terminal portion of the subunit. In contrast, 5-azido-[3H]trimethisoquin, a photoaffinity label of the high-affinity site for noncompetitive blockers, labels a 47,000-dalton NH2-terminal fragment of the delta subunit. These results suggest that (-)-[3H]ANMC binds to sites completely distinct from the binding sites for acetylcholine. The high-affinity-binding site for (-)-ANMC and that for phencyclidine and 5-azidotrimethisoquin are allosterically coupled but are regulated differently and are probably physically distinct.
Novel activity of human angiotensin I converting enzyme: release of the NH2- and COOH-terminal tripeptides from the luteinizing hormone-releasing hormone.
Authors: Skidgel R A , Erdös E G
Published: May 2006

Angiotensin I converting enzyme (ACE; kininase II; peptidyldipeptide hydrolase, EC 3.4.15.1) cleaves COOH-terminal dipeptides from active peptides containing a free COOH terminus. We investigated the hydrolysis of luteinizing hormone-releasing hormone (LH-RH) by homogeneous human ACE. Although this decapeptide is blocked at both the NH2 and COOH termini, it was metabolized to several peptides, which were separated by HPLC and identified by amino acid analysis. A major product was the NH2-terminal tripeptide, less than Glu-His-Trp, and another was LH-RH-(4-10) heptapeptide, indicating that the Trp-Ser bond is cleaved to release the NH2-terminal tripeptide. ACE also released the COOH-terminal tripeptide, Arg-Pro-Gly-NH2, and then sequentially the dipeptides Gly-Leu and Ser-Try, leaving less than Glu-His-Trp intact. Thus, less than Glu-His-Trp was formed by both NH2- and COOH-terminal hydrolysis. The cleavage of LH-RH was inhibited by specific ACE inhibitors and by antibody to ACE but not by inhibitors of other enzymes, showing that the hydrolysis was indeed due to ACE. In the absence of chloride, the hydrolysis proceeded at only 16% of the maximal rate (in 500 mM NaCl), but in 10 mM NaCl it increased to 64%. In 500 mM NaCl solution, 86% of the hydrolysis was accounted for by the release of the NH2-terminal tripeptide, whereas in 10 mM NaCl, the COOH-terminal and NH2-terminal cleavage occurred about equally. The Km of LH-RH in 500 mM NaCl was 167 microM and the catalytic constant kcat was 210 min-1. When the NH2-terminal pyroglutamic acid was replaced with glutamic acid ([Glu1]LH-RH), ACE liberated almost exclusively the COOH-terminal tripeptide in 10 mM NaCl. Thus, human ACE, although it is named peptidyl dipeptidase or dipeptidyl carboxypeptidase, can cleave a protected peptide at the NH2 or COOH terminus. The enzyme could be involved in the in vivo metabolism of LH-RH and possibly other blocked peptides.
Feedback regulation of rRNA and tRNA synthesis and accumulation of free ribosomes after conditional expression of rRNA genes.
Authors: Gourse R L , Takebe Y , Sharrock R A , Nomura M
Published: May 2006

We have constructed a conditional rRNA gene expression system by fusing a plasmid-encoded rrnB operon to the lambda PL promoter/operator. It was thereby possible to study the events that lead to the regulation of chromosomal rRNA and tRNA synthesis after overproduction of rRNA. rRNA induction resulted in a 2-fold increase in 30S and 50S free ribosomal subunits, whereas the polysome fraction was unaffected. Overproduction of rRNA and "free" ribosomes produced a large repression of rRNA and tRNA synthesis from chromosomal genes and a smaller increase in the concentration of guanosine tetraphosphate. These results lend support to the ribosome feedback regulation model: rRNA and tRNA operons are negatively regulated, either directly or through some intermediate, by free, nontranslating ribosomes.
Calcium currents in GH3 cultured pituitary cells under whole-cell voltage-clamp: inhibition by voltage-dependent potassium currents.
Authors: Barros F , Katz G M , Kaczorowski G J , Vandlen R L , Reuben J P
Published: May 2006

To isolate inward Ca2+ currents in GH3 rat pituitary cells, an inward Na+ current as well as two outward K+ currents, a transient voltage-dependent current (IKV) and a slowly rising Ca2+-activated current (IKCa), must be suppressed. Blockage of these outward currents, usually achieved by replacement of intracellular K+ with Cs+, reveals sustained inward currents. Selective blockage of either K+ current can be accomplished in the presence of intracellular K+ by use of quaternary ammonium ions. When IKCa and Na+ currents are blocked, the net current elicited by stepping the membrane potential (Vm) from -60 to 0 mV is inward first, becomes outward and peaks in 10-30 msec, and finally becomes inward again. Under this condition, in which both IKV and Ca2+ currents should be present throughout the duration of the voltage step, the Ca2+ current was not detected at the time of peak outward current. That is, plots of peak outward current vs. Vm are monotonic and are not modified by nisoldipine or low external Ca2+ as would be expected if Ca2+ currents were present. However, similar plots at times other than at peak current are not monotonic and are altered by nisoldipine or low Ca2+ (i.e., inward currents decrease and plots become monotonic). When K+ channels are first inactivated by holding Vm at -30 mV, a sustained Ca2+ current is always observed upon stepping Vm to 0 mV. Furthermore, substitution of Ba2+ for Ca2+ causes blockage of IKV and inhibition of this current results in inward Ba2+ currents with square wave kinetics. These data indicate that the Ca2+ current is completely inhibited at peak outward IKV and that Ca2+ conductance is progressively disinhibited as the transient K+ current declines due to channel inactivation. This suggests that in GH3 cells Ca2+ channels are regulated by IKV.
Activated protein C stimulates the fibrinolytic activity of cultured endothelial cells and decreases antiactivator activity.
Authors: Sakata Y , Curriden S , Lawrence D , Griffin J H , Loskutoff D J
Published: May 2006

The effects of bovine activated protein C (APC) on the fibrinolytic activity of cultured bovine aortic endothelial cells were investigated. Confluent monolayers were incubated with purified APC under various conditions and changes in total fibrinolytic activity and in the level of plasminogen activator and plasminogen activator inhibitor (antiactivator) were monitored. The addition of APC to the cells in the absence of other blood or plasma components led to a rapid, dose-dependent increase of fibrinolytic activity both in the media and in cellular extracts. For example, 3.4 micrograms of APC per ml resulted in a 15-fold increase of fibrinolytic activity in the medium within 1 hour. The enhanced fibrinolytic activity reflected increases in both the urokinase-related and tissue-type plasminogen activators produced by these cells. Interestingly, treatment of cells with APC also caused a rapid, dose-dependent decrease in antiactivator activity. Diisopropyl fluorophosphate-inactivated APC did not decrease antiactivator or increase plasminogen activator. Although a small but significant direct (i.e., cell-independent) effect of APC on both fibrinolytic activity and antiactivator activity could be demonstrated, the major portion of these changes appeared to be cell-mediated. These observations indicate that the fibrinolytic potential of cultured endothelial cells is increased by APC and that the enzyme active site is essential for this change. Moreover, the results suggest that one of the primary mechanisms for this stimulation of endothelial cell fibrinolytic activity involves an APC-mediated decrease in antiactivator.
Sex determination: a hypothesis based on noncoding DNA.
Authors: Chandra H S
Published: May 2006

Certain recent models of sex determination in mammals, Drosophila melanogaster, Caenorhabditis elegans, and snakes are examined in the light of the hypothesis that the relevant genetic regulatory mechanisms are similar and interrelated. The proposed key element in each of these instances is a noncoding DNA sequence, which serves as a high-affinity binding site for a repressor-like molecule regulating the activity of a major "sex-determining" gene. On this basis it is argued that, in several eukaryotes, (i) certain DNA sequences that are sex-determining are noncoding, in the sense that they are not the structural genes of a sex-determining protein; (ii) in some species these noncoding sequences are present in one sex and absent in the other, while in others their copy number or accessibility to regulatory molecules is significantly unequal between the two sexes; and (iii) this inequality determines whether the embryo develops into a male or a female.
Interaction of synaptic modification rules within populations of neurons.
Authors: Finkel L H , Edelman G M
Published: May 2006

A theoretical account is given for long- and short-term changes in populations of neurons subject to independent rules for pre- and postsynaptic modification. The postsynaptic rule proposes that coactivated heterosynaptic inputs to a neuron alter the states of ion channels at a given synapse, thereby changing the susceptibility of these channels to local biochemical alterations. The resultant change in the population distribution of local channel states affects the postsynaptic potential produced at the synapse by subsequent inputs. This postsynaptic rule applies, in general, to short-term changes at specific individual synapses. In contrast, the presynaptic rule applies in general to long-term changes in the whole neuron, resulting in an altered probability of transmitter release. Because of neuroanatomical constraints, the presynaptic rule affects large numbers of synapses defined by the connectivity of that neuron and distributed nonspecifically over the population. We show that the combined action of the two independent rules upon populations of neurons arrayed in interconnected neuronal groups leads to consistent alterations of the probability of firing of certain circuits while maintaining variability in the response of the population to novel input. This "dual rules" model fulfills the requirements of the theory of neuronal group selection.
Sequence-specific cleavage of single-stranded DNA: oligodeoxynucleotide-EDTA X Fe(II).
Authors: Dreyer G B , Dervan P B
Published: May 2006

The synthesis of a DNA hybridization probe 19 nucleotides in length, equipped with the metal chelator EDTA at C-5 of thymidine in position 10 (indicated by T*) is described. DNA-EDTA 1 has the sequence 5'-T-A-A-C-G-C-A-G-T*-C-A-G-G-C-A-C-C-G-T-3', which is complementary to a 19-nucleotide sequence in the plasmid pBR322. In the presence of Fe(II), O2, and dithiothreitol, DNA-EDTA 1 affords specific cleavage (25 degrees C, pH 7.4, 60 min) at its complementary sequence in a heat-denatured 167-base-pair restriction fragment. Cleavage occurs over a range of 16 nucleotides at the site of hybridization of 1, presumably due to a diffusible reactive species. No other cleavage sites are observed in the 167-base-pair restriction fragment. The procedure used to synthesize DNA-EDTA probes is based on the incorporation of a thymidine modified at C-5 with the triethyl ester of EDTA. By using routine phosphoramidite procedures, thymidine-EDTA can be incorporated into oligodeoxynucleotides of any desired length and sequence. Because the efficiency of the DNA cleavage reaction is dependent on the addition of both Fe(II) and reducing agent (dithiothreitol), the initiation of the cleavage reaction can be controlled. These DNA-EDTA X Fe(II) probes should be useful for the sequence-specific cleavage of single-stranded DNA (and most likely RNA) under mild conditions.
Specific modulation of the transcription of cloned avian vitellogenin II gene by estradiol-receptor complex in vitro.
Authors: Jost J P , Geiser M , Seldran M
Published: May 2006

Avian vitellogenin-cauliflower mosaic virus hybrid gene is effectively transcribed in vitro in the homologous embryonic liver nuclei system. The transcription of the hybrid gene is modulated by the addition of an estradiol-receptor preparation that has been shown to bind selectively to an upstream region of cloned vitellogenin gene. Stimulation of the transcription of cloned vitellogenin hybrid gene by estradiol receptor is alpha-amanitin sensitive, hormone dependent, and promoter specific. Simian virus 40 and Escherichia coli promoters are not stimulated by the estradiol-receptor complex. The endogenous silent vitellogenin II gene (wild type) present in the nuclei is not turned on by the addition of estradiol-receptor complex. Deletion or inversion of the DNA sequence where the estradiol-receptor complex binds results in the complete suppression of the in vitro stimulation of transcription by estradiol receptor. Correct initiation of the transcription was demonstrated by primer extension studies of the newly synthesized RNA.
Comparative analysis of repeated sequences in rat apolipoproteins A-I, A-IV, and E.
Authors: Boguski M S , Elshourbagy N , Taylor J M , Gordon J I
Published: May 2006

To understand the structural, functional, and evolutionary relationships among the principal protein components of rat high density lipoprotein particles, we undertook a systematic comparative analysis of the primary structures of apolipoproteins (apo)-A-I, -A-IV, and -E. Human apo-A-I and rat apo-A-IV have been shown previously to contain repeated sequences that presumably arose by intragenic duplication of 11- or 22-amino acid amphipathic segments. For apo-A-I, these segments are thought to be the structures responsible for lipid binding and activation of lecithin:cholesterol acyltransferase. From an analysis of the sequence of a full-length cDNA clone, rat apo-A-I is shown to contain eight tandem repetitions of a 22-amino acid segment. However, compared with human apo-A-I, the rat protein has undergone three deletions, two of which involve multiple amino acids in the repeated sequence domain. This disruption of the periodic structure of the protein raises the possibility of species-specific variation in the ability of rat apo-A-I to interact with high density lipoproteins and activate lecithin:cholesterol acyltransferase. Statistical analysis of the structure and organization of repeated sequences in apo-A-I, -A-IV, and -E demonstrates that all three proteins are paralogous members of a dispersed gene family. Despite overall similarity in sequence organization, different portions of these sequences have evolved at different rates. Diversification of a duplicated ancestral sequence has resulted in three lipid-binding proteins with distinct and shared functions.
Detection and characterization of a mouse alpha-spectrin cDNA clone by its expression in Escherichia coli.
Authors: Cioe L , Curtis P
Published: May 2006

A cloned segment of mouse alpha-spectrin mRNA has been identified by immunological techniques. Double-stranded cDNA derived from spleens of anemic mice was introduced into a bacterial expression vector, pUC, and transformed Escherichia coli colonies were screened by using an antiserum to erythrocyte membrane ghost proteins. Of 17 positive colonies, 2 bound antibody to mouse spectrin, and these 2 colonies contained 750-base-pair inserts that cross-hybridized. Transfer of the 750-base-pair insert to an expression vector containing the PL promoter of phage lambda produced larger amounts of peptides that were bound by antibody to mouse spectrin. The spectrin-like peptides made in E. coli elicited antibody that reacted only with the alpha-spectrin subunit of erythrocyte membranes. This clone will be useful for the study of the structure and expression of the spectrin gene, particularly in understanding the role of spectrin in human inherited hemolytic anemias.
Enzyme-controlled scavenging of ascorbyl and 2,6-dimethoxy-semiquinone free radicals in Ehrlich ascites tumor cells.
Authors: Pethig R , Gascoyne P R , McLaughlin J A , Szent-Györgyi A
Published: May 2006

The rate of scavenging by Ehrlich ascites cells of anionic ascorbyl and 2,6-dimethoxy-p-semiquinone free radicals has been investigated by electron spin resonance spectroscopy both for viable cells and for subcellular fractions obtained by differential centrifugation. The scavenging activity is concluded to be associated with an NAD(P)H enzyme containing an active sulfhydryl group. Attempts to identify the enzyme with the reported properties of either semi-dehydro-ascorbate reductase or DT-diaphorase have not been successful. The overall free-radical scavenging activity for viable cells is dextrose dependent and is controlled by the coulombic barrier associated with the cell-surface charge. The cytotoxicity of the mixture of ascorbic acid with 2,6-dimethoxy-p-benzoquinone is concluded to result from a loss of NAD(P)H reducing power in the cells.
Identification and partial characterization of hepatocyte-stimulating factor from leukemia cell lines: comparison with interleukin 1.
Authors: Woloski B M , Fuller G M
Published: May 2006

Leukemia cell lines of the monocytic series (HL-60, U-937, and P388D1) produce a hepatocyte-stimulating factor (HSF) following induction of differentiation with phorbol diester. In 24-72 hr, these leukemia cells produce 2-30% the amount of HSF as human peripheral blood monocytes. Cells of the series at earlier stages of differentiation produced greater amounts of HSF. Fractionation of the medium from each cell type by HPLC reveals much of the HSF activity in the 25- to 30-kilodalton range. Under the same culture conditions, interleukin 1 is produced; however, its bioactivity is in the 7- to 15-kilodalton range. Neither monokine shows reciprocal bioactivity. Superinducing culture conditions that greatly increase interleukin 1 production completely eliminate HSF production, suggesting that there is different stability of the mRNA coding for each protein or that there are different temporal events important to the induction of synthesis of these proteins.
Mutations in direct repeat sequences and in a conserved sequence adjacent to the repeats result in a defective replication origin in plasmid R6K.
Authors: McEachern M J , Filutowicz M , Helinski D R
Published: May 2006

Plasmid pMM3 is a pBR322 derivative carrying the gamma origin of replication of the naturally occurring plasmid R6K. We have produced a gamma-origin mutant bank of this plasmid using the single-strand-specific mutagen sodium bisulfite. Members of this bank contain single or multiple mutations in the seven direct repeats and the flanking sequences in the gamma origin. Three mutants with defective gamma origins have been isolated from this mutant bank. Two of these direct repeat mutants, gamma 117 and gamma 120, are unable to replicate and also have lost the ability to bind the R6K initiation protein pi in vitro at one of the seven 22-base-pair direct repeats within their respective origins. Precise deletion of the damaged repeat of either of these mutants restores origin function, suggesting that the primary defect of these mutants involves a disruption of the normal spacing of pi binding and flanking sequences within the gamma origin. The third mutant, gamma 111, binds pi normally but replicates at a greatly reduced copy number due to a mutation near the seventh repeat. This mutation falls within a short sequence that appears to be conserved among a number of other plasmids that contain direct repeats within their origins of replication.
Affinity-purified interleukin 2 induces proliferation of large but not small B cells.
Authors: Mond J J , Thompson C , Finkelman F D , Farrar J , Schaefer M , Robb R J
Published: May 2006

Immunoaffinity-purified interleukin 2 (IL2) stimulated proliferation of large but not small B cells. Stimulation was observed even when B cells were cultured at very low cell densities (3 X 10(4) per microwell containing 0.2 ml of medium). Addition of small numbers of purified splenic T cells did not enhance the IL2-induced B-cell proliferative response. These results suggest that IL2 was not operating through contaminating T cells. B cells cultured with anti-Ig antibody in vitro showed enhanced proliferation when cultured with EL4 thymoma-derived B-cell growth factor but not when cultured with IL2. A direct role for IL2 in B-cell activation is discussed.
The beta 1 domain of the mouse E beta chain is important for restricted antigen presentation to helper T-cell hybridomas.
Authors: Folsom V , Gay D , Tonegawa S
Published: May 2006

We have constructed a hybrid E beta gene by replacing the second exon of the Ed beta gene (which encodes the majority of the beta 1 protein domain) with the corresponding exon from the Eb beta gene. The hybrid gene has been introduced into a d haplotype host, the lymphoma beta-cell line A20-2J, and an E alpha E beta dimer composed of the endogenous Ed alpha chain and the product of the hybrid Ed beta/Eb beta gene was immunoprecipitated from extracts of transfected cells with an Ed alpha Eb beta-specific monoclonal antibody. Transfected cells have acquired the ability to present antigen to Ed alpha Eb beta-restricted helper T-cell hybridomas, indicating that the second exon of the gene for the E beta chain encodes sequences required for the restricted recognition of the antigen-presenting cell by the class II-restricted responder T-cell.