bluuebunny/crossref_metadata_2025 huggingface dataset:
Insulin, insulin-like growth factor II, and nerve growth factor effects on tubulin mRNA levels and neurite formation.
Authors: Mill J F , Chao M V , Ishii D N
Published: May 2006

We have previously shown that insulin and the insulin-like growth factors share some important neurotrophic properties with nerve growth factor (NGF), including the capacity to enhance neurite formation. In this study, we have examined the effects of these neuritogenic agents on the expression of genes coding for important cytoskeletal proteins of axons and dendrites. Insulin specifically and coordinately increased the levels of alpha- and beta-tubulin mRNAs in human neuroblastoma SH-SY5Y cells. The dose-response curves for these increases were very similar to that for enhancement of neurite formation. Tubulin transcripts reached a transient maximum in approximately 1 day, suggesting that higher levels are important during initiation of neurites and that high levels are not required to sustain neurites once formed. Insulin-like growth factor II shared with insulin the capacity to substantially increase tubulin mRNA levels. NGF had but a small effect. Complementary mechanisms for these neurotrophic agents are suggested, because other studies show NGF and insulin can synergistically potentiate neurite formation. None of the factors altered the levels of actin mRNA. Thus, neurite formation does not seem to require a coordinate increase in actin and tubulin transcripts in SH-SY5Y cells.
The Ca channel in skeletal muscle is a large pore.
Authors: McCleskey E W , Almers W
Published: May 2006

The permeability of Ca channels to various foreign cations has been investigated in the absence of external Ca2+. All physiological metal cations are clearly permeant, including Mg2+. The large organic cation n-butylamine+ is sparingly permeant or impermeant, but its larger derivative 1,4-diaminobutane2+ is highly permeant. Among the cations of the methylated ammonium series, permeability diminishes in a graded fashion as ion size increases. Tetramethylammonium, the largest cation found to be permeant, has a diameter of about 6 A; hence, the aqueous pore of the Ca channel at its narrowest point can be no smaller. That the pore is so large strengthens our view that, under physiologic conditions, the high selectivity of Ca channels is due to selective binding of Ca2+ rather than to rejection of other cations by, for example, a sieving mechanism.
Cloning of yeast TOP1, the gene encoding DNA topoisomerase I, and construction of mutants defective in both DNA topoisomerase I and DNA topoisomerase II.
Authors: Goto T , Wang J C
Published: May 2006

Rabbit antibodies specific to yeast DNA topoisomerase I were used in immunological screening of a Saccharomyces cerevisiae genomic DNA library in Escherichia coli. One of the clones identified by its expression of antigenic determinants of the yeast enzyme is shown to contain the coding sequence of the enzyme: no active DNA topoisomerase I is detectable in cell extracts when insertion or deletion mutations are introduced into a 2-kilobase-pair (kb) region of the sequence in a haploid yeast genome. Blot hybridizations show that there is a single copy of the cloned sequence per haploid and that the sequence is transcribed to give a 2.7-kb poly(A)+ message. Mutants in which 1.7 kb of the sequence is deleted are viable. Temperature-shift experiments using synchronously grown cells of a delta top1 top2 temperature-sensitive (ts) double mutant and its isogenic top2 ts strain show that, whereas mitotic blocks can prevent killing of the top2 ts mutant at a nonpermissive temperature, the same treatments are ineffective in preventing cell death of the delta top1 top2 ts double mutant. These experiments suggest that in yeast DNA topoisomerase I serves a role auxiliary to DNA topoisomerase II.
A model for actin polymerization and the kinetic effects of ATP hydrolysis.
Authors: Pantaloni D , Hill T L , Carlier M F , Korn E D
Published: May 2006

A model for actin polymerization is proposed in which the rate of elongation of actin filaments depends on whether adenosine 5'-triphosphate or adenosine 5'-diphosphate is bound to the two terminal subunits of the filament. This model accounts quantitatively for the experimental data on the kinetics of filament elongation and explains the effect of ATP hydrolysis on actin polymerization.
Localization of Ca2+ release channels with ryanodine in junctional terminal cisternae of sarcoplasmic reticulum of fast skeletal muscle.
Authors: Fleischer S , Ogunbunmi E M , Dixon M C , Fleer E A
Published: May 2006

The mechanism of Ca2+ release from sarcoplasmic reticulum, which triggers contraction in skeletal muscle, remains the key unresolved problem in excitation-contraction coupling. Recently, we have described the isolation of purified fractions referable to terminal and longitudinal cisternae of sarcoplasmic reticulum. Junctional terminal cisternae are distinct in that they have a low net energized Ca2+ loading, which can be enhanced 5-fold or more by addition of ruthenium red. The loading rate, normalized for calcium pump protein content, then approaches that of longitudinal cisternae of sarcoplasmic reticulum. We now find that the ruthenium red-enhanced Ca2+ loading rate can be blocked by the previous addition of ryanodine. The inhibition constant is in the nanomolar range (20-180 nM). Ryanodine and ruthenium red have no effect on the Ca2+ loading rate of longitudinal cisternae. Direct binding studies with [3H]ryanodine localized the receptors to the terminal cisternae and not to longitudinal cisternae. Scatchard analysis of the binding data gives a dissociation constant for ryanodine in the range of the drug action on the terminal cisternae (approximately 100 nM range) with approximately 4 to 20 pmol bound per mg of protein. Ryanodine is known to be toxic in animals, leading to irreversible muscle contractures. These studies provide evidence on the mode of action of ryanodine and its localization to the terminal cisternae. The low concentration at which the drug is effective appears to account for its toxicity. Ryanodine locks the Ca2+ release channels in the "open state," so that Ca2+ is not reaccumulated and the muscle fiber cannot relax.
Transforming and nontransforming growth factors are present in medium conditioned by fetal rat calvariae.
Authors: Centrella M , Canalis E
Published: May 2006

Conditioned medium recovered from fetal rat calvarial cultures contains an autocrine factor termed bone-derived growth factor (BDGF); this factor has been purified by acid extraction, gel-permeation chromatography, and two reversed-phase HPLC steps and examined for mitogenicity on normal rat kidney fibroblasts (NRK, clone 49F). HPLC-purified BDGF caused a dose-related increase in cell number, DNA content, and [3H]thymidine incorporation into acid-insoluble material. Since highly purified BDGF appeared less mitogenic than cruder preparations, the latter were tested for additional growth factors, with particular attention to those required for anchorage-independent colony formation in soft agar. BDGF did not displace 125I-labeled epidermal growth factor (EGF) in a radioligand-receptor assay, indicating the absence of EGF and transforming growth factor alpha (TGF-alpha). Without EGF, no BDGF preparation induced NRK cells to form soft agar colonies. However, calvarial conditioned medium contained a factor which, like TGF-beta, induced large soft-agar colonies in the presence of EGF; this TGF-beta-like factor did not copurify with BDGF. Polyclonal antibodies against platelet-derived growth factor did not neutralize the effects of BDGF on NRK cells. BDGF is a potent mitogen for nonskeletal-tissue-derived fibroblasts. Although crude BDGF preparations do contain TGF-beta, BDGF is distinct from this factor and others necessary for NRK cell transformation to anchorage-independent growth.
Interaction of purified type IIB von Willebrand factor with the platelet membrane glycoprotein Ib induces fibrinogen binding to the glycoprotein IIb/IIIa complex and initiates aggregation.
Authors: De Marco L , Girolami A , Zimmerman T S , Ruggeri Z M
Published: May 2006

Von Willebrand factor (vWF) was purified from the plasma of a patient with type IIB von Willebrand disease (vWF from such a patient, IIB vWF) who had a normal platelet count and showed no evidence of spontaneous platelet aggregation. Large multimers of IIB vWF were absent from purified preparations and from plasma. Ristocetin-induced platelet aggregation was enhanced by purified IIB vWF. The aggregation of washed normal platelets mixed with IIB vWF (0.4 microgram/ml) required lower amounts of ristocetin than the aggregation of normal platelets mixed with the same concentrations of normal vWF. Moreover, purified IIB vWF alone induced aggregation of platelet-rich plasma at concentrations as low as 10 micrograms of IIB vWF/ml in the absence of any other agonist. Aggregation was blocked by a monoclonal antibody against the platelet membrane glycoprotein, GPIb, as well as by an anti-GPIIb/IIIa antibody. Washed platelet suspensions were promptly aggregated by IIB vWF only when fibrinogen and CaCl2 were added to the mixture. Purified IIB vWF induces the binding of fibrinogen to platelets. Such binding was blocked by the anti-GPIb monoclonal antibody as well as by the anti-GPIIb/IIIa monoclonal antibody that inhibited aggregation. A second anti-GPIIb/IIIa antibody, which has the property of blocking vWF but not fibrinogen binding to platelets, blocked neither aggregation nor fibrinogen binding induced by IIB vWF. These studies demonstrate that platelet aggregation is triggered by the initial interaction of IIB vWF with GPIb which is followed by exposure of fibrinogen binding sites on GPIIb/IIIa. Fibrinogen binds to these sites and acts as a necessary cofactor for the aggregation response.
Expression of preproenkephalin-like mRNA and its peptide products in mammalian testis and ovary.
Authors: Kilpatrick D L , Howells R D , Noe M , Bailey L C , Udenfriend S
Published: May 2006

The distribution of preproenkephalin mRNA and proenkephalin-derived peptides have been examined in gonadal tissues from rats, hamsters, and cattle. A preproenkephalin mRNA band was detected in the ovaries of all three species and in hamster testis that is identical in size to the 1450-nucleotide mRNA typically found in tissues that express proenkephalin. Rat testis, on the other hand, expresses at least one preproenkephalin-like mRNA that is substantially greater in size (1900 nucleotides). [Met]enkephalin-containing peptides were also detected in each of the gonadal tissues examined. Although the abundance of preproenkephalin-like mRNA in rat testis was comparable to that in rat brain, the testicular content of proenkephalin-derived [Met]enkephalin sequences was less than 4% of the rat brain content. Together these data suggest that preproenkephalin-like mRNA in rat testis is not efficiently translated, proenkephalin-derived peptides undergo rapid turnover in this tissue, or the mRNA in rat testis has a frameshift resulting in an altered coding sequence.
A human histone H4 gene exhibits cell cycle-dependent changes in chromatin structure that correlate with its expression.
Authors: Chrysogelos S , Riley D E , Stein G , Stein J
Published: May 2006

By use of synchronized human HeLa S3 cells, a site sensitive to both DNase I and nuclease S1 was identified 50-150 base pairs upstream of the ATG codon of a cell cycle-dependent histone H4 gene. This site expanded to include a broad region of approximately equal to 300 base pairs sensitive to DNase I throughout S phase and then narrowed again to the original site after the completion of DNA replication. The level of nuclease S1 sensitivity was greatest during early S phase, when the gene is replicated and its transcription rate is maximal. The chromatin structure of the human beta-globin gene, which is not expressed in HeLa cells, was also analyzed throughout the cell cycle, and in no case was a sub-band seen as a result of DNase I or nuclease S1 digestion, nor were there any changes in nuclease sensitivity correlated with its replication. Thus the cell cycle-dependent chromatin alterations in this histone H4 gene appear to be due to the coupled replication and expression of this gene rather than simply its replication. These results suggest that histone genes, as compared with developmentally regulated genes, exhibit an "intermediate" level of regulation whereby the gene is never in a completely inactive conformation, but changes in chromatin structure occur as a function of the cell cycle and expression.
Genetic complementation of UV-induced DNA repair in Chinese hamster ovary cells by the denV gene of phage T4.
Authors: Valerie K , de Riel J K , Henderson E E
Published: May 2006

The denV gene of phage T4, encoding the pyrimidine dimer-specific DNA repair enzyme endonuclease V, has been introduced by DNA transfection into the UV-sensitive DNA repair-deficient Chinese hamster ovary (CHO) cell line UV5. Transformants were first selected for resistance to the antibiotic G418 conferred by the neo gene from Tn5 carried by the same plasmid. A majority of the isolated G418-resistant UV5 clones also showed an increased resistance to 254-nm UV light. One clone, designated I-A1, was found to have an intermediate level of colony-forming ability after UV irradiation when compared to UV5 and wild-type AA8 cells. A Southern blot showed that I-A1 carries a single integrated intact copy of the denV gene. Alkaline sucrose gradients revealed a dose-dependent appearance of breaks in the DNA of I-A1 cells following UV-irradiation, while unirradiated cells did not exhibit any significant breaks. Analysis of DNA repair by isopycnic sedimentation showed that DNA excision repair by I-A1 was at least equal to the level of repair in AA8 cells. These results show that the prokaryotic denV gene can restore UV repair capabilities in vivo to CHO UV5 cells defective in repair of UV-induced damage.
Shared idiotopes among antibodies encoded by heavy-chain variable region (VH) gene members of the J558 VH family as basis for cross-reactive regulation of clones with different antigen specificity.
Authors: Victor-Kobrin C , Manser T , Moran T M , Imanishi-Kari T , Gefter M , Bona C A
Published: May 2006

A wide idiotype cross-reactivity was observed among six groups of monoclonal antibodies specific for arsonate and nitrophenyl haptens, hemagglutinin of PR8 and X31 influenza viruses, dextran, A48-idiotype, and a set of six monoclonal antibodies with unknown antigenic specificity. All of these antibodies are encoded by heavy-chain variable region (VH) genes belonging to the J558 VH family. This idiotypic cross-reactivity was determined by studying the binding of these antibodies to a panel of six monoclonal anti-idiotype antibodies, each one raised against a member of the six groups of monoclonal antibodies. The administration at birth of two such monoclonal anti-idiotype antibodies induced a long-lasting suppression not only of the corresponding idiotype but also of VH-related idiotypes with different antigenic specificities. These results suggest that the idiotypes encoded by VH genes that belong to the same VH gene family are interactive one with another. The possible physiological consequences of this immunochemical cross-reactivity are discussed.
Biochemical and immunological evidence for a cardiodilatin-like substance in the snail neurocardiac axis.
Authors: Nehls M , Reinecke M , Lang R E , Forssmann W G
Published: May 2006

Cardiac hormones, which have been isolated recently from mammalian atria, are potent regulatory peptides of blood pressure and blood volume. By using biochemical and immunological methods to determine cardiac hormones of the cardiodilatin family, this type of peptide hormone was detected in a neurosecretory system projecting from the subesophageal ganglion to the heart of the snail. The cardiodilatin-like molecule was characterized by its biological effects on mammalian vascular smooth muscle, by radioimmunoassay combined with high-performance liquid chromatography, and by immunocytochemistry. In mammals cardiodilatin-like peptides appear to serve purely endocrine functions. In contrast, in the snail they are present in a neuroendocrine system, the function of which remains to be established.
Diethyl pyrocarbonate: a chemical probe for secondary structure in negatively supercoiled DNA.
Authors: Herr W
Published: May 2006

Purine residues located within regions of DNA that have the potential to form left-handed Z-helical structures are modified preferentially by diethyl pyrocarbonate; this hyperreactivity is dependent on the degree of negative superhelicity of the circular DNA molecules. As negative superhelical density increases, guanosines in a 32-base-pair alternating G-C sequence and adenosines (but not guanosines) in a 64-base-pair alternating A-C/G-T sequence become 5- to 10-fold more reactive to diethyl pyrocarbonate. The negative superhelical densities at which enhanced reactivity occurs are similar to those reported for the point at which left-handed helices form within plasmids carrying these DNA sequences. Probing of negatively supercoiled pBR322 with diethyl pyrocarbonate reveals a hyperreactive region 31 base pairs in length of which only 9 base pairs are a perfect alternating purine and pyrimidine sequence; the reactivity of purines within this sequence indicates that purines in the anti conformation, or guanosines in the syn conformation with neighboring 3' thymidines, are not hyperreactive in the Z-DNA form.
Regulation of ribulose bisphosphate carboxylase activity
in vivo
by a light-modulated inhibitor of catalysis
Authors: Seemann Jeffrey R. , Berry Joseph A. , Freas Suzan M. , Krump Martha A.
Published: May 2006

The activity of ribulose 1,5-bisphosphate carboxylase [RuBPCase; 3-phospho-D-glycerate carboxylyase (dimerizing), EC 4.1.1.39] in leaf extracts of a number of species kept in the dark overnight was found to be very low. This was not the result of a change in the activation state or in the amount of enzyme that could be extracted from “dark” leaves. Rather, in
Phaseolus vulgaris
it was due to an inhibitor of catalysis that occupied the catalytic site of the enzyme. This inhibitor was compartmentalized in the chloroplast and its maximum concentration in both dark leaves and in intact chloroplasts made from such leaves was slightly in excess of the RuBPCase catalytic site concentration. The inhibitor (a phosphate ester) was bound preferentially to the activated form of the enzyme, apparently functioning as a positive effector of activation. Treatment of the enzyme-inhibitor complex
in vitro
with alkaline phosphatase could restore RuBPCase activity.
In vivo
, both the initial rate of disappearance and the final concentration of inhibitor in intact leaves was found to vary with light intensity, and these changes could account for observed light-dependent changes in RuBPCase activity, indicating that light modulation of inhibitor concentration controlled RuBPCase activity. Recovery of activity
in vivo
could be inhibited by 3-(3′,4′,4-dichlorophenyl)-1,1-dimethylurea.
Characterization of toposomes from sea urchin blastula cells: a cell organelle mediating cell adhesion and expressing positional information.
Authors: Noll H , Matranga V , Cervello M , Humphreys T , Kuwasaki B , Adelson D
Published: May 2006

Cell adhesion in the sea urchin blastula is mediated by a 22S genus-specific glycoprotein complex consisting initially of six 160-kDa subunits that are processed proteolytically as development proceeds. Noncytolytic removal of the 22S particle from the surface with either 2.5% butanol or trypsin renders dissociated cells reaggregation incompetent, and addition restores reaggregation and development. Polyclonal antibodies against the 22S complex prevent reaggregation in a genus-specific manner while monoclonal antibodies stain cell surface structures in a pattern consistent with a code that specifies the position of a cell in the embryo by a unique combination of subunits in its cell adhesion particles. The existence of similar particles in Drosophila and amphibian embryos suggests that these glycoprotein complexes are a general class of organelles, the toposomes, that in the embryo mediate cell adhesion and express positional information.
Cellular and viral DNA hypomethylation associated with induction of Epstein-Barr virus lytic cycle.
Authors: Szyf M , Eliasson L , Mann V , Klein G , Razin A
Published: May 2006

Epstein-Barr virus (EBV) producer and nonproducer cell lines have been treated with a combination of phorbol 12-myristate 13-acetate and n-butyrate (sodium salt). These inducers caused a massive hypomethylation of the EBV producer line P3HR-1 DNA (about 30%) at the time when DNA replication was inhibited. The viral DNA in these cells is heavily methylated as judged by digestion with Hpa II and probing with the Bam HI H fragment of EBV. However, upon induction with phorbol 12-myristate 13-acetate and n-butyrate, total hypomethylation of this viral DNA region was observed within 24 hr. This hypomethylation preceded EBV amplification, which became apparent only 32-36 hr after induction. When induction was carried out in the presence of retinoic acid, hypomethylation of cellular and viral DNA, viral DNA amplification, and production of the viral early antigen and viral capsid antigen were substantially inhibited. EBV DNA in another producer line (Jijoye nude) and in the nonproducer line Raji was hypomethylated and did not undergo further hypomethylation in response to induction. The observed hypomethylation of P3HR-1 and EBV DNA in the absence of DNA replication suggests that it is achieved by an active demethylation mechanism. This changes our perception of the DNA methylation phenomenon, since it has been generally accepted that hypomethylation of DNA takes place by a passive mechanism that involves DNA replication in the absence of methylation.
DNA methylation at asymmetric sites is associated with numerous transition mutations.
Authors: Selker E U , Stevens J N
Published: May 2006

We describe two unusual 5S RNA regions from Neurospora crassa that are tightly linked. Sequence analysis suggests that these genes or pseudogenes, which we designate zeta (zeta) and eta (eta), arose by a 794-base-pair tandem duplication followed by hundreds of exclusively cytosine to thymine mutations. The duplication was most likely generated by nonhomologous recombination involving a DNA segment having a striking purine-pyrimidine strand asymmetry. Restriction analysis of genomic DNA from tissue grown in the presence or absence of 5-azacytidine indicates that many, and perhaps all, cytosines in the duplicated region are methylated in most cells. This is in contrast to the situation typically observed in eukaryotes, where 5-methylcytosine is found only at positions one or two nucleotides preceding guanine residues. No DNA methylation was detected in the unique DNA flanking the zeta-eta duplication. Thus the "signal" for methylation may be the duplication itself. The numerous transition mutations in this region probably occurred by deamination of 5-methylcytosines. Our results suggest that DNA methylation can have important evolutionary consequences in eukaryotes.
Mutations that impair a posttranscriptional step in expression of HLA-A and -B antigens.
Authors: DeMars R , Rudersdorf R , Chang C , Petersen J , Strandtmann J , Korn N , Sidwell B , Orr H T
Published: May 2006

Mutations can interfere with posttranscriptional expression of the HLA-A and -B genes. B-lymphoblastoid cells that contain one copy of the major histocompatibility complex (MHC) were subjected to mutagenesis and immunoselection for MHC antigen-loss mutants. Some mutations partially reduced surface expression of HLA-A and eliminated HLA-B expression concurrently, although the HLA-A and -B genes were present and transcribed. Antigen expression was fully restored in hybrids of these mutants with other B-lymphoblastoid cells. Therefore, normal cell surface expression of the HLA-A and -B antigens on B lymphoblasts requires (i) execution of at least one trans-active step in the production of the antigens after transcription of the HLA-A and -B genes or (ii) association of the class I antigens with other molecules. DNA analysis of one mutant suggests the possibility that a locus required for the normal expression of the HLA-A and -B antigens is located between the MHC complement genes and the HLA-DP alpha II locus.
Expression of rat apolipoprotein A-IV and A-I genes: mRNA induction during development and in response to glucocorticoids and insulin.
Authors: Elshourbagy N A , Boguski M S , Liao W S , Jefferson L S , Gordon J I , Taylor J M
Published: May 2006

Rat apolipoproteins (apo-) A-IV and A-I share many structural similarities, the most notable of which is a domain of repeated docosapeptides with amphipathic helical potential. Although the genes for apo-A-IV and apo-A-I probably diverged from a common ancestor, these proteins seem to have developed different functions in their evolution. In the present study, cloned cDNAs were used to characterize the expression of apo-A-IV and apo-A-I mRNAs in a wide variety of adult rat tissues, as well as in small intestine and liver obtained from fetal, suckling, and weanling animals; comparisons were made to the expression of apo-E mRNA. The apo-A-IV and apo-A-I mRNAs were most abundant in adult small intestine and liver, with trace amounts detected in other tissues. Substantial amounts of these mRNAs were detected in the yolk sac, suggesting that this fetal tissue plays an important role in lipid metabolism during gestation. Noncoordinate accumulation of apo-A-IV and apo-A-I mRNAs was observed within and between the liver and small intestine during neonatal development. The apo-A-IV mRNA levels in the developing small intestine and liver appeared to correlate with their triglyceride secretion rates, suggesting that this protein plays an important role in the metabolism of triglyceride-rich lipoproteins. When dexamethasone (0.1 microM), insulin (0.01 microM), or insulin and dexamethasone together were incubated with primary cultures of nonproliferating adult rat hepatocytes, apo-A-IV mRNA levels were 4-, 7-, and 11-fold higher, respectively, than in non-hormone-treated control hepatocytes. Hormone administration resulted in a 2-fold greater amount of apo-A-I mRNA in each case, with no significant change in the level of apo-E mRNA. The overall results suggest that these structurally related apolipoproteins are regulated in substantially different ways.
Depolarization regulates adrenal preproenkephalin mRNA.
Authors: LaGamma E F , White J D , Adler J E , Krause J E , McKelvy J F , Black I B
Published: May 2006

The regulation of neuropeptide gene expression has been investigated by using rat adrenal medullae grown in explant culture. After 3 days in culture the (now denervated) explants exhibited a 10-fold increase in leucine-enkephalin (leu-EK) content. Inhibition of protein synthesis with cycloheximide completely blocked the rise, whereas inhibition of RNA synthesis with actinomycin D or alpha-amanitin inhibited the increase by 50%. Inhibition of DNA synthesis with 1-beta-D-arabinofuranosylcytosine (cytosine arabinoside) had no discernible effect. To determine whether the rise in leu-EK was associated with an increase in specific mRNA coding for the opiate peptide precursor, blot hybridization analysis was performed. A single species of preproenkephalin mRNA was detected after various culture periods. The amount of mRNA increased 34-fold after 2 days in culture and 74-fold after 4 days. Consequently, the rise in mRNA levels preceded the increase in the amount of leu-EK. Depolarization of the adrenal medullae with either elevated potassium or veratridine, which prevents leu-EK accumulation, inhibited the increase in the amount of preproenkephalin mRNA. Moreover, the effect of veratridine was blocked by tetrodotoxin, suggesting that transmembrane sodium ion influx affects the increase in the amount of message. Our studies suggest that elevation of leu-EK in explanted (denervated) medullae is associated with increased amounts of mRNA coding for the peptide precursor and that these processes can be regulated by depolarization.
Semisynthesis of horse heart cytochrome c analogues from two or three fragments.
Authors: ten Kortenaar P B , Adams P J , Tesser G I
Published: May 2006

Horse heart cytochrome c was cleaved with cyanogen bromide. The largest fragment, [Hse65]cytochrome c-(1-65)-pentahexacontapeptide lactone, was used in condensations involving four analogues of the complementary cytochrome c-(66-104)-nonatriacontapeptide. Two of the latter compounds were obtained from a semisynthesis starting with a partially protected fragment N epsilon 86-88,99,100-penta(methylsulfonylethyloxycarbonyl)cytochrome c-(81-104)-tetracosapeptide (also arising from a cyanogen bromide-mediated degradation) and analogues of the middle part, cytochrome c-(66-80)-pentadecapeptide, which were prepared by organochemical synthesis. Two other analogues of the cytochrome c-(66-104)-nonatriacontapeptide were prepared entirely by organochemical synthesis. Each of the covalently recombined analogous cytochromes c could retain an electron in the presence of oxygen and transfer it to cytochrome c oxidase, although with different reaction rates and Michaelis constants. Their redox potentials varied over a broad range. The exchanges Tyr67----Phe(F) and Thr78----Val gave rise to analogues with a lower redox potential than native cytochrome c, while the exchange Phe82----Leu or Tyr97----Leu led to analogues with the same and a higher redox potential, respectively.
Femtomole analysis of prostaglandin pharmaceuticals.
Authors: McGuffin V L , Zare R N
Published: May 2006

An analytical method is described whereby the major classes of prostaglandins are fully resolved by microcolumn liquid chromatography and detected at the subfemtomole level by laser-induced fluorescence. The prostaglandins are labeled with the fluorescent reagent 4-bromo-methyl-7-methoxycoumarin and are subsequently separated on a high-efficiency fused-silica microcolumn (0.2 mm i.d., 1.06 m length, 150,000 theoretical plates). The optimal chromatographic conditions consist of a 3-micron octadecylsilica packing material and an isocratic mobile phase of 47.6% methanol, 23.8% acetonitrile, and 28.6% water. The prostaglandin derivatives are detected directly on the microcolumn by laser fluorimetry, using a helium/cadmium laser (325 nm, 15 mW) as the excitation source together with a simple filter/photo-multiplier optical detection system. In real sample matrices, the prostaglandin PGF2 alpha is readily quantifiable from the detection limit (0.3 fmol) to the formulation strength of the therapeutic agent Lutalyse (Upjohn), spanning more than six orders of magnitude in concentration. The simplicity and general applicability of the present analytical methodology and instrumentation suggest that this technique can be used to attack a wide variety of biomedically important problems with exceptional sensitivity and selectivity.
Specific regions of the intervening sequences of beta-globin RNA are resistant to nuclease in 50S heterogeneous nuclear RNA-protein complexes.
Authors: Patton J R , Chae C B
Published: May 2006

The specific assembly of heterogeneous nuclear RNA-protein complexes (hnRNPs) containing precursor beta-globin RNA was investigated by using the 50S hnRNP released from chicken reticulocyte nuclei by endogenous nuclease. The nuclease-resistant regions were mapped on adult beta-globin intervening sequences (IVS) at the resolution of nucleotides with an RNA mapping method [Patton, J. R. and Chae, C.-B. (1983) J. Biol. Chem. 258, 3991-3995]. We found that there is one 28-nucleotide-long nuclease-resistant region in the first IVS and there are four nuclease-resistant regions in the second IVS. Of particular interest is the presence in 50S hnRNP of a nuclease-resistant region (24-28 nucleotides long) in both IVS immediately upstream from the putative lariat branch site in an RNA splicing intermediate. Our results demonstrate that hnRNPs containing precursor beta-globin RNA are, like those containing mature beta-globin RNA, assembled in a site-specific manner.
Naturally occurring poly(dA-dT) sequences are upstream promoter elements for constitutive transcription in yeast.
Authors: Struhl K
Published: May 2006

pet56, his3, and ded1 are adjacent but unrelated genes located on chromosome XV of the yeast Saccharomyces cerevisiae. his3 and pet56 are transcribed in opposite directions from initiation sites separated by approximately equal to 200 base pairs. Under normal growth conditions, both genes are transcribed at a similar basal level. Deletion analysis of the his3 gene indicates that the upstream promoter element for constitutive expression is defined by a 17-base-pair region that contains 15 thymidine residues in the coding strand. Sequential deletions of the pet56 gene indicate that this same region is required for wild-type transcription levels. Thus, this poly(dA-dT) sequence acts bidirectionally to activate transcription of two unrelated genes. Transcription of the ded1 gene is initiated approximately equal to 300 base pairs downstream from the his3 gene, and it occurs at a 5-fold higher level. This gene contains a 34-base-pair region containing 28 thymidine residues in the coding strand located upstream from the ded1 TATA box. Deletion of this dA-dT stretch significantly reduces transcription below the wild-type level. Thus, for at least three different yeast genes, naturally occurring stretches of poly(dA-dT) serve as upstream promoter elements for constitutive expression. In addition, it appears that longer stretches of poly(dA-dT) are more effective upstream promoter elements. These transcriptional effects may be due to exclusion of nucleosomes from poly(dA-dT) regions.
Monoclonal antibodies to human vitamin D-binding protein.
Authors: Pierce E A , Dame M C , Bouillon R , Van Baelen H , DeLuca H F
Published: May 2006

Monoclonal antibodies to vitamin D-binding protein isolated from human serum have been produced. The antibodies obtained have been shown to be specific for human vitamin D-binding protein by three independent assays. The antibodies recognize human vitamin D-binding protein specifically in an enzyme-linked immunosorbent assay. Human vitamin D-binding protein is detected specifically in both pure and crude samples by a radiometric immunosorbent assay (RISA) and by an immunoprecipitation assay. The anti-human vitamin D-binding protein antibodies cross-react with monkey and pig vitamin D-binding protein, but not with vitamin D-binding protein from rat, mouse, or chicken, as determined by the RISA and immunoprecipitation assays.
Inhibition of gonadotropin-induced granulosa cell differentiation by activation of protein kinase C.
Authors: Shinohara O , Knecht M , Catt K J
Published: May 2006

The induction of granulosa cell differentiation by follicle-stimulating hormone (FSH) is characterized by cellular aggregation, expression of luteinizing hormone (LH) receptors, and biosynthesis of steroidogenic enzymes. These actions of FSH are mediated by activation of adenylate cyclase and cAMP-dependent protein kinase and can be mimicked by choleragen, forskolin, and cAMP analogs. Gonadotropin releasing hormone (GnRH) agonists inhibit these maturation responses in a calcium-dependent manner and promote phosphoinositide turnover. The phorbol ester phorbol 12-myristate 13-acetate (PMA) also prevented FSH-induced cell aggregation and suppressed cAMP formation, LH receptor expression, and progesterone production, with an ID50 of 0.2 nM. In FSH-treated cells, PMA did not reduce the initial increase in cAMP formation during the first 24 hr of culture but prevented its secondary increase from 24 to 48 hr. PMA also inhibited LH receptor induction by cholera toxin, forskolin, and 8-bromo-cAMP, but it did not impair cAMP responses to the former two agents, indicating that the site of action of the phorbol ester is distal to adenylate cyclase. The early stimulation of cAMP-dependent protein kinase activity by FSH was also unaffected by PMA, consistent with its lack of effect on the initial cAMP response to FSH. However, PMA caused a marked decrease in cytosolic protein kinase C activity within 1 min of its addition to the cells. The permeant diacylglycerols, 1-oleoyl-2-acetoyl-sn-glycerol and sn-1,2-dioctanoyl glycerol, also inhibited LH receptor formation, while the nonpermeant diacylglycerol, diolein, was inactive. These results indicate that in situ activation of protein kinase C by PMA or permeant diacylglycerols inhibits cAMP-dependent granulosa cell differentiation, and suggest that the inhibitory actions of GnRH agonists on granulosa cell maturation are also mediated by protein kinase C.
Induction of surface exclusion (entry exclusion) by Streptococcus faecalis sex pheromones: use of monoclonal antibodies to identify an inducible surface antigen involved in the exclusion process.
Authors: Dunny G M , Zimmerman D L , Tortorello M L
Published: May 2006

The Streptococcus faecalis plasmid pCF-10 is representative of a class of plasmids that enables its host cells to respond to sex pheromones produced by other S. faecalis cells. The pheromone response has been previously shown to result in increased conjugal plasmid transfer, cell clumping, and multiple cell-surface antigenic changes. To test for other effects of pheromone induction, cells carrying pCF-10 were used as recipients in matings with an isogenic donor strain carrying a derivative of pCF-10, tagged with a transposon to provide an additional selective marker. Pheromone induction of the "male recipients" decreased their recipient ability by a factor of 10-300 in comparison to uninduced cells or plasmid-free recipients. These results indicate that an entry exclusion (surface exclusion) function, similar to that described in studies of plasmids in Gram-negative bacteria, is induced during the S. faecalis pheromone response process. The exclusion operates only against homologous plasmids. Immunological, biochemical, and genetic experiments using monoclonal antibodies reactive with C130, the predominant protein antigen associated with the pheromone response of cells carrying pCF-10, indicate that this antigen is involved in surface exclusion. The data also support the notion that synthesis of C130 involves a posttranslational modification of a precursor of C130 to a final product of higher molecular weight form.
Isolation and biochemical and functional characterization of perforin 1 from cytolytic T-cell granules.
Authors: Podack E R , Young J D , Cohn Z A
Published: May 2006

The Ca2+-dependent cytolytic activity of isolated T-lymphocyte granules was purified to apparent homogeneity by high-salt extraction, gel filtration, and ion-exchange chromatography. The lytic activity resided in a 72- to 75-kDa protein of cytolytic granules. Incubation of the isolated protein with erythrocytes in the presence of Ca2+ ions resulted in hemolysis and the formation of membrane lesions of 160 A in diameter, corresponding in size and morphology to membrane lesions formed on target cells by cloned, intact natural killer (NK) and cytolytic T lymphocytes. Hence, the 75-kDa granule protein is identified as monomeric perforin 1 (P1), postulated previously from the analysis of membrane lesions formed during NK and T-cell-mediated cytolysis. P1-mediated hemolysis is Ca2+-dependent and is inhibited by Zn2+ ions. Lysis is accompanied by the polymerization of P1 to membrane-associated tubular complexes (poly-P1) that form large transmembrane pores. P1 causes a rapid membrane depolarization of J774 cells in the presence of Ca2+. Purified P1 also induces transmembrane monovalent and divalent ion flow across lipid vesicles only in the presence of Ca2+. Whole-cell patch-clamp recordings of S49 lymphoma cells show a P1-dependent inward membrane current flow in the presence but not in the absence of Ca2+. The current increase can be dissected as a summation of discrete current events, indicative of formation of functional channels by polymerization of P1.
A pertussis toxin-sensitive GTP-binding protein in the human neutrophil regulates multiple receptors, calcium mobilization, and lectin-induced capping.
Authors: Lad P M , Olson C V , Grewal I S , Scott S J
Published: May 2006

Human neutrophils treated with pertussis toxin had decreased functional responses to several agents including zymosan-treated serum, heat-aggregated immunoglobulin, platelet-activating factor, and fMet-Leu-Phe. Responses affected include superoxide generation and release of lysozyme. The degree and type of inhibition was dependent on the individual receptor and the cellular response studied. Measurement of intracellular calcium levels with quin-2 showed that both fMet-Leu-Phe- and platelet-activating factor-mediated increases in quin-2 fluorescence were diminished as a result of pertussis toxin treatment. fMet-Leu-Phe-mediated calcium uptake was also inhibited. However, under conditions where fMet-Leu-Phe-mediated effects on cell function were completely abolished, only a partial inhibition of 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8) sensitive calcium uptake was observed. A study of the linked reactions of chemotaxis, capping, and shape change revealed that chemotaxis was inhibited regardless of the chemoattractant utilized (zymosan-treated serum, fMet-Leu-Phe, and platelet-activating factor) and the associated reactions of Con A capping and fMet-Leu-Phe- or Con A-mediated shape change were reduced in pertussis toxin-treated cells. Our results suggest that multiple mediators of inflammation act through a pertussis toxin-sensitive GTP-binding protein that regulates the mobilization of internal calcium as well as calcium uptake and is, in addition, a key control element of shape change, capping, and chemotaxis.
Role of an RNA cleavage/poly(A) addition site in the production of membrane-bound and secreted IgM mRNA.
Authors: Danner D , Leder P
Published: May 2006

The switch from membrane-bound to secreted IgM is accomplished by producing alternative forms of mRNA from a single mu heavy-chain gene. This process might be controlled at any of three steps--transcription termination, RNA splicing, or RNA cleavage/poly(A) addition. To distinguish between these possibilities, we have constructed a model human mu gene and observed its expression in early- and late-stage murine B cells. In each case, expression of the model gene reflects the state of development of the host cell; i.e., more of the mRNA for membrane-bound IgM is made in early B cells and more of the secreted form is made in late B cells. Using systematic deletions and analyses of RNA products of the model gene, we implicate RNA cleavage/poly(A) addition as the governing reaction. Removal of the cleavage/poly(A) signal for secreted mu mRNA by a series of BAL-31 deletions produces not only a decrease in secreted mu mRNA but also a compensatory increase in the membrane-bound form. Further, transcripts that do not terminate to the 5' side of the membrane anchor exons are found in cells producing only secreted IgM. As defined by these deletions, we establish that the RNA cleavage signal spans at least 35 bases and speculate that it forms an RNA stemloop that may be important in 3' end formation.
Plasmid pT181 replication is regulated by two countertranscripts.
Authors: Kumar C C , Novick R P
Published: May 2006

A transcription map of the replication control region of the Staphylococcus aureus plasmid pT181 has been constructed. Two major leftward transcripts, RNA III and RNA IV, start at positions 339 and 413, respectively. These two RNAs can serve as mRNAs for a plasmid-specific replication protein RepC. Two short rightward transcripts, RNA I and RNA II, approximately 85 and 150 nucleotides long, respectively, start at position 246. These rightward transcripts (referred to as countertranscripts) do not appear to be translated but act directly as negative regulators of plasmid replication, probably by interfering with translation of the RepC mRNAs. There is no significant base sequence homology among the countertranscripts of pT181, ColE1, and R1/NR1/R6-5, suggesting that the structural parallelism has risen by convergent molecular evolution.
Identification of the components necessary for adenovirus translational control and their utilization in cDNA expression vectors.
Authors: Kaufman R J
Published: May 2006

A transient expression system was used to study the role of the adenovirus late and simian virus 40 (SV40) early mRNA leader sequences and adenovirus virus-associated (VA) RNAs in mRNA translation. Hybrid transcription units containing the adenovirus late and SV40 early promoters fused to various coding regions were introduced into monkey COS cells on plasmids containing a SV40 origin of replication. The translational efficiencies of the mRNAs produced from these plasmids were determined after alterations in the viral leader sequences or in the presence of VA RNAs provided by adenovirus infection of the transfected cells or by cotransfection with plasmids containing the VA genes. Efficient translation of mRNA with either adenovirus or SV40 leader sequences is dependent upon the presence of VA RNA. Translational stimulation by VA RNA of mRNAs containing the adenovirus tripartite leader sequences is dramatically reduced if leader exons 2 and 3 are removed or if their orientation is altered. Sequence analysis has indicated a homology between the nontranslated 5' end of SV40 early mRNA and sequences at the border of the 2nd and 3rd tripartite leader exons, which may be responsible for the increased translation of these mRNAs in the presence of VA RNA.
Adult and fetal human globin genes are expressed following chromosomal transfer into MEL cells.
Authors: Papayannopoulou T H , Lindsley D , Kurachi S , Lewison K , Hemenway T , Melis M , Anagnou N P , Najfeld V
Published: May 2006

Somatic cell hybridization of mouse erythroleukemia (MEL) cells and HEL cells, a human erythroleukemia line that produces fetal (gamma) but fails to express adult (beta) globin, was used to test whether the expression of the two human globin genes is regulated cis or trans. An experimental approach using anti-human globin monoclonal antibodies for detection, efficient cloning, and monitoring of hybrids of interest was employed. Further characterization of hybrids used isoelectric focusing for detection of human globins and S1 nuclease mapping. In contrast to the parental HEL line, all chromosome 11-retaining HEL-MEL hybrids expressed human beta-globin, suggesting that the HEL beta-globin genes (i) are transcriptionally competent, (ii) become activated in response to a positive trans-acting element within the MEL environment, and (iii) fail to express into the HEL environment because of either the absence of a positive trans-acting element or the presence of a trans-acting inhibitor of beta-globin gene expression. In addition to beta-globin, the primary HEL-MEL hybrids co-expressed gamma-globin; however, gamma-globin expression segregated by subcloning so that secondary and tertiary clones either expressed only beta-globin or co-expressed gamma- and beta-globin. The results of subcloning can be explained by assuming that gamma-globin gene expression is controlled by a HEL cell-derived transacting element encoded by a gene not syntenic to chromosome 11 or by postulating that the HEL gamma-globin genes become randomly modified during the continuous proliferation of hybrids.