bluuebunny/crossref_metadata_2025 huggingface dataset:
Phosphorylation/dephosphorylation of the beta-adrenergic receptor regulates its functional coupling to adenylate cyclase and subcellular distribution.
Authors: Sibley D R , Strasser R H , Benovic J L , Daniel K , Lefkowitz R J
Published: May 2006

Prolonged exposure of cells or tissues to drugs or hormones such as catecholamines leads to a state of refractoriness to further stimulation by that agent, known as homologous desensitization. In the case of the beta-adrenergic receptor coupled to adenylate cyclase, this process has been shown to be intimately associated with the sequestration of the receptors from the cell surface through a cAMP-independent process. Recently, we have shown that homologous desensitization in the frog erythrocyte model system is also associated with increased phosphorylation of the beta-adrenergic receptor. We now provide evidence that the phosphorylation state of the beta-adrenergic receptor regulates its functional coupling to adenylate cyclase, subcellular translocation, and recycling to the cell surface during the process of agonist-induced homologous desensitization. Moreover, we show that the receptor phosphorylation is reversed by a phosphatase specifically associated with the sequestered subcellular compartment. At 23 degrees C, the time courses of beta-adrenergic receptor phosphorylation, sequestration, and adenylate cyclase desensitization are identical, occurring without a lag, exhibiting a t1/2 of 30 min, and reaching a maximum at approximately 3 hr. Upon cell lysis, the sequestered beta-adrenergic receptors can be partially recovered in a light membrane vesicle fraction that is separable from the plasma membranes by differential centrifugation. The increased beta-adrenergic receptor phosphorylation is apparently reversed in the sequestered vesicle fraction as the sequestered receptors exhibit a phosphate/receptor stoichiometry that is similar to that observed under basal conditions. High levels of a beta-adrenergic receptor phosphatase activity appear to be associated with the sequestered vesicle membranes. The functional activity of the phosphorylated beta-adrenergic receptor was examined by reconstituting purified receptor with its biochemical effector the guanine nucleotide regulatory protein (Ns) in phospholipid vesicles and assessing the receptor-stimulated GTPase activity of Ns. Compared to controls, phosphorylated beta-adrenergic receptors, purified from desensitized cells, were less efficacious in activating the Ns GTPase activity. These results suggest that phosphorylation of the beta-adrenergic receptor leads to its functional uncoupling and physical translocation away from the cell surface into a sequestered membrane domain. In the sequestered compartment, the phosphorylation is reversed thus enabling the receptor to recycle back to the cell surface and recouple with adenylate cyclase.
Alternative splicing generates two distinct Eip28/29 gene transcripts in Drosophila Kc cells.
Authors: Schulz R A , Cherbas L , Cherbas P
Published: May 2006

The Drosophila Eip28/29 gene encodes two primary translation products, ecdysone-inducible polypeptide (EIP) 28III and EIP 29III. When cells of the Kc cell line are treated with the steroid hormone ecdysone, the number of Eip28/29 transcripts and the synthesis of the various forms of EIP 28 and 29 increase rapidly. We have reported the sequence of the Eip28/29 gene and of its major transcript. Here we describe a minor or short-form transcript that is about 25% of the total Eip28/29 gene transcripts in both untreated and hormone-treated cells. This transcript is formed by the use of an alternative splice donor sequence 12 nucleotides upstream from the major donor site at the end of the second exon. Evidently the relative abundance of the two products is not hormonally regulated. The short form translation product should lack only an internal dibasic tetrapeptide. The long and short forms probably represent distinct mRNAs for EIP 28III and EIP 29III, respectively.
Actin-severing activity copurifies with phosphofructokinase.
Authors: Füchtbauer A , Jockusch B M , Leberer E , Pette D
Published: May 2006

Microinjection of muscle 6-phosphofructokinase (PFK; EC 2.7.1.11) into tissue culture cells led to a reversible disintegration of microfilament bundles (stress fibers). The mode of disruption as well as of recovery of stress fibers was very similar to that found previously in experiments performed with the actin-severing protein brevin, an extracellular variant of gelsolin. PFK, like brevin, was also capable of disrupting stress fibers in detergent-extracted cells and in ethanol-fixed cells, in a Ca2+-dependent manner. When compared with heart muscle gelsolin, PFK comigrated with the 85- to 90-kDa band. Antibodies against PFK crossreacted with gelsolin from the same species. These results point to a tight association between polypeptides with similar biochemical and immunological parameters present in both preparations. They suggest hitherto unexpected cellular control mechanisms for both microfilament functions and glycolysis.
Expression of human HSP70 during the synthetic phase of the cell cycle.
Authors: Milarski K L , Morimoto R I
Published: May 2006

Expression of the major heat shock and stress-induced protein, HSP70, is under complex regulatory control in human cells. In addition to being induced by physiological stress such as heat shock or transition metals, the HSP70 gene is induced by serum stimulation and immortalizing products of the adenovirus E1A 13S and polyoma large tumor antigen genes. Here we show that expression of the human HSP70 gene is tightly regulated during the cell cycle. Using selective mitotic detachment, a noninductive method to obtain synchronous populations of HeLa cells, we show that levels of HSP70 mRNA rapidly increase 10- to 15-fold upon entry into S phase and decline by late S and G2. A transient increase in HSP70 synthesis is detected during early S phase. The subcellular localization of HSP70 varies throughout the cell cycle; the protein is diffusely distributed in the nucleus and cytoplasm in G1, localized in the nucleus in S, and again diffusely distributed in G2 cells. We suggest that the temporal pattern of HSP70 expression during S phase, the nuclear localization, and activation by trans-acting immortalizing proteins indicate a role for HSP70 in the nucleus of replicating cells.
Total synthesis of a gene for bovine rhodopsin.
Authors: Ferretti L , Karnik S S , Khorana H G , Nassal M , Oprian D D
Published: May 2006

To carry out systematic structure-function studies of bovine rhodopsin by specific amino acid replacements, we have accomplished the total synthesis of its gene, which is 1057 base pairs long. The synthetic gene contains 28 unique restriction sites that are on the average 60 base pairs apart. Replacement of specific restriction fragments by synthetic counterparts containing the desired nucleotide changes permits specific mutagenesis in all parts of the gene. The synthesis of the gene involved enzymatic joining of a total of 72 synthetic oligonucleotides, 15-40 nucleotides long, to form DNA duplexes. The total gene was assembled from three synthetic fragments that were cloned. All synthetic oligonucleotides were characterized by 5'-end analysis, and the accuracy of the joining reactions was confirmed by sequencing the three fragments as well as the complete gene.
RecA protein filaments can juxtapose DNA ends: an activity that may reflect a function in DNA repair.
Authors: Register J C , Griffith J
Published: May 2006

To further characterize the role of RecA protein-DNA filaments in general recombination and DNA repair, we have examined interactions of these filaments with themselves following formation. When linear double-stranded DNA was incubated with RecA in the presence of Mg2+ and adenosine 5'-[gamma-thio]triphosphate, monomer-length (1n) nucleoprotein filaments were observed. Following continued incubation, filaments having 2n, 3n, ... lengths were observed, indicating that an end-to-end joining of the monomer-length filaments had occurred. When linear single-stranded DNA was covered by RecA protein under several conditions, the ends of the resulting filaments joined together rapidly, producing circular filaments. The end-to-end joining of single-stranded DNA-RecA filaments appeared to require that 3' DNA ends be juxtaposed with 5' DNA ends, because double-stranded DNA molecules having long single-stranded DNA tails with only 3' or 5' termini did not join end-to-end. However, when both 5' and 3' ends were present in the reaction, joining was observed. We suggest that this end-to-end joining activity may help explain the role of RecA protein in both the protection of damaged DNA ends and the repair of double-stranded DNA breaks.
The nature of enzyme catalysis in trypsin.
Authors: Weiner S J , Seibel G L , Kollman P A
Published: May 2006

We present a combined quantum/molecular mechanical study of the trypsin-catalyzed hydrolysis of a specific tripeptide substrate, including the entire enzyme in the calculation, as well as 200 H2O molecules. The results illustrate how the enzyme and nearby H2O molecules stabilize the ionic intermediates in peptide hydrolysis, such that the reaction is calculated to have a barrier that is significantly smaller than the calculated and experimental base-catalyzed barrier of formamide hydrolysis in aqueous solution. This enables us to understand how serine proteases increase the rates for reactions that take place in their active sites, compared to the corresponding rates for analogous solution reactions.
Effects of the external pH on Ca channels: experimental studies and theoretical considerations using a two-site, two-ion model.
Authors: Iijima T , Ciani S , Hagiwara S
Published: May 2006

Some effects of the external pH on Ca channels were studied in a hybridoma cell line (mAb-7B), by using the whole-cell configuration of the patch-clamp technique. As the pH was lowered, both the activation and the inactivation curves shifted toward less negative membrane potentials, suggesting a pH-induced decrease of an external negative surface potential, sensed by the mechanism of gating. The potential for half-activation, V1/2, and that for half-inactivation, Vh, were related by a straight line with a slope of one. The inward current varied exponentially with V1/2, as would be expected if the field inside the channel and the Ca2+ concentration at the entrance were sensitive to the surface potential. However, the reversal potential and the outward current were unaltered by changes in the pH. Under the hypothesis that the channel senses the surface potential, all these results, as well as the nernstian behavior of the reversal potential with respect to Ca2+, observed in previous studies, are accounted for by a three-barrier, two-ion model for a channel, provided it is assumed that the potential in the channel drops almost entirely across the barrier adjacent to the external solution.
Secondary, tertiary, and quaternary structure of T-cell-specific immunoglobulin-like polypeptide chains.
Authors: Novotný J , Tonegawa S , Saito H , Kranz D M , Eisen H N
Published: May 2006

To explore the possibility that the difference in antigen recognition between B and T cells derives from a structural difference in their respective antigen-specific receptors (immunoglobulins on B cells and immunoglobulin-like molecules on T cells), we compared the extracellular segments of the T-cell receptor alpha, beta, and gamma polypeptide chains and the N-terminal segment of the T-cell T8 (Lyt-2) antigen chain with the corresponding regions of immunoglobulins whose three-dimensional structures are known. The results indicate that the four T-cell polypeptide chains are organized into immunoglobulin-like domains consisting of multistranded antiparallel beta-sheet bilayers. Invariant amino acid side chains that are conserved in diverse immunoglobulins, including those that mediate domain-domain interactions and form a constant scaffold for antibody binding sites, are also conserved in the chains encoded by the T-cell receptor genes and in the N-terminal domain of T8 (Lyt-2). It appears that the binding sites of the antigen-specific T-cell alpha beta-chain receptors and of antibodies are very similar in their overall dimensions and geometry: a T-cell alpha beta receptor molecule probably has an antigen-specific binding site that is fundamentally no different than the conventional binding site of an antibody.
Lineage-related polypeptide markers in acute lymphoblastic leukemia detected by two-dimensional gel electrophoresis.
Authors: Hanash S M , Baier L J , McCurry L , Schwartz S A
Published: May 2006

We have utilized two-dimensional polyacrylamide gel electrophoresis, coupled with ultrasensitive silver staining, to identify lineage-related polypeptide markers in lymphoblasts from children with acute lymphoblastic leukemia. Twelve polypeptides were detected that could distinguish between the major subgroups of acute lymphoblastic leukemia. These included a new marker for common acute lymphoblastic leukemia and markers for cells of B and T lineages. Analysis of the two-dimensional patterns also allowed the tentative identification of T-cell lineage in two cases with an otherwise undifferentiated non-T-cell non-B-cell phenotype. Two-dimensional electrophoresis thus provides a powerful tool for the delineation of the cell of origin in leukemia.
Insulin-like growth factor I/somatomedin C: a potent inducer of oligodendrocyte development.
Authors: McMorris F A , Smith T M , DeSalvo S , Furlanetto R W
Published: May 2006

Cell cultures established from cerebrum of 1-day-old rats were used to investigate hormonal regulation of the development of oligodendrocytes, which synthesize myelin in the central nervous system. The number of oligodendrocytes that developed was preferentially increased by insulin, or by insulin-like growth factor I (IGF-I), also known as somatomedin C. High concentrations (5 micrograms/ml) of insulin were required for substantial induction of oligodendrocyte development, whereas only 3.3 ng of IGF-I per ml was needed for a 2-fold increase in oligodendrocyte numbers. At an IGF-I concentration of 100 ng/ml, oligodendrocyte numbers were increased 6-fold in cultures grown in the presence of 10% fetal bovine serum, or up to 60-fold in cultures maintained in serum-free medium. IGF-I produced less than a 2-fold increase in the number of nonoligodendroglial cells in the same cultures. Type I IGF receptors were identified on oligodendrocytes and on a putative oligodendrocyte precursor cell population identified by using mouse monoclonal antibody A2B5. These results indicate that IGF-I is a potent inducer of oligodendrocyte development and suggest a possible mechanism based on IGF deficiency for the hypomyelination that results from early postnatal malnutrition.
A monoclonal antibody that cross-reacts with phosphorylated epitopes on two microtubule-associated proteins and two neurofilament polypeptides.
Authors: Luca F C , Bloom G S , Vallee R B
Published: May 2006

A monoclonal antibody is described that was raised against bovine brain microtubule-associated protein (MAP) 1B. Immunoblot analysis revealed that immunoreactivity was abolished by dephosphorylation of the antigen. The antigen/antibody reaction was also directly inhibited by sodium phosphate. In whole brain tissue, MAP 1B was the primary immunoreactive species. However, the antibody was also found to react with MAP 1A as well as with the high and middle molecular weight neurofilament polypeptides. No cross-reaction with MAP 2, which is known to be extensively phosphorylated, other MAPs, or the low molecular weight neurofilament polypeptide was observed. This evidence suggests at least some sequence homology between these different polypeptide components of the neuronal cytoskeleton and points to a common mechanism for their phosphorylation.
Tumor necrosis factor enhances eosinophil toxicity to Schistosoma mansoni larvae.
Authors: Silberstein D S , David J R
Published: May 2006

Tumor necrosis factor (TNF) is a monocyte product that kills or inhibits the growth of certain tumor cells. Its cell source and physical characteristics suggest that it is similar to a monokine, eosinophil-cytotoxicity-enhancing factor (M-ECEF), that activates human eosinophil toxicity to Schistosoma mansoni larvae. The availability of recombinant human TNF allowed us to test this possibility. The data show that TNF has no direct effect on the parasites but enhances eosinophil toxicity to the parasites in a dose-dependent fashion. This effect is specific for eosinophils and not neutrophils. TNF and the eosinophil-specific activity of TNF are coeluted with M-ECEF in reversed-phase HPLC. Further, M-ECEF activity in HPLC fractions is reduced by treatment with rabbit anti-TNF antibody and protein A-Sepharose. This demonstrates physical similarity and at least partial immunological identity of TNF and a fraction of M-ECEF. Thus, TNF can enhance eosinophil function and may constitute an important immunological regulatory mechanism. This effect should be considered when TNF is applied clinically.
Similarity between the interleukin 1 receptors on a murine T-lymphoma cell line and on a murine fibroblast cell line.
Authors: Dower S K , Call S M , Gillis S , Urdal D L
Published: May 2006

Interleukin 1 beta (IL-1 beta), one of two different polypeptide hormones with interleukin 1 (IL-1) biological activity, produced by activated human monocytes, is a 17.5-kDa protein. IL-1 beta binds specifically to a variety of cells; the cellular distribution of binding is consistent with reported biological responsiveness. In this report we show that two unrelated, but IL-1-responsive, cell lines, LBRM-33-1A5, a T-lymphoma line, and BALB/3T3, a fibroblast line, bind 125I-labeled IL-1 beta via similar plasma membrane receptor molecules. The T-lymphoma cells possess 238 +/- 16 plasma membrane receptors per cell and bind 125I-labeled IL-1 beta with an affinity of 3.6 +/- 0.9 X 10(9) M-1. The IL-1 receptor has a molecular size of approximately equal to 79.5 kDa, as estimated by affinity cross-linking. The fibroblasts possess 4.8 +/- 0.5 X 10(3) IL-1 receptors per cell and bind 125I-labeled IL-1 beta with an affinity of 2.6 +/- 0.5 X 10(9) M-1. The molecular size of the receptor molecule on the fibroblasts is approximately equal to 78 kDa. Despite the similarity in the characteristics of the ligand-receptor system on the two different cell types, the biological responses of the two cell types to IL-1 beta occur at IL-1 beta concentrations that differ by four orders of magnitude.
Magnesium sulfate: rationale for its use in preeclampsia.
Authors: Watson K V , Moldow C F , Ogburn P L , Jacob H S
Published: May 2006

Preeclampsia is a disorder of pregnancy characterized clinically by hypertension, proteinuria, and edema and characterized pathologically in its late stages by widespread microvascular thrombi. There is evidence from a number of studies that production of prostacyclin (prostaglandin I2, PGI2), a potent vasodilator and inhibitor of platelet aggregation, is deficient in preeclamptic compared to normal pregnancy. Traditional therapy utilizes infusions of large amounts of MgSO4, but the physiologic basis for this is not clear. We studied the effect of MgSO4 on PGI2 release by cultured human umbilical vein endothelial cells (HUVEC) by several methods. By platelet aggregometry, the known antiaggregatory effect of intact HUVEC was enhanced by MgSO4. By radioimmunoassay for 6-keto-PGF1 alpha, the stable metabolite of PGI2, it was shown that MgSO4 amplifies release of PGI2 by HUVEC in a dose-dependent manner, with a peak occurring between 2 and 3 mM. In separate experiments, MgSO4 overcame the enhanced adherence of platelets to HUVEC exhausted by repeated exposure to thrombin. Finally, PGI2 production was 2- to 5-fold greater by HUVEC incubated with plasma obtained from preeclamptic patients undergoing MgSO4 therapy than by HUVEC incubated with pretherapy plasma. We conclude that MgSO4 mediates enhanced production of PGI2 by vascular endothelium, thereby potentially enhancing its thromboresistant properties.
Decreased content of integral membrane calcium-binding protein (IMCAL) in tissues of the spontaneously hypertensive rat.
Authors: Kowarski S , Cowen L A , Schachter D
Published: May 2006

Prior studies report that plasma membranes of the spontaneously hypertensive rat (Okamoto-Aoki strain) bind less calcium than do the corresponding preparations from Wistar Kyoto controls. The possibility that the differences result from a decrease in the content of integral membrane calcium-binding protein (IMCAL) was explored by the application of immunoassays with polyclonal antisera and a mouse monoclonal antibody. IMCAL binds calcium with relatively high affinity, and its content in many rat tissues is regulated by vitamin D and the level of dietary calcium. Immunoassays of tissue IMCAL demonstrate significant reductions in content in the erythrocyte ghost, intestinal mucosa, kidney, heart, testis, and liver of the spontaneously hypertensive rat as compared to the control strains. The decreases are observed both at 4-5 weeks of age, before the onset of severe hypertension and at 8-9 weeks in the presence of severe hypertension. Moreover, the magnitude of the decrease in erythrocyte IMCAL can account for much of the decrease in the calcium-binding capacity of erythrocyte membranes reported by others. The results are especially significant because an abnormality in the membrane binding of calcium and in the regulation of cytosolic calcium ion concentration could underlie the pathogenesis of the hypertension.
Nerve injury stimulates the secretion of apolipoprotein E by nonneuronal cells.
Authors: Snipes G J , McGuire C B , Norden J J , Freeman J A
Published: May 2006

Nerve trauma initiates significant changes in the composition of proteins secreted by nonneuronal cells. The most prominent of these proteins is a 37-kDa protein, whose expression correlates with the time course of nerve development, degeneration, and regeneration. We now report that the 37-kDa protein is apolipoprotein E (apoE). We produced a specific antiserum against the 37-kDa protein isolated from previously crushed nerves. This antiserum recognizes a 36-kDa protein in rat serum that we have purified and identified as apoE. The anti-37-kDa antiserum also recognizes apoE on electrophoretic transfer blots of authentic samples of high and very low density lipoproteins. The nerve 37-kDa protein comigrates with apoE by two-dimensional electrophoresis, shares a similar amino acid composition, and reacts with an antiserum against authentic apoE. The purified apoE specifically blocks the immunoprecipitation of [35S]methionine-labeled 37-kDa protein synthesized by nonneuronal cells. Thus, on the basis of its molecular mass, isoelectric point, amino acid composition, and immunological properties, we conclude that the 37-kDa protein is apoE. We also used light microscopic immunohistochemistry to localize apoE following nerve injury. In rats with optic nerve lesions, the 37-kDa antiserum bound specifically to the degenerating optic tracts and to the retino-recipient layers of the lateral geniculate nucleus and the superior colliculus. We propose that apoE is synthesized by phagocytic cells in response to nerve injury for the purpose of mobilizing lipids produced as a consequence of axon degeneration.
Curved DNA: design, synthesis, and circularization.
Authors: Ulanovsky L , Bodner M , Trifonov E N , Choder M
Published: May 2006

Curved DNA molecules and unusually small circles have been obtained by ligation of synthetic 21-base precursors: (sequence in text). The ligation resulted in the formation of double-stranded oligo-(precursor)s possessing a strong 10.5-base-pair (bp) periodicity of the runs of adenines. Two-dimensional polyacrylamide gel electrophoresis of the ligation products showed two distinct families of spots: (i) noncircular oligo(precursor)s of 21 to 231 bp (1- to 11-mers) and (ii) four circles from 105 to 168 bp (eluted and analyzed by denaturing gel electrophoresis). The noncircular oligomers exhibited anomalously slow migration, as if they were as much as three times longer than they actually are. The amount of circular products peaked sharply at approximately equal to 126 bp, near which size the circles have been estimated to be nonconstrained both torsionally and in terms of bending. The nonconstrained circularization provides a technique for the direct measurement of the inherent curvature of DNA in solution. From the size of the circles, an estimate of 8.7 degrees is obtained for the absolute value of the AA X TT wedge angle (roll and tilt combined).
Evolution in biosynthetic pathways: two enzymes catalyzing consecutive steps in methionine biosynthesis originate from a common ancestor and possess a similar regulatory region.
Authors: Belfaiza J , Parsot C , Martel A , de la Tour C B , Margarita D , Cohen G N , Saint-Girons I
Published: May 2006

The metC gene of Escherichia coli K-12 was cloned and the nucleotide sequence of the metC gene and its flanking regions was determined. The translation initiation codon was identified by sequencing the NH2-terminal part of beta-cystathionase, the MetC gene product. The metC gene (1185 nucleotides) encodes a protein having 395 amino acid residues. The 5' noncoding region was found to contain a "Met box" homologous to sequences suggestive of operator structures upstream from other methionine genes that are controlled by the product of the pleiotropic regulatory metJ gene. The deduced amino acid sequence of beta-cystathionase showed extensive homology with that of the MetB protein (cystathionine gamma-synthase) that catalyzes the preceding step in methionine biosynthesis. The homology strongly suggests that the structural genes for the MetB and MetC proteins evolved from a common ancestral gene.
Role of proline peptide bond isomerization in unfolding and refolding of ribonuclease.
Authors: Schmid F X , Grafl R , Wrba A , Beintema J J
Published: May 2006

The isomerization of the proline peptide bond between tyrosine-92 and proline-93 in bovine pancreatic ribonuclease A has been investigated in the unfolded protein as well as during the slow refolding process. This bond is in the cis state in the native protein. By comparison of various homologous ribonucleases we show that isomerization of proline-93 is associated with a change in fluorescence of tyrosine-92. This provides a spectroscopic probe to monitor this process in the disordered chain after unfolding as well as its reversal in the course of slow refolding. In unfolded ribonuclease incorrect trans isomers of proline-93 are found in both slow-folding species. trans----cis reversal of isomerization of this proline peptide bond during refolding shows kinetics that are identical with the time course of formation of native protein. Isomerization of proline-93 is slower than the formation of a native-like folded intermediate that accumulates on the major slow refolding pathway. Models to explain these results are discussed.
Low density lipoprotein receptor-dependent prostaglandin synthesis in Swiss 3T3 cells stimulated by platelet-derived growth factor.
Authors: Habenicht A J , Dresel H A , Goerig M , Weber J A , Stoehr M , Glomset J A , Ross R , Schettler G
Published: May 2006

We studied the effects of human plasma lipoproteins on the synthesis of prostaglandin (PG) E2 in Swiss 3T3 mouse fibroblasts. Quiescent cells, maintained in medium deficient in both platelet-derived growth factor (PDGF) and lipoproteins, synthesized less than 8 ng of PGE2 per 10(6) cells per 22 hr, and this rate did not change in response to the addition of lipoproteins. In contrast, PDGF-stimulated cells, incubated in medium deficient in lipoproteins, synthesized 45-110 ng of PGE2 per 10(6) cells during the same period of time, and this rate increased 2- to 5-fold in the presence of added low density lipoproteins (LDL). This stimulatory effect of LDL seemed to depend on LDL receptor-mediated binding, uptake, and degradation of the lipoproteins because: both LDL and very low density lipoproteins were active, whereas high density lipoproteins were not; low concentrations of LDL were effective; the effect of native LDL was blocked by acetylation of the LDL; PDGF increased both the expression of LDL receptors and the cellular uptake of LDL; chloroquine blocked the effect of LDL but not that of exogenous arachidonic acid. These results provide evidence that the LDL pathway is critically linked to PG synthesis in PDGF-stimulated cells.
Molecular cloning and DNA sequence of the Arabidopsis thaliana alcohol dehydrogenase gene.
Authors: Chang C , Meyerowitz E M
Published: May 2006

Arabidopsis thaliana provides an excellent experimental plant system for molecular genetics because of its remarkably small genome size, near absence of dispersed middle repetitive DNA, and short life cycle. We have cloned and determined the nucleotide sequence of a single-copy gene from A. thaliana likely to be the gene encoding alcohol dehydrogenase (ADH; alcohol:NAD+ oxidoreductase, EC 1.1.1.1). The gene was isolated from a random recombinant library by cross-hybridization with a maize Adh1 gene probe. The DNA sequence contains an open reading frame capable of encoding a polypeptide the same length as maize ADH1 and ADH2 (379 amino acids) and having approximately equal to 80% homology with both maize enzymes. This open reading frame is interrupted by six introns whose positions are conserved with six of the nine intron positions present in both maize genes. The 5' and 3' untranslated regions are, respectively, 58 and 204 base pairs long. Sequences important for eukaryotic gene expression such as the TATA box, polyadenylylation signal, and intron splicesite sequences are found in the expected locations. The gene hybridizes to a specific anaerobically induced RNA in Arabidopsis whose appearance correlates with the anaerobic induction of Arabidopsis ADH protein.
Dihydropyridine-sensitive Ca2+ channels in mammalian skeletal muscle cells in culture: electrophysiological properties and interactions with Ca2+ channel activator (Bay K8644) and inhibitor (PN 200-110).
Authors: Cognard C , Romey G , Galizzi J P , Fosset M , Lazdunski M
Published: May 2006

The whole-cell patch-clamp technique has been used to analyze the properties of the dihydropyridine-sensitive Ca2+ channel in rat skeletal muscle cells (myoballs) in culture. The potential dependence of Ca2+-channel activation is similar to that observed in cardiac cells. However, the skeletal muscle Ca2+ channel is activated more slowly (by a factor of about 10). The voltage dependence of Ca2+-channel inactivation indicates a half-maximal inactivation (Vh0.5) at -72 mV as compared to Vh0.5 = -35 mV for cardiac cells. Blockade of the skeletal muscle Ca2+ channel by the dihydropyridine (+)-PN 200-110 is voltage dependent, with a half-maximal effect (K0.5) of 13 nM for an application of the drug to the myoball membrane held at -90 mV and of 0.15 nM for an application at a potential of -65 mV. The 100-fold difference in apparent affinity is interpreted as a preferential association of PN 200-110 with the inactivated form of the Ca2+ channel. The K0.5 value found from electrophysiological experiments for the binding to the inactivated state (K0.5 = 0.15 nM) is nearly identical to the equilibrium dissociation constant found from binding experiments with (+)-[3H]PN 200-110 using transverse-tubular membranes (Kd = 0.22 nM). The dihydropyridine activator Bay K8644 acts by increasing Ca2+ current amplitude and by slowing down deactivation.
Autoradiographic comparison of the distribution of the neutral endopeptidase "enkephalinase" and of mu and delta opioid receptors in rat brain.
Authors: Waksman G , Hamel E , Fournié-Zaluski M C , Roques B P
Published: May 2006

The neutral endopeptidase EC 3.4.24.11, also designated enkephalinase, has been visualized by in vitro autoradiography using the tritiated inhibitor [3H]-N-[(2RS)-3-hydroxyaminocarbonyl-2-benzyl-1-oxopropyl] glycine, ([3H]HACBO-Gly). Specific binding of [3H]HACBO-Gly (Kd = 0.4 +/- 0.05 nM) corresponding to 85% of the total binding to brain slices was inhibited by 1 microM thiorphan, a selective inhibitor of enkephalinase, but remained unchanged in the presence of captopril, a selective inhibitor of angiotensin-converting enzyme. Very high levels of [3H]HACBO-Gly binding were found in the choroid plexus and the substantia nigra. High levels were present in the caudate putamen, globus pallidus, nucleus accumbens, olfactory tubercle, and in the substantia gelatinosa of the spinal cord. Moderate densities were found in parts of the amygdala, the periaqueductal gray matter, the interpeduncular nucleus, and the molecular layer of the cerebellum. The distribution of enkephalinase was compared to that of mu and delta opioid receptors, selectively labeled with [3H]Tyr-D-Ala-Gly-MePhe-glycinol and [3H]Tyr-D-Thr-Gly-Phe-Leu-Thr, respectively. In the caudate putamen, [3H]HACBO-Gly binding overlapped the clustered mu sites but appeared more closely related to the diffusely distributed delta sites. High levels of enkephalinase and mu opioid binding sites were present at the level of the periaqueductal gray matter and in the substantia gelatinosa of the spinal cord, regions where only sparse delta opioid receptors could be detected. The association of enkephalinase with delta and mu opioid receptors in these areas is consistent with the observed role of the enzyme in regulating the effects of opioid peptides in striatal dopamine release and analgesia, respectively. Except for the choroid plexus and the cerebellum, the close similarity observed in numerous rat brain areas between the distribution of enkephalinase and that of mu and/or delta opioid binding sites could account for most of the pharmacological effects elicited by enkephalinase inhibitors.
DNA binding site for a factor(s) required to initiate simian virus 40 DNA replication.
Authors: Yamaguchi M , DePamphilis M L
Published: May 2006

Efficient initiation of DNA replication in the absence of nonspecific DNA repair synthesis was obtained by using a modification of the system developed by J.J. Li and T.J. Kelly [(1984) Proc. Natl. Acad. Sci. USA 81, 6973-6977]. Circular double-stranded DNA plasmids replicated in extracts of CV-1 cells only when the plasmids contained the cis-acting origin sequence for simian virus 40 DNA replication (ori) and the extract contained simian virus 40 large tumor antigen. Competition between plasmids containing ori and plasmids carrying deletions in and about ori served to identify a sequence that binds the rate-limiting factor(s) required to initiate DNA replication. The minimum binding site (nucleotides 72-5243) encompassed one-half of the simian virus 40 ori sequence that is required for initiation of replication (ori-core) plus the contiguous sequence on the late gene side of ori-core containing G + C-rich repeats that facilitates initiation (ori-auxiliary). This initiation factor binding site was specific for the simian virus 40 ori region, even though it excluded the high-affinity large tumor antigen DNA binding sites.
ATP inhibits nuclear and mitochondrial type I topoisomerases from human leukemia cells.
Authors: Castora F J , Kelly W G
Published: May 2006

Type I topoisomerases have been purified from nuclei and mitochondria of human acute lymphoblastic leukemia cells. Both of these ATP-independent enzymes are actually found to be inhibited by ATP at physiologically significant concentrations. Other adenine nucleotides showed varying effects: ADP inhibited only at high concentrations; AMP had no effect on either topoisomerase. Both enzymes were also inhibited by dATP. The importance of the adenine ring structure was confirmed by the lack of an inhibitory effect observed with equivalent levels of GTP, UTP, CTP, or their deoxy counterparts. Assays performed in the presence of nonhydrolyzable analogs of ATP suggest that hydrolysis of ATP does not accompany this enzyme inhibition. This was supported by direct determination of the ATPase activity of the purified enzymes. Type I topoisomerase from calf thymus and HeLa cells were also found to be sensitive to ATP. These results suggest that mammalian type I topoisomerases in general may possess a nucleotide-binding site that may be involved in regulation of enzyme activity.
Cytolytic effects of neutrophils: role for a membrane-bound neutral proteinase.
Authors: Pontremoli S , Melloni E , Michetti M , Sacco O , Sparatore B , Salamino F , Damiani G , Horecker B L
Published: May 2006

A neutral serine proteinase, purified 250-fold from the plasma membrane fraction of human neutrophils, differs in its catalytic and molecular properties from the well-known neutral proteinases present in azurophil (primary) granules. Stimulation of neutrophils with low concentrations of phorbol 12-myristate 13-acetate (PMA) results in the release into the medium of the membrane-bound proteinase and the concomitant production of oxygen radicals. These concentrations of PMA also induce full cytolytic activity measured with 51Cr-labeled ox erythrocytes. A role for the neutral serine proteinase in the cytolytic activity of PMA-stimulated neutrophils is supported by the following observations: (i) the lytic activity of the stimulated neutrophils is correlated with the quantity of neutral proteinase present in the membranes; (ii) the extracellular medium from PMA-stimulated neutrophils causes the cytolysis of 51Cr-labeled erythrocytes that have been exposed to nonlytic concentrations of H2O2; (iii) cytolysis of H2O2-treated erythrocytes is also observed with the crude proteinase solubilized from neutrophil membranes or with the purified proteinase from the same source; and (iv) in each case the cytolytic activity is proportional to the proteinase activity present and is prevented by the addition of serine proteinase inhibitors. We conclude that cytolysis of target cells by PMA-activated neutrophils can result from the cooperative effects of oxygen radicals and the membrane-bound neutral serine proteinase. The participation of enzymes from specific (secondary) granules is excluded because, with the low concentrations of PMA employed, very little release of secondary granule constituents is observed.
Assignment of the gene for the beta subunit of thyroid-stimulating hormone to the short arm of human chromosome 1.
Authors: Dracopoli N C , Rettig W J , Whitfield G K , Darlington G J , Spengler B A , Biedler J L , Old L J , Kourides I A
Published: May 2006

The chromosomal locations of the genes for the beta subunit of human thyroid-stimulating hormone (TSH) and the glycoprotein hormone alpha subunit have been determined by restriction enzyme analysis of DNA extracted from rodent-human somatic cell hybrids. Human chorionic gonadotropin (CG) alpha-subunit cDNA and a cloned 0.9-kilobase (kb) fragment of the human TSH beta-subunit gene were used as hybridization probes in the analysis of Southern blots of DNA extracted from rodent-human hybrid clones. Analysis of the segregation of 5- and 10-kb EcoRI fragments hybridizing to CG alpha-subunit cDNA confirmed the previous assignment of this gene to chromosome 6. Analysis of the patterns of segregation of a 2.3-kb EcoRI fragment containing human TSH beta-subunit sequences permitted the assignment of the TSH beta-subunit gene to human chromosome 1. The subregional assignment of TSH beta subunit to chromosome 1p22 was made possible by the additional analysis of a set of hybrids containing partially overlapping segments of this chromosome. Human TSH beta subunit is not syntenic with genes encoding the beta subunits of CG, luteinizing hormone, or follicle-stimulating hormone and is assigned to a conserved linkage group that also contains the structural genes for the beta subunit of nerve growth factor (NGFB) and the proto-oncogene N-ras (NRAS).
Sodium-23 magnetic resonance imaging of the eye and lens.
Authors: Garner W H , Hilal S K , Lee S W , Spector A
Published: May 2006

In order to develop a better understanding of cataract and to evaluate the effectiveness of potential drugs, noninvasive techniques must be devised to detect early metabolic changes. As a prelude to these goals, sodium-23 imaging experiments operating at 29.8 MHz (2.7 teslas) were performed on the bovine eye and lens. A spatially localized transverse relaxation time (T2)-weighted spin-density map of the sodium-23 within the lens is presented, with a resolution better than 250 micron. Due to the presence of short-T2 (3 msec) components within the lens, only the use of the planar-integral projection reconstruction (PPR) imaging scheme allowed sufficiently short echo-times (1 msec) to permit sodium-23 signal detection. These noninvasive imaging results show differences in the apparent sodium concentration within the lens that are consistent with separate, invasive measurements of sodium concentration. Separate analysis (with no spatial localization) at 79.4 MHz (7.2 teslas), using a shift reagent (dysprosium) to distinguish extracellular from intracellular sodium, indicates that approximately 62% of the detected sodium-23 signal is intracellular. These results are consistent with observations based on invasive measurements and further support the existence of the pump-leak system and a sodium gradient within the lens.
Cytochrome
c
2
is not essential for photosynthetic growth of
Rhodopseudomonas capsulata
Authors: Daldal Fevzi , Cheng Shinta , Applebaum Joy , Davidson Edgar , Prince Roger C.
Published: May 2006

The structural gene for cytochrome
c
2
(
cycA
) of the photosynthetic bacterium
Rhodopseudomonas capsulata
has been cloned, and the nucleotide and deduced polypeptide sequences have been determined. Compared with the known amino acid sequence of the purified cytochrome
c
2
, the nucleotide sequence corresponding to the N-terminal part of the
cycA
gene product indicates the presence of a putative 21 amino acid signal sequence. Thus, cytochrome
c
2
may be synthesized as a precursor which is processed during its secretion to the periplasm. Insertion and insertion-deletion mutations were constructed
in vitro
and the chromosomal
cycA
+
allele of a wild-type strain was replaced with these mutations by homologous recombination to yield
c
2
-
mutants of
R. capsulata
. The
c
2
-
mutants are stable, and they can grow by photosynthesis and by respiration. Since cytochrome
c
2
is the primary electron donor to the reaction center during photosynthesis, the ability of these mutants to grow photosynthetically indicates that an alternative way(s) of reducing the oxidized reaction center must exist in
R. capsulata
. One candidate for this role may be the membrane-bound cytochrome
c
1
.
Elongation factor Tu.guanosine 3'-diphosphate 5'-diphosphate complex increases the fidelity of proofreading in protein biosynthesis: mechanism for reducing translational errors introduced by amino acid starvation.
Authors: Dix D B , Thompson R C
Published: May 2006

Complexes of elongation factor Tu (EF-Tu) with guanosine 3'-diphosphate 5'-diphosphate (ppGpp) bind to ribosomes where they slow the incorporation of aminoacyl-tRNAs into protein by inhibiting both the binding of aminoacyl-tRNA.EF-Tu.GTP ternary complexes and the formation of peptide bonds. The latter action increases the time available for aminoacyl-tRNA rejection by the ribosome and, therefore, increases the effectiveness of proofreading. Synthesis of ppGpp and the formation of EF-Tu.ppGpp occur in vivo in response to amino acid starvation. Our finding, therefore, suggests an explanation for the otherwise puzzling observation that amino acid starvation has, at most, a moderate effect on the fidelity of protein synthesis in wild-type Escherichia coli. We suggest that an EF-Tu.ppGpp-induced increase in the effectiveness of proofreading buffers the overall translational fidelity of these cells against amino acid starvation-induced errors in initial selection of aminoacyl-tRNA ternary complexes.
Pertussis toxin or phorbol 12-myristate 13-acetate can distinguish between epidermal growth factor- and angiotensin-stimulated signals in hepatocytes.
Authors: Johnson R M , Connelly P A , Sisk R B , Pobiner B F , Hewlett E L , Garrison J C
Published: May 2006

Epidermal growth factor (EGF) causes rapid increases in free intracellular Ca2+ and stimulates the phosphorylation of 11 cytosolic proteins in hepatocytes. Ten of the 11 cytosolic proteins altered by EGF are identical to those affected by angiotensin II, a hormone that stimulates the breakdown of phosphatidylinositol 4,5-bisphosphate. An increase in the phosphorylation of the other protein, spot c (Mr = 36,000, pI = 5.5), is observed only with EGF. Treatment of intact rats with pertussis toxin to ADP-ribosylate Ni, the inhibitory GTP-binding protein of the adenylate cyclase complex, abolished the effect of EGF on Ca2+ mobilization and on the phosphorylation of the 10 proteins affected in common with angiotensin II. This treatment had minimal effects on the ability of EGF to stimulate the phosphorylation of its unique substrate, spot c. In marked contrast, modification of Ni did not block the ability of angiotensin II to stimulate Ca2+ mobilization or protein phosphorylation. Pretreatment of normal hepatocytes with 4 beta-phorbol 12-myristate 13-acetate blocked all responses to EGF, including the increased phosphorylation of spot c, but had no effect on the responses to angiotensin II. These results imply that Ni or a similar pertussis toxin substrate may mediate the apparent effects of EGF on phosphatidylinositol breakdown and that protein kinase C may regulate a site in the transduction pathway. Angiotensin II appears to use a different signal transduction mechanism to stimulate phosphatidylinositol metabolism in hepatocytes.
Cloning and screening with nanogram amounts of immunopurified mRNAs: cDNA cloning and chromosomal mapping of cystathionine beta-synthase and the beta subunit of propionyl-CoA carboxylase.
Authors: Kraus J P , Williamson C L , Firgaira F A , Yang-Feng T L , Münke M , Francke U , Rosenberg L E
Published: May 2006

We have developed conditions for efficient cDNA cloning of nanogram amounts of purified mRNAs coding for cystathionine beta-synthase [L-serine hydro-lyase (adding homocysteine), EC 4.2.1.22] and for the cytosolic precursors of mitochondrial ornithine transcarbamylase (carbamoylphosphate:L-ornithine carbamoyltransferase, EC 2.1.3.3) and the beta subunit of propionyl-CoA carboxylase [propanoyl-CoA: carbon-dioxide ligase (ADP-forming), EC 6.4.1.3]. The three mRNAs, prepared by sequential immunoselection from the same batch of rat liver polysomes, were pooled (20 ng each), and cDNA was synthesized by using avian reverse transcriptase. The second DNA strand was prepared by "nick-translation repair" of the cDNA . mRNA hybrid with RNase H, polymerase I, and DNA ligase from Escherichia coli. The double-stranded (ds) DNA was tailed with deoxycytidine residues, annealed with Pst I-cut/dG-tailed pBR322, and used to transform E. coli. The library generated by this three-step procedure contained 5000 independent colonies. A 550-base-pair (bp) cDNA clone of the beta subunit of propionyl-CoA carboxylase was detected by hybrid-selected translation; it was then used to screen the library for longer cDNAs. Two hybridizing cDNAs, 1200 and 1000 bp long with a 200-bp overlap, representing together a full-length copy of the coding region and 446 bp of 3' untranslated sequence, were recovered. Each plasmid mapped to the region q13.3----q22 of human chromosome 3. Cystathionine beta-synthase clones were obtained by screening the library with a single-stranded [32P]cDNA prepared directly from the highly purified synthase mRNA by reverse transcriptase. The longest hybridizing cDNA of 1700 bp was used in hybrid-selected translation and detected a polypeptide of 63 kDa, identical in size to rat liver synthase. In situ hybridization of this cDNA to q22 of human chromosome 21 confirmed two previous tentative assignments of the synthase locus to this chromosome.