bluuebunny/crossref_metadata_2025 huggingface dataset:
RNA from the yeast transposable element Ty1 has both ends in the direct repeats, a structure similar to retrovirus RNA.
Authors: Elder R T , Loh E Y , Davis R W
Published: May 2006

The RNA homologous to the yeast transposable element Ty1 is one of the more abundant poly(A)+ RNAs in many strains of the yeast Saccharomyces cerevisiae. The 5' and 3' ends of Ty1 RNA have been determined from analysis of cDNA. The 5' end is 245 bases into the left delta sequence measured from the left side of the Ty1 element. The delta sequence is a direct repeat of about 340 base pairs present at each end of the Ty1 element. The Ty1 transcription includes 93-97 bases of the left delta sequence and continues through the entire internal portion of the element and through about 295 bases of the right delta sequence before reaching the 3' end located 38-46 bases from the right side of the right delta sequence. Because the delta sequences present at each end of a single Ty1 element have identical or very similar DNA sequences, these end points for Ty1 RNA raise several questions about the expression of Ty1 elements. First, what are the initiation and termination signals, because the Ty1 transcript must read through a DNA sequence that is identical to the 3' end at about 50 bases from the 5' end? Second, why is the direction of transcription of the Ty1 element opposite to that of genes that are overexpressed after the insertion of a Ty1 element? Third, because the Ty1 RNA itself has direct repeats of about 45 bases, a structure analogous to retrovirus RNAs, is the Ty1 RNA an intermediate in the transposition of Ty1?
Conformational flexibility of junctions between contiguous B- and Z-DNAs in supercoiled plasmids.
Authors: Singleton C K , Klysik J , Wells R D
Published: May 2006

The junctions between contiguous right- and left-handed helical DNA segments in supercoiled plasmids are shown to exhibit both sequence- and superhelical-dependent conformational flexibility. The nonalternating (purine-pyrimidine) tetranucleotide d(G-A-T-C), which is imbedded within a (dC-dG) tract and exists as a junction at low-density values, may be adopting a left-handed helix under the influence of higher negative supercoiling. The effect of supercoiling on the transition from a right- to a left-handed state for (dC-dG)n inserts, where n = 16, 13, and 5, also was evaluated. Only moderate amounts of superhelicity (-0.03 to -0.075) are required for formation and stabilization of left-handed DNA even for the (dC-dG) region of 10 dC X dG base pairs. Finally, we demonstrate that junction formation within a BamHI restriction site causes a drastic inhibition (greater than 80%) of cleavage by this restriction endonuclease.
Antigenicity of metallothionein.
Authors: Winge D R , Garvey J S
Published: May 2006

The antigenic determinants of vertebrate metallothionein have been determined by a competitive-binding double-antibody radioimmunoassay to consist of two immunologically dominant regions in the NH2-terminal domain (residues 1-29). The COOH-terminal domain (residues 30-61) exhibited trivial immunoreactivity in competitive binding assays. The tryptic peptide encompassing residues 1-25 of the molecule competed with 125I-labeled metallothionein as effectively as the native protein. The crossreactivity was unaffected whether the protein was native or denatured. The antigenicity is thus independent of the degree of folding of the protein and, although all antigenic sites depend to some degree on conformation or topography, this favors a sequential (or continuous) rather than a discontinuous nature of the determinants. The two regions in metallothionein that appear to be important in the interaction of the molecule with the antisera include the NH2-terminal acetylated methionine and the cluster of lysines in the sequence from residues 20-25. These regions are homologous in the various vertebrate metallothioneins known to crossreact with rabbit anti-rat metallothionein antiserum. This implies that the induction in rabbits of antiserum to rat metallothionein is an autoimmune phenomenon. The results support the two-cluster model of metallothionein and are compatible with predictions of sites of antigenicity based on regions of high hydrophilicity.
Neutral metal-bound water is the base catalyst in liver alcohol dehydrogenase.
Authors: Makinen M W , Maret W , Yim M B
Published: May 2006

The catalytic role of the active site metal-water complex in horse liver alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) is investigated on the basis of a comparative analysis of the pH dependence of steady-state kinetic parameters of the native and active-site-specific Co2+-reconstituted enzyme and on the basis of assignment of the coordination environment of the Co2+ by electron paramagnetic resonance methods. The pH dependence of the kinetic parameters for the oxidation of benzyl alcohol reveals two ionizations (pK1 approximately equal to 6.7; pK2 approximately equal to 10.6) that govern kcat and belong to the ternary enzyme-NAD+-alcohol complex and two ionizations (pK1' approximately equal to 7.5; pK2' approximately equal to 8.9) that govern kcat/Km and belong to the binary enzyme-NAD+ complex. The ionizations pK2 and pK2' decrease by 0.5-1 pK alpha unit upon replacement of the active site Zn2+ by Co2+. A similar metal ion dependence of pK2 and pK2' is observed for the oxidation of 2-propanol. We attribute these ionizations to a metal-bound water molecule. The zero-field splitting energy of the Co2+ in the binary enzyme-NADH complex and the ternary enzyme-NADH-CF3CH2OH complex is approximately equal to 22 cm-1, indicative of a pentacoordinate species. Binding of a water molecule to the metal ion as the fifth ligand in the ternary enzyme-NADH-CF3CH2OH complex is confirmed on the basis of magnetic interactions of H2(17)O with Co2+. The results indicate that the active site metal ion in catalytically competent ternary enzyme-coenzyme-substrate complexes is pentacoordinate and is ligated by a neutral water molecule in the physiological pH range. We suggest that the neutral metal-bound water molecule serves as the base catalyst for proton abstraction in alcohol oxidation.
Cerebral metabolic studies in vivo by 31P NMR.
Authors: Prichard J W , Alger J R , Behar K L , Petroff O A , Shulman R G
Published: May 2006

31P NMR studies on the brains of living rabbits were carried out at 32 MHz in a spectrometer having a 200-mm clear bore. Paralyzed pump-ventilated animals under nitrous oxide analgesia were inserted into the 1.89-T field and signals were focused in the brain by using a 4-cm surface coil. Several conventional physiological variables were monitored together with 31P spectra during induction and reversal of insulin shock and hypoxic hypoxia sufficient to abolish the electroencephalogram and during status epilepticus. A reversible decrease in phosphocreatine stores accompanied by an increase in Pi was detected during hypoglycemia and hypoxia. Similar changes were observed in prolonged status epilepticus but were not reversed. ATP levels fell about 50% in hypoglycemia but only slightly in the other two metabolic stresses. Intracellular pH rose in hypoglycemia; in status epilepticus and hypoxia it fell, but only when cardiovascular function was severely impaired. From the measured NMR parameters and the assumptions (i) that creatine kinase was at equilibrium and (ii) that the creatine/phosphocreatine pool was constant, it was possible to calculate the relative changes in cytoplasmic ADP levels associated with these metabolic disturbances.
Prosomatostatin-specific antigen in rat brain: localization by immunocytochemical staining with an antiserum to a synthetic sequence of preprosomatostatin.
Authors: Lechan R M , Goodman R H , Rosenblatt M , Reichlin S , Habener J F
Published: May 2006

Using an antiserum to a 15-amino acid synthetic peptide corresponding to amino acids 63-77 of rat preprosomatostatin (rat somatostatin cryptic peptide, RSCP), we have compared the distribution of immunoreactive RSCP (IR-RSCP) with that of immunoreactive somatostatin-14 in the rat brain. IR-RSCP was present in neuronal cell bodies, processes, and axon terminals in the hypothalamic tuberoinfundibular system as well as in diverse regions of the central nervous system in an identical distribution to immunoreactive somatostatin. These observations indicate that in neurons the somatostatin prohormone or the NH2-terminal extension peptide of somatostatin-28 (or both) is stored and transported intracellularly along with somatostatin 14. In addition, the presence of IR-RSCP in nerve terminals suggests that this material may be secreted as a hormone or neuromodulator and may serve as a biologic marker of somatostatin secretion.
Bacterio-opsin mRNA in wild-type and bacterio-opsin-deficient
Halobacterium halobium
strains
Authors: DasSarma Shiladitya , RajBhandary Uttam L. , Khorana H. Gobind
Published: May 2006

We have examined transcripts corresponding to the
Halobacterium halobium
bacterio-opsin (
BO
) gene in wild-type and in
BO
-deficient mutant strains containing the insertion elements ISH1 or ISH2 in the
BO
gene. BO mRNA from the wild-type strain was purified by hybrid selection using single-stranded cDNA. Labeling by vaccinia virus capping enzyme and [α-
32
P]GTP showed that it contains the 5′-terminal nucleotide of the primary transcript. Sequence analysis showed that transcription begins only two nucleotides upstream of the initiator codon for BO. Two species of BO mRNA were found; the major species has a ragged 3′ terminus ≈45 nucleotides downstream from the terminator codon for BO, while the minor species is about 170 nucleotides longer at the 3′ end. Analysis of the transcripts in several
BO
gene mutant strains by RNA gel-transfer hybridization showed that (
i
) mutants with ISH1 insertions within the NH
2
-terminal coding region of the gene contain no detectable transcripts, (
ii
) mutants with ISH2 near the middle of the coding region of the gene contain multiple incomplete transcripts, and (
iii
) a mutant that is partially
BO
deficient due to an insertion of ISH2 100 base pairs upstream of the site of initiation of transcription contains a decreased level of BO mRNA.
Morphologic characterization of the pathway of transferrin endocytosis and recycling in human KB cells.
Authors: Willingham M C , Hanover J A , Dickson R B , Pastan I
Published: May 2006

The pathway of transferrin uptake and recycling was investigated in KB cells to attempt to identify the organelles involved in the return of transferrin to the cell surface. Comparison was made with the pathway of internalization of epidermal growth factor (EGF), which has been shown to terminate in lysosomes. A horseradish peroxidase conjugate of transferrin (TF-HRP) was incubated with KB and at 4 degrees C and, at various times after warming to 37 degrees C, the location of TF-HRP was examined at the electron microscopic level. Transferrin, like EGF, was found to enter cells via coated pits and to move to the Golgi region in receptosomes (endosomes). Transferrin was located in the tubular elements of the transreticular portion of the Golgi but not in Golgi stacks. Interestingly, transferrin was not concentrated in the coated pits of the Golgi. In contrast, EGF was highly concentrated there. Transferrin was next detected in tubular elements (approximately equal to 600 A in width and up to 5,000 A in length) that were closely associated with microtubules and in dumbbell-shaped structures. In contrast, EGF was not detected in these structures. These results suggest that those organelles containing transferrin, but not EGF, participate in the return of receptor-bound transferrin to the cell surface.
Keratolinin: the soluble substrate of epidermal transglutaminase from human and bovine tissue.
Authors: Zettergren J G , Peterson L L , Wuepper K D
Published: May 2006

Substrates of human and bovine epidermal transglutaminase (glutaminyl-peptide gamma-glutamyltransferase, R-glutaminyl-peptide:amine-gamma-glutamyltransferase, EC 2.3.2.13) were isolated and purified by ion exchange chromatography and preparative zone electrophoresis. These substrates of Mr 36,000, which we propose to call keratolinin, incorporated dansylcadaverine and were precipitated by antibody. Keratolinin is ultimately polymerized on the inner leaflet of the keratinocyte membrane to form the cornified envelope. Each Mr 36,000 substrate was dissociated by chaotropic agents or detergents into noncovalent subunits; the Mr of these subunits was 6,000-6,200 on electrophoresis in 15% acrylamide/1% NaDodSO4/6 M urea gels. Isoelectric focusing of human or bovine keratolinin revealed two moieties separated by 0.3-0.4 pH unit (human, 5.4/5.0; bovine, 6.3/6.0). The two proteins were readily resolved by chromatofocusing and each isoelectric moiety of bovine keratolinin incorporated dansylcadaverine by epidermal transglutaminase and calcium and reacted with identity to antiserum to soluble Mr 36,000 keratolinin. Antiserum to human keratolinin failed to crossreact with its bovine counterpart. Antiserum to involucrin did not crossreact with either keratolinin or epidermis by immunodiffusion. Human and bovine epidermal keratolinins are biochemically similar but immunochemically distinct proteins from the epidermis. Involucrin appears only in significant quantities in cell culture.
Differentiation of myoblast cell lines and biological methylation: 3-deazaadenosine stimulates formation of multinucleated myofibers.
Authors: Scarpa S , Strom R , Bozzi A , Aksamit R R , Backlund P S , Chen J , Cantoni G L
Published: May 2006

Treatment of myoblast cell lines with 3-deazaadenosine stimulates differentiation into myofibers. Myoblast clone L5/ 3B5 , which does not form myofibers after 6 days in fusion medium, was stimulated to form myofibers after 5 days of culture in fusion medium containing 50 microM 3-deazaadenosine. Myoblast clone L5/ 3C4 , which normally begins to form myofibers after 4 days in fusion medium, was stimulated by 50 microM 3-deazaadenosine to form myofibers after 3 days in culture and the extent of fusion was also increased. In the presence of 100 microM homocysteine thiolactone, the concentration of 3-deazaadenosine that stimulated maximal fusion was reduced by a factor of 10, from 50 microM to 5 microM 3-deazaadenosine. Stimulation of myofiber formation by 3-deazaadenosine suggests a requirement for one or more methylation reactions in myoblast differentiation and the potentiation by homocysteine thiolactone indicates that myofiber formation is specifically stimulated by an intracellular accumulation of 3-deazaadenosylhomocysteine.
Relationship between parathyroid hormone secretion and cytosolic calcium concentration in dispersed bovine parathyroid cells.
Authors: Shoback D M , Thatcher J , Leombruno R , Brown E M
Published: May 2006

The parathyroid cell is unusual among exocytotic systems in that low extracellular Ca2+ concentrations stimulate, while high Ca2+ concentrations inhibit, parathyroid hormone (PTH) release, suggesting that this cell might have unique secretory mechanisms. In the present studies, we used the Ca2+-sensitive fluorescent dye QUIN -2 to examine the relationship between cytosolic Ca2+ concentration and PTH release in dispersed bovine parathyroid cells. The secretagogue dopamine, which enhances PTH release 2- to 3-fold in association with 20- to 30-fold increases in cellular cAMP, had no effect on the cytosolic Ca2+ level (261 +/- 28 vs. 236 +/- 22 nM for control cells at 1 mM extracellular Ca2+; P greater than 0.05). Dibutyryl-cAMP, which produces a comparable stimulation of PTH release, likewise did not modify the level of cytosolic Ca2+. Removal of extracellular Ca2+ produced a further decrease of the cytosolic Ca2+ to 82 +/- 10 nM. However, PTH secretion persisted at a near maximal rate despite this decrease of extracellular and cytosolic Ca2+ and was 95 +/- 2.5% of the rate of hormonal release at 0.5 mM extracellular Ca2+. In contrast, addition of the divalent cation ionophore ionomycin to parathyroid cells at 1.0 mM extracellular Ca2+ inhibited PTH secretion in association with an increase in cytosolic Ca2+ from 230 +/- 13 nM to 570 +/- 50 nM. Moreover, the magnitude of the ionomycin-induced reduction in PTH secretion (64 +/- 4% relative to the secretory rate at 0.5 mM Ca2+) was equivalent to the inhibition of PTH release caused by 1.5 mM extracellular Ca2+ (64 +/- 6%), which increased the cytosolic Ca2+ to similar levels (450 +/- 48 nM). Thus, the parathyroid cell differs from secretory cells thought to operate by stimulus-secretion coupling in the following ways: changes in PTH release can occur without detectable alterations in the cytosolic Ca2+ concentration, maximal rates of PTH secretion occur at cytosolic Ca2+ concentrations that fail to support exocytosis in other cell types, and increases in the cytosolic Ca2+ concentration due to ionomycin inhibit rather than stimulate PTH release. Therefore, the control of PTH secretion by Ca2+ and other secretagogues may involve previously undefined mechanisms whereby hormonal release is relatively independent of the cytosolic Ca2+ at low levels of this parameter and is inversely related to cytosolic Ca2+ at higher levels of intracellular Ca2+.
Production of human monoclonal IgG antibodies against Rhesus (D) antigen.
Authors: Bron D , Feinberg M B , Teng N N , Kaplan H S
Published: May 2006

An Epstein-Barr virus (EBV)-transformed human B-cell line ( LB4r ) producing anti-Rhesus [Rho(D) antigen] antibody was fused with a non-immunoglobulin-producing mouse-human heteromyeloma ( SHM - D33 ) and selected in hypoxanthine/aminopterin/thymidine medium containing 0.5 microM ouabain. Surviving hybrids found to secrete specific anti-Rho(D) antibody were cloned by limiting dilution. Two clones (D4-B2 and E10-C1) producing high levels (12 and 20 micrograms/ml per 10(6) cells per 24 hr, respectively) of monospecific antibody (IgG3, lambda chain) were selected for expansion and further characterization. Compared to the parental cell line ( LB4r ), these hybridoma cell lines presented several advantages: antibody production was increased 10-fold, cloning efficiency was improved, and the EBV genome was not retained. Antibody production has been stable for greater than 8 months. These human monoclonal anti-Rho(D) antibodies have demonstrated utility in routine blood-group typing. They may also prove useful in the biochemical and genetic characterization of the Rh antigen system. Most important, they offer a source of Rh-immune globulin for the prevention of Rh immunization and alloimmune hemolytic disease of the newborn.
Inhibition of Na+/Ca2+ exchange in membrane vesicle and papillary muscle preparations from guinea pig heart by analogs of amiloride.
Authors: Siegl P K , Cragoe E J , Trumble M J , Kaczorowski G J
Published: May 2006

Na+/Ca2+ exchange is inhibited in both guinea pig cardiac membrane vesicles and papillary muscles in a concentration-dependent fashion by several analogs of the pyrazine diuretic amiloride. Structure/activity studies based on transport measurements in vesicles prepared from guinea pig left ventricle indicate that hydrophobic substitutions at the terminal nitrogen atom of the guanidinium moiety of amiloride improved the inhibitory potency almost 100-fold over that of the parent compound. 3',4'- Dichlorobenzamil ( DCB ) is one of the most active inhibitors (IC50 = 17 microM). In electrically stimulated papillary muscles isolated from guinea pig heart, 10-40 microM DCB decreases contractile force. At 100 microM inhibitor, diastolic tension is significantly increased. The positive inotropic responses to veratridine and ouabain are inhibited by 20 and 40 microM DCB . Since the responses to these interventions were a consequence of increased intracellular Na+ concentration, these data indicate that DCB is an inhibitor of Na+-dependent Ca2+ influx in the intact tissue. Interpretation of mechanical responses elicited by paired pulses suggests that 40 microM but not 100 microM DCB decreases release of Ca2+ from the sarcoplasmic reticulum. The mechanical data obtained with concentrations of DCB that inhibited Na+/Ca2+ exchange in vesicles suggest that a significant amount of Ca2+ can enter the cardiac cell via Na+/Ca2+ exchange under normal conditions and that this transport system may be an important source of Ca2+ supplying the sarcoplasmic reticulum in guinea pig heart. Moreover, these amiloride analogs function as potent inhibitors of the positive inotropic effect caused by increased intracellular Na+ concentration.
In Z-DNA the sequence G-C-G-C is neither methylated by Hha I methyltransferase nor cleaved by Hha I restriction endonuclease.
Authors: Vardimon L , Rich A
Published: May 2006

Plasmids carrying 24- or 32-base-pair inserts of alternating (dG-dC) residues were used to analyze the level of methylation of the G-C-G-C sites by Hha I DNA methyltransferase and their cleavage by Hha I endonuclease in the B-DNA or Z-DNA conformation. In supercoiled plasmids in which the inserts formed Z-DNA, the extent of methylation at the insert G-C-G-C sites was dramatically lower than the level of methylation at the G-C-G-C sites located outside the insert in the same plasmid. Similarly, cleavage by Hha I endonuclease was sharply lowered when the insert was in the Z-DNA form. In the relaxed plasmid, all its G-C-G-C sites were methylated to the same extent and the unmethylated sites were readily cleaved. After treatment with the methylase, the supercoiled plasmid was linearized and then digested with Hha I restriction endonuclease. This exposed unmethylated G-C-G-C sites from the insert that had been protected against cleavage in the Z conformation. A chemical reaction was used to study the distribution of the unmethylated cytosine residues. No accumulation of unmethylated cytosine residues was found anywhere along the entire 32-base-pair insert, which is consistent with a cooperative B-Z transition.
Nucleic acid-binding molecules with high affinity and base sequence specificity: intercalating agents covalently linked to oligodeoxynucleotides.
Authors: Asseline U , Delarue M , Lancelot G , Toulmé F , Thuong N T , Montenay-Garestier T , Hélène C
Published: May 2006

Oligodeoxyribonucleotides covalently linked to an intercalating agent via a polymethylene linker were synthesized. Oligothymidylates attached to an acridine dye (Acr) through the 3'-phosphate group [(Tp)n(CH2) mAcr ] specifically interact with the complementary sequence. The interaction is strongly stabilized by the intercalating agent. By using absorption and fluorescence spectroscopies, it is shown that complex formation between (Tp)n(CH2) mAcr and poly(rA) involves the formation of n A X T base pairs, where n is the number of thymines in the oligonucleotide. The acridine ring intercalates between A X T base pairs. Fluorescence excitation spectra reveal the existence of two environments for the acridine ring, whose relative contributions depend on the linker length (m). The binding of (Tp)4(CH2) mAcr to poly(rA) is analyzed in terms of site binding and cooperative interactions between oligonucleotides along the polynucleotide lattice. Thermodynamic parameters show that the covalent attachment of the acridine ring strongly stabilizes the binding of the oligonucleotide to its complementary sequence. The stabilization depends on the linker length; the compound with m = 5 gives a more stable complex than that with m = 3. These results open the way to the synthesis of a family of molecules exhibiting both high-affinity and high-specificity for a nucleic acid base sequence.
Copper metallothionein of yeast, structure of the gene, and regulation of expression.
Authors: Butt T R , Sternberg E J , Gorman J A , Clark P , Hamer D , Rosenberg M , Crooke S T
Published: May 2006

Addition of copper to yeast cells leads to the induction of a low molecular weight, cysteine-rich protein that binds copper. This protein, termed copper chelatin or thionein, is related to the metallothionein family of proteins that are induced in response to cadmium and zinc in vertebrate cells. We have determined the structure of the yeast copper-binding protein by DNA sequence analysis of the gene. Although the 6573-dalton yeast protein is substantially divergent from vertebrate metallothioneins, the arrangement of 12 cysteine residues, which is a hallmark of metal-binding proteins, is partially conserved. We analyzed the regulatory DNA sequence of the gene by fusing it with the Escherichia coli galactokinase gene and assaying the levels of enzyme activity in yeast in response to copper. The transcriptional activation has a specific requirement for copper. Zinc, cadmium, and gold were unable to regulate the galactokinase activity. The yeast copper metallothionein regulatory sequences represent a previously unreported class of yeast promoter that is regulated by copper.
In vitro transformation of mesenchymal cells derived from embryonic muscle into cartilage in response to extracellular matrix components of bone.
Authors: Sampath T K , Nathanson M A , Reddi A H
Published: May 2006

Subcutaneous implantation of demineralized diaphyseal bone matrix into rats induces cartilage and bone formation in vivo. When minced skeletal muscle is cultured on hemicylinders of demineralized bone in vitro, mesenchymal cells are transformed into chondrocytes. In the present investigation, the potential of extracellular matrix components of bone to trigger cartilage differentiation in vitro was examined. Extraction of bone hemicylinders with 6 M guanidine X HCl resulted in the absence of chondrogenesis in vitro and endochondral bone formation in vivo. Biologically inactive hemicylinders of bone were then reconstituted with the guanidine extract and also with partially purified components extracted from bone matrix and bioassayed. Reconstitution completely restored the ability to elicit chondrogenesis in vitro and endochondral bone differentiation in vivo. Reconstitution of the whole guanidine extract on Millipore filters coated with gels of tendon collagen (type I) and subsequent culture with minced skeletal muscle also resulted in cartilage induction in vitro. These observations show that the extracellular matrix of bone is a repository of factors that govern local cartilage and bone differentiation.
Growth hormone promotes the differentiation of myoblasts and preadipocytes generated by azacytidine treatment of 10T1/2 cells.
Authors: Nixon B T , Green H
Published: May 2006

Previous studies have shown that growth hormone promotes the differentiation of preadipose 3T3 cells into adipose cells. This action of the hormone is not mediated by insulin-like growth factor 1 (IGF-1), but is exerted directly on the preadipocytes. In order to determine whether growth hormone promotes the differentiation of other mesenchymal cell types, we have examined its effect on the formation of multinucleated muscle cells from myoblasts. Clone mu cells, generated from line 10T1/2 by treatment with azacytidine, were known to be capable of undergoing myogenesis. When these cells were cultivated in a medium not able alone to support their differentiation, the addition of physiological concentrations of growth hormone strongly promoted myogenesis. When fetal calf serum was used instead of growth hormone to promote myogenesis, the effectiveness of the serum was reduced by the addition of a specific antiserum to growth hormone. IGF-1 could not substitute for growth hormone in promoting myogenesis. When another 10T1/2 line not ordinarily able to form myotubes was subjected to treatment with 5-azacytidine, it was subsequently able to give rise to muscle and adipose cell colonies; the number of such colonies was much increased by the presence of growth hormone. We have not been able to demonstrate a similar dependence on growth hormone for myogenesis by the established lines L6E9 and C2.
Decrease of tyrosine-O-sulfate-containing proteins found in rat fibroblasts infected with Rous sarcoma virus or Fujinami sarcoma virus.
Authors: Liu M C , Lipmann F
Published: May 2006

Our interest was aroused by the recent report by Huttner [ Huttner , W. B. (1982) Nature (London) 299, 273-276] on general sulfation of tyrosine residues of proteins in normal and malignantly transformed tissues. Here we report on the reduction of sulfation in embryonic rat fibroblasts, line 3Y1, infected with Rous sarcoma virus or Fujinami sarcoma virus. In view of the instability of tyrosine O-sulfate in strong acid, the protein sulfation was tested for after incubation with [35S]sulfate and exhaustive Pronase hydrolysis. We found in general a reduction of sulfation in transformed tissue. It was greatest in the fibroblasts permanently transformed with Rous sarcoma virus. When fibroblasts transformed by the temperature-sensitive Fujinami sarcoma virus, line ts225 -3Y1, were used for comparison of sulfation at nonpermissive and permissive temperatures, the latter showed a strong reduction. Furthermore, we tested these cells for the uptake of inorganic [35S]sulfate. Uptake appeared highly reduced in the permanently infected fibroblasts, but ts225 -3Y1 grown at permissive and nonpermissive temperatures exhibited no difference. Uptake at both temperatures was comparable to uptake by normal 3Y1 cells. A recently much investigated cell surface protein, fibronectin, was reported to be lost on malignant transformation and to contain sulfate in an undetermined location. We found that ts225 -3Y1 cells grown at permissive temperature released fibronectin that contained tyrosine O-sulfate.
Wound signals in plants: A systemic plant wound signal alters plasma membrane integrity
Authors: Walker-Simmons Mary , Holländer-Czytko Heike , Andersen Julie K. , Ryan Clarence A.
Published: May 2006

Within 4 hr after wounding the lower leaves of young potato and tomato plants, a rapid and remarkable change is induced in the cells of upper undamaged leaves that results in extensive lysis of protoplasts during their isolation. Protoplast yields from unwounded upper leaves, 4 hr after wounding a lower leaf by crushing with a hemostat, decreased 25% below yields from leaves of unwounded plants. From 8 to >20 hr after wounding, protoplast yields were less than half of those from control plants. Multiple woundings decreased yields even further, as did chewing of the lower leaves by tobacco hornworms over a period of several minutes. In addition, within 4 hr of excising young tomato plants at their base with a razor blade, a 90% decrease in leaf protoplast yields was recorded. The major loss of protoplasts induced by wounding was primarily due to an increased cell lysis during protoplast isolation. Cell lysis was apparently due to a weakened cell membrane, because newly recovered protoplasts released from leaves of wounded plants were extremely fragile and exhibited 70% lysis during low speed centrifugation, compared to 20% lysis of protoplasts recovered from control plants. We conclude that a signal is released by wounding that is rapidly transmitted or transported through the plants to induce a profound change in the leaf cell membranes that renders them fragile during protoplast isolation. It is proposed that this signal may play a role in inducing cellular changes in the plant cells as part of their responses to environmental stress such as pest attacks.
Prevention of rejection of murine islet allografts by pretreatment with anti-dendritic cell antibody.
Authors: Faustman D L , Steinman R M , Gebel H M , Hauptfeld V , Davie J M , Lacy P E
Published: May 2006

Previously we have demonstrated that islets of Langerhans treated with donor-specific anti-Ia serum and complement survive when transplanted across the major histocompatibility complex of the mouse. In this study, using immunofluorescence, we demonstrate two morphologically distinct populations of Ia-positive cells scattered within the Ia-negative islet tissue. A large irregularly shaped Ia-positive subset of cells were identified as dendritic cells by using the 33D1 antibody specific for a mouse dendritic cell antigen. The other small, round Ia-positive subset was 33D1 negative. Islets pretreated with anti-dendritic cell antibody and complement prior to transplantation survived in their histoincompatible recipients for greater than 200 days. Rejection of stable islet allografts promptly occurred when transplant recipients were challenged with 1 X 10(5) donor dendritic cells 60 days after transplantation. These results demonstrate an important in vivo role for donor dendritic cells in the stimulation of allograft rejection.
RU 486: a steroid with antiglucocorticosteroid activity that only disinhibits the human pituitary-adrenal system at a specific time of day.
Authors: Gaillard R C , Riondel A , Muller A F , Herrmann W , Baulieu E E
Published: May 2006

RU 486 is a synthetic steroid hormone antagonist which acts at the receptor level. It has both intrinsic anti-progesterone and antiglucocorticosteroid properties in animals. We investigated the antiglucocorticosteroid activity in humans by evaluating the pituitary-adrenal response to RU 486 in men and in pregnant and nonpregnant women. In non-pregnant women, RU 486 (approximately equal to 1 mg/kg of body weight per day) produced an interruption of the luteal phase without affecting the pituitary-adrenal axis, thereby indicating a more potent anti-progesterone than antiglucorticosteroid effect. In the course of pregnancy interruption by RU 486 (approximately equal to 4 mg/kg per day), there was a significant increase in plasma corticotropin, beta-lipotropin, and cortisol concentrations. In normal men, RU 486 administration led to a dose-dependent stimulation of plasma corticotropin, beta-endorphin, and cortisol. This disinhibition of the pituitary-adrenal axis was only observed during the morning hours of the circadian rhythm. When administered concomitantly with 1 mg of dexamethasone at midnight, 6 mg of RU 486 per kg completely suppressed the dexamethasone inhibitory effect on the pituitary-adrenal axis. These results indicate that RU 486 is an antiglucocorticosteroid that disrupts the negative pituitary feedback of both the morning cortisol rise and administered dexamethasone. Furthermore, they demonstrate the possibility of optimizing the anti-progestational effect of the compound and its potential use for human fertility control by modifying the dose and the time of administration of the drug and thereby minimizing the antiglucocorticosteroid effect.
Gamma-interferon induces expression of the HLA-D antigens on normal and leukemic human myeloid cells.
Authors: Koeffler H P , Ranyard J , Yelton L , Billing R , Bohman R
Published: May 2006

Gamma-Interferon (IFN-gamma) is a lymphokine produced by T lymphocytes. We find that recombinant human IFN-gamma induces expression of HLA-D antigens on human promyelocytic leukemia cells (HL-60) and enhances expression of HLA-D antigens on normal human monocytes and macrophages. Induction of both HLA-D antigen expression and HLA-D mRNA accumulation occurs within 1 day of exposure of HL-60 to IFN-gamma and is maximal by day 5. Maximal antigen expression occurs in the presence of 100-500 units of IFN-gamma per ml. IFN-gamma induces expression of DR but not DC antigens on HL-60, as confirmed by using four different murine monoclonal antibodies or one rabbit heterologous antibody. RNA blot data show that IFN-gamma-exposed HL-60 cells contain mRNA for DR alpha and DR beta polypeptides but not for DC alpha and DC beta polypeptides, which also suggests that HLA-DR and HLA-DC gene regions can be independently regulated. IFN-gamma induces 20% of HL-60 cells to differentiate to macrophage-like cells. However, IFN-gamma dose-response studies using both HL-60 cells and a nondifferentiation variant of HL-60 cells (HL-60 blast) clearly show that induction of transcription and expression of HLA-D gene products by IFN-gamma can be uncoupled from expression of other monocyte-macrophage characteristics. Further studies show that IFN-gamma enhances expression of HLA-D antigens on normal human monocytes and macrophages. Expression of the HLA-D antigens is necessary for the interaction of macrophages and T lymphocytes; IFN-gamma may play a fundamental role in this interaction.
Regulation of the araC gene of Escherichia coli: catabolite repression, autoregulation, and effect on araBAD expression.
Authors: Miyada C G , Stoltzfus L , Wilcox G
Published: May 2006

The araC gene encodes a positive regulatory protein required for L-arabinose utilization in Escherichia coli. Transcription from the araC promoter has been shown to be under positive control by cAMP receptor protein and under negative control by its protein product (autoregulation). This work describes the identification of the region of the araC promoter that interacts with the cAMP receptor protein to mediate catabolite repression. A 3-base-pair deletion centered 60 base pairs from the transcriptional initiation site results in a mutant araC promoter that, in the absence of araC protein, reduces transcriptional activity when compared with the wild-type promoter and is unresponsive to various concentrations of intracellular cAMP in vivo. The same deletion results in a lowered affinity of the araC promoter for cAMP receptor protein in vitro. However, this lowered affinity for the mutant araC promoter does not result in substantial reduction of intracellular araC protein because autoregulation of the araC gene dominates catabolite repression. The 3-base-pair deletion in the cAMP receptor protein binding site of the araC promoter does not affect catabolite repression of the adjacent araBAD operon. The implications of these results on current models for expression of the araBAD operon and the araC gene are discussed.
Lignoceric acid is oxidized in the peroxisome: implications for the Zellweger cerebro-hepato-renal syndrome and adrenoleukodystrophy.
Authors: Singh I , Moser A E , Goldfischer S , Moser H W
Published: May 2006

The deficient oxidation and accumulation of very-long-chain fatty acids in the Zellweger cerebro-hepato-renal syndrome (CHRS) and X chromosome-linked adrenoleukodystrophy (ALD), coupled with the observation that peroxisomes are lacking in CHRS, prompted us to investigate the subcellular localization of the catabolism of lignoceric acid (C24:0). Peroxisomal and mitochondrial-rich fractions were separated from rat liver crude mitochondria by sucrose density gradient centrifugation. Enzyme activity for the oxidation of [1-14C]palmitic acid to water-soluble acetate was 2- to 3-fold higher in the mitochondrial than in the peroxisomal-rich fraction whereas [1-14C]lignoceric acid was oxidized at a 2- to 3-fold higher rate in the peroxisomal than in the mitochondrial fraction. Moreover, unlike palmitic acid oxidation, lignoceric acid oxidation was not inhibited by potassium cyanide in either rat liver fractions or human skin cultured fibroblasts, showing that lignoceric acid is mainly and possibly exclusively oxidized in peroxisomes. We also conducted studies to clarify the striking phenotypic differences between CHRS and the childhood form of ALD. In contrast to CHRS, we found normal hepatocellular peroxisomes in the liver biopsy of a childhood ALD patient. In addition, in the presence of potassium cyanide, the oxidation of palmitic acid in cultured skin fibroblasts was inhibited by 62% in control and X chromosome-linked ALD patients compared with 88% in CHRS and neonatal ALD. This differential effect may be related to differences in peroxisomal morphology in those disorders.
Solubilization and characterization of mu, delta, and kappa opioid binding sites from guinea pig brain: physical separation of kappa receptors.
Authors: Itzhak Y , Hiller J M , Simon E J
Published: May 2006

Sucrose density gradient centrifugation of digitonin-solubilized opioid binding sites from guinea pig brain and cerebellum was carried out. Centrifugation of extracts of whole brain into a gradient devoid of sodium and low in digitonin revealed the presence of two well-separated peaks of opioid binding activity. Peak A was shown to have the binding characteristics of kappa sites, whereas peak B seems to be a mixture of mu and delta sites. When extracts of guinea pig cerebellum were treated in the same manner, a single peak of binding activity was obtained that coincided with peak A from guinea pig brain and exhibited the characteristics of kappa binding sites. All three sites closely resemble their membrane-bound counterparts, retaining good affinity and selectivity for their appropriate ligands. The apparent sedimentation coefficients (S20,w) of the digitonin-solubilized binding sites present in the two peaks are 19 s for peak A and 34-39 s for peak B, and the estimated apparent molecular weights are 400,000 for kappa sites and 750,000-875,000 for the mixture of mu and delta sites. Our results suggest that kappa sites constitute separate molecular entities from mu and delta sites.
Single channel recordings of calcium-activated potassium channels in the apical membrane of rabbit cortical collecting tubules.
Authors: Hunter M , Lopes A G , Boulpaep E L , Giebisch G H
Published: May 2006

Recordings of single potassium channels from the apical membrane of rabbit cortical collecting tubule have been achieved using the patch-clamp technique. The conductive properties of the channel have been studied in inside-out patches. The slope conductance of the open channel is approximately equal to 90 pS. The channel is selective to potassium over sodium, with a selectivity ratio of 9:1. Decreasing the calcium concentration of the solution bathing the cytoplasmic face of the patch results in a decrease of the open-channel probability. Decreasing the calcium concentration to 10 nM or less completely inhibited channel activity. The channel is also inhibited by barium in a dose-dependent fashion.
Requirement for alanine in the amino acid control of deprivation-induced protein degradation in liver.
Authors: Pösö A R , Mortimore G E
Published: May 2006

Protein degradation in liver is actively controlled by a small group of inhibitory amino acids--leucine, tyrosine (or phenylalanine), glutamine, proline, histidine, tryptophan, and methionine. Other evidence, however, suggests that one or more of the remaining 12 noninhibitory amino acids is also required for suppression of proteolysis at normal concentrations. This question was investigated in livers of fed rats perfused in the single-pass mode. The deletion of alanine at normal (1x), but not at 4x or 10x normal, plasma amino acid concentrations evoked a near-maximal acceleration of protein degradation. No other noninhibitory amino acid was effective. Because alanine alone was not directly inhibitory and its omission was not associated with a decrease in inhibitory amino acid pools, alanine was presumed to act as a coregulator in the expression of inhibitory activity. When tested alone, the inhibitory group was as effective as the complete mixture at 0.5x and 4x levels, but it lost its suppressive ability within a narrow zone of concentration centered slightly above 1x. The addition of 1x (0.48 mM) alanine completely restored the inhibition. Pyruvate and lactate could be effectively substituted, but only at concentrations 10-20 times greater than that of alanine. These, together with earlier findings, indicate the existence of a regulatory complex that recognizes specific amino acids and transmits positive and negative signals to proteolytic sites. The results also suggest that alanine can provide an important regulatory link between energy demands and protein degradation.
Primary structure of the Escherichia coli ribonucleoside diphosphate reductase operon.
Authors: Carlson J , Fuchs J A , Messing J
Published: May 2006

The nucleotide sequence of the Escherichia coli K-12 DNA comprising the operon for the structural genes of the subunits of ribonucleotide diphosphate reductase has been determined. The DNA sequenced maps at 48.5 minutes on the E. coli chromosome and includes a total length of 8557 nucleotides. An open reading frame between nucleotides 3506 and 5834, encoding a 776-amino acid polypeptide chain with a molecular weight of 87,532, has been identified as the nrdA gene. An open reading frame between nucleotides 6012 and 7139, encoding a 375-amino acid polypeptide with a molecular weight of 43,466, has been identified as the nrdB gene. The sequences reveal not only the primary structures for both subunits, but also some interesting aspects of potential regulatory sites.
Structures of cysteine-containing peptides in isosafrole-inducible rat hepatic microsomal cytochrome P-450d: sequence homology with 3-methylcholanthrene-induced cytochrome P-450c.
Authors: Haniu M , Ryan D E , Levin W , Shively J E
Published: May 2006

Six cysteine-containing tryptic peptides were isolated from rat liver cytochrome P-450d, the major isosafrole-induced isozyme, by reversed-phase high performance liquid chromatography. The six peptides contained a total of seven cysteine residues. Five of the peptides have significant sequence homology (20/22, 10/16, 8/13, 13/18, and 5/9 identical residues) to cysteine-containing peptides in cytochrome P-450c, the major isozyme induced by 3-methylcholanthrene. One of the peptides (partial sequence, Cys-Ile-Gly-Glu-Ile-Pro-Ala-Lys-Trp-Glu-Val-Phe-Leu-) can be included in the highly conserved COOH-terminal domain found in all cytochromes P-450 that have been sequenced. Although this domain has been postulated as the heme-binding domain by some investigators, it is not homologous to the heme-binding region in cytochrome P-450cam. A second peptide (partial sequence, Asp-Pro-Thr-Ser-Val-Ser-Ser-Cys-Tyr-Leu-Glu-Glu-His-Val-Ser-Lys) is, however, a possible candidate for the heme attachment site to cysteine because of its weak homology to the heme-binding site in cytochrome P-450cam. These results indicate that either the location of the heme-binding site is different in various forms of cytochromes P-450 or the amino acid sequence surrounding the heme-binding cysteine is not highly conserved among these proteins.
Inhibitors of CDP-choline synthesis, action potential calcium channels, and stimulus-secretion coupling.
Authors: de Blas A , Adler M , Shih M , Chiang P K , Cantoni G L , Nirenberg M
Published: May 2006

The effects of putative transmethylation inhibitors were tested on stimulus-secretion coupling and neurotransmitter secretion at synapses between neuroblastoma X glioma hybrid cells and myotubes. 5'-Deoxy-5'-isobutylthio-3-deazaadenosine or 5'-deoxy-5'-isobutylthioadenosine inhibited CDP-choline synthesis catalyzed by cholinephosphate cytidylyltransferase (CTP:cholinephosphate cytidylyltransferase, EC 2.7.7.15) and thereby decreased the rate of phosphatidylcholine synthesis from CDP-choline, but did not affect the transmethylation pathway for phosphatidylcholine synthesis. These compounds also inhibited 45Ca2+ uptake by hybrid cells mediated by voltage-sensitive Ca2+ channels, acetylcholine secretion at synapses, and signal transduction through cell membranes mediated by myotube nicotinic acetylcholine receptors. In contrast, 3-deazaadenosine or adenosine inhibited the transmethylation pathway for phosphatidylcholine synthesis, but had no effect on Ca2+ action potentials, acetylcholine secretion, or signal transduction through cell membranes mediated by nicotinic acetylcholine receptors. These results show that the stimulus-secretion coupling and secretion reactions studied are not dependent on phospholipid methylation and suggest that the activity of action potential Ca2+ channels and the rate of neurotransmitter secretion are functionally coupled to the rate of phosphatidylcholine synthesis via the CDP-choline pathway.