bluuebunny/crossref_metadata_2025 huggingface dataset:
Binding of [3H]forskolin to rat brain membranes.
Authors: Seamon K B , Vaillancourt R , Edwards M , Daly J W
Published: May 2006

[12-3H]Forskolin (27 Ci/mmol) has been used to study binding sites in rat brain tissue by using both centrifugation and filtration assays. The binding isotherm measured in the presence of 5 mM MgCl2 by using the centrifugation assay is described best by a two-site model: Kd1 = 15 nM, Bmax1 (maximal binding) = 270 fmol/mg of protein; Kd2 = 1.1 microM; Bmax2 = 4.2 pmol/mg of protein. Only the high-affinity binding sites are detected when the binding is determined by using a filtration assay; Kd = 26 nM, Bmax = 400 fmol/mg of protein. Analogs of forskolin that do not activate adenylate cyclase (EC 4.6.1.1) do not compete effectively for [3H]forskolin binding sites. Analogs of forskolin that are less potent than forskolin in activating adenylate cyclase are also less potent in competing for forskolin binding sites. The presence of 5 mM MgCl2 or MnCl2 was found to enhance binding. In the presence of 1 mM EDTA the amount of high-affinity binding is reduced to 110 fmol/mg of protein with no change in Kd. There is no effect of CaCl2 (20 mM) or NaCl (100 mM) on the binding. No high-affinity binding can be detected in membranes from ram sperm, which contains an adenylate cyclase that is not activated by forskolin. It is proposed that the high-affinity binding sites for forskolin are associated with the activated complex of catalytic subunit and stimulatory guanine nucleotide binding protein.
Rearranged immunoglobulin heavy chain variable region (VH) pseudogene that deletes the second complementarity-determining region.
Authors: Takahashi N , Noma T , Honjo T
Published: May 2006

We have cloned two rearranged heavy chain variable region (VH) genes from the IgG-producing human cell line CESS. The VH gene, which is linked to the mu chain constant region (C mu) gene, has two deletions at residues 45-62 and 82A-90, the former of which corresponds closely to the second complementarity-determining region (CDR2). These results could indicate that translocation of CDR2 occurred and could give support to the argument that reassortment of the V mini-genes is involved in the generation of hypervariability during evolution. However, the rearranged pseudogene could have also arisen by fortuitous deletion. The other VH gene of CESS is an expressed form and is probably linked to the C gamma gene. The diversity region (D) segments used in these rearranged V genes are less than 38% homologous to known human germline D segments, indicating the presence of more unknown germline D segments.
Protein synthesis in rabbit reticulocytes: a study of the mechanism of action of the protein factor RF that reverses protein synthesis inhibition in heme-deficient reticulocyte lysates.
Authors: Grace M , Bagchi M , Ahmad F , Yeager T , Olson C , Chakravarty I , Nasrin N , Banerjee A , Gupta N K
Published: May 2006

A eukaryotic initiation factor 2 (eIF-2)-ancillary protein factor Co-eIF-2 promotes displacement of GDP from eIF-2 X GDP and facilitates ternary complex (Met-tRNAf X eIF-2 X GTP) formation in the presence of Mg2+. Heme-regulated protein synthesis inhibitor, HRI, phosphorylates the alpha-subunit of eIF-2 and thus inhibits ternary complex formation as Co-eIF-2 does not displace GDP from eIF-2 alpha (P) X GDP. RF, a high molecular weight cell supernatant factor, reverses protein synthesis inhibition in heme-deficient reticulocyte lysates and also reverses HRI inhibition of ternary complex formation. RF contains Co-eIF-2 activity. In addition, an active RF preparation contains excess alpha-subunit of eIF-2 in the free and unphosphorylated form and this alpha-subunit of eIF-2 is not phosphorylated by HRI and ATP. In this paper we report (i) an active RF preparation contains excess alpha-subunit of eIF-2 and this alpha-subunit can be phosphorylated by HRI and ATP in the presence of GDP; (ii) RF promotes ternary complex formation by eIF-2 X [3H]GDP with accompanying GDP displacement; (iii) in the presence of HRI and ATP, RF promotes ternary complex formation by eIF-2 X [3H]GDP without accompanying GDP displacement; (iv) in the presence of HRI and ATP, the ternary complex formed using RF is active in Met-tRNAf X 40S initiation complex formation; (v) both the ternary complex and the Met-tRNAf X 40S complex formation in the presence of HRI and ATP are completely inhibited by prior incubation of RF with GDP; (vi) upon further fractionation of an active RF fraction, a preparation can be obtained that contains HRI-sensitive Co-eIF-2 activity. However, this preparation does not efficiently reverse protein synthesis inhibition in heme-deficient reticulocyte lysates and does not contain excess alpha-subunit of eIF-2. Based on these observations, we have suggested (a) RF provides the unphosphorylated alpha-subunit to eIF-2 alpha (P) X GDP and restores eIF-2 activity. This RF activity is inhibited as the alpha-subunit in the RF preparation becomes phosphorylated by HRI and ATP in the presence of GDP; (b) RF contains Co-eIF-2 activity, which has dual functions: (i) stimulation of ternary complex formation by eIF-2 and (ii) GDP displacement from eIF-2 X GDP during ternary complex formation. In the presence of HRI and ATP, Co-eIF-2 but does not displace GDP from eIF-2 alpha(P) X GDP.
Reconstitution of purified halorhodopsin.
Authors: Bogomolni R A , Taylor M E , Stoeckenius W
Published: May 2006

Asolectin lipid vesicles containing halorhodopsin show light-induced acidification in the presence of proton ionophores. This effect is abolished by triphenyltin chloride, a chloride/hydroxyl antiporter, and is greatly diminished by valinomycin in the presence of potassium ions, which collapse the membrane potential. This indicates that halorhodopsin orients in the lipid vesicles preferentially inside out, pumping chloride into the extravesicular compartment. The absorption maximum of halorhodopsin in asolectin vesicles in 3 M NaCl is at 567 nm, and the action spectrum for the light-induced pH changes followed closely the absorption spectrum. Replacement of chloride by acetate or sulfate causes a shift in the absorption maximum to approximately equal to 559 nm and renders the pump inactive. The different photocycles of the two forms were used to show that 80% of the molecules have the extracellular side exposed to the vesicle interior and that the halide-binding site(s) associated with the spectral transition is accessible from the extracellular side of the molecule. The data presented demonstrate that the purified chromoprotein is the light-driven chloride pump in Halobacterium halobium.
Water flow and coral colony size: Interhabitat comparisons of the octocoral
Alcyonium siderium
Authors: Sebens K. P.
Published: May 2006

Colonies of the octocoral
Alcyonium siderium
Verrill form aggregations on subtidal vertical rock walls in the Gulf of Maine. Maximum and mean colony sizes increase dramatically from protected embayments to sites exposed to ocean swells and from the bottom to the top of each vertical wall studied. Water movement along the substratum, generated by surface waves and tides, also increases along both gradients. Although plankton concentrations were similar at all sites during a 2-yr period, increased encounter rates of zooplankton with coral tentacles will result in greater energy intake and, thus, faster growth rates at the more exposed sites with greater flow. Other hypotheses for the proximate cause of the size gradient, such as differential mortality of large colonies or generally higher mortality rates at the protected site, are not consistent with available data.
Receptors for human alpha and beta interferon but not for gamma interferon are specified by human chromosome 21.
Authors: Raziuddin A , Sarkar F H , Dutkowski R , Shulman L , Ruddle F H , Gupta S L
Published: May 2006

We examined the proposed role of human chromosome 21 in determining the cellular sensitivity to human alpha, beta, and gamma interferons (HuIFN-alpha, -beta, and -gamma) and the expression of the receptors for the HuIFNs with the use of mouse-human hybrid cells containing human chromosome 21. Hybrid cells (WA17) containing three copies of human chromosome 21 showed specific displaceable binding of 125I-labeled HuIFN-alpha 2 (125I-HuIFN-alpha 2), which was not observed with mouse parent (A9) cells. Crosslinking of 125I-HuIFN-alpha 2 bound to WA17 cells with disuccinimidyl suberate yielded a complex of Mr approximately equal to 150,000 similar to the 125I-HuIFN-alpha 2-receptor complex obtained with human cells as described earlier. Such a complex was not obtained with mouse parent (A9) cells or with hybrid cells containing certain other human chromosomes but not chromosome 21. Mice inoculated with mouse-human hybrid cells containing human chromosome 21 produce antibodies that block the antiviral action of HuIFN-alpha and -beta on human cells. Such antibodies could immunoprecipitate the 125I-HuIFN-alpha 2-receptor complex obtained from human cells but not free 125I-HuIFN-alpha 2, indicating that these antibodies were directed against the receptor. WA17 hybrid cells were highly sensitive to the antiviral action of HuIFN-alpha 2, -alpha (Le) and -beta but were completely insensitive to HuIFN-gamma. Furthermore, 125I-HuIFN-gamma showed specific binding to human WISH cells but not to WA17 hybrid cells or A9 mouse cells. The results indicate that the receptors for HuIFN-alpha and -beta but not for HuIFN-gamma are specified by human chromosome 21. Hybrid cells containing one, two, or three copies of human chromosome 21 were found to be increasingly sensitive to HuIFN-alpha 2, indicating that a chromosome 21-specified component (possibly the HuIFN-alpha receptor) may be a limiting factor in the cellular sensitivity to HuIFN-alpha.
Inducible transcription and puffing in Drosophila melanogaster transformed with hsp70-phage lambda hybrid heat shock genes.
Authors: Cohen R S , Meselson M
Published: May 2006

A series of hsp70-phage lambda hybrid genes having various amounts of 5' flanking DNA was introduced into the germ line of Drosophila melanogaster by P-element-mediated transformation. Heat-induced transcription was normal in lines transformed with hsp70-lambda genes having 194 and 146 base pairs of DNA upstream from the mRNA initiation site. Lines transformed with genes having 70 base pairs of upstream DNA accumulated correctly initiated transcripts in response to heat shock, but the amount was somewhat reduced and minor amounts of incorrectly initiated transcripts were observed. No transcription, with or without heat shock, was seen in lines transformed with hsp70-lambda genes having only 52, 44, or 25 base pairs of upstream DNA. Heat shock induced polytene chromosome puffing at the site of integration in a line transformed with a gene having 194 base pairs of upstream DNA. The hsp70-lambda gene and a cotransformed Adh gene closely linked on the same integrating fragment were expressed independently, each apparently responding only to its own normal control signals.
Isolation and primary structure of two peptides with cardioacceleratory and hyperglycemic activity from the corpora cardiaca of Periplaneta americana.
Authors: Scarborough R M , Jamieson G C , Kalish F , Kramer S J , McEnroe G A , Miller C A , Schooley D A
Published: May 2006

Two cardioacceleratory peptides from the corpora cardiaca of Periplaneta americana have been purified by gel filtration and reversed-phase liquid chromatography, Based on analysis of the intact factors and their chymotryptic fragments, we have assigned the primary structure of these octapeptides as pGlu-Val-Asn-Phe-Ser-Pro-Asn-Trp-NH2, designated periplanetin CC-1, and pGlu-Leu-Thr-Phe-Thr-Pro-Asn-Trp-NH2, designated periplanetin CC-2. They represent new members of a family of invertebrate peptides that includes locust adipokinetic hormone and crustacean red-pigment concentrating hormone. Both peptides show adipokinetic activity in grasshoppers and hyperglycemic activity in cockroaches. One of these peptides (CC-2) has provocative sequence homology with the NH2-terminal portion of glucagon.
Cloning and sequencing of liver cDNA coding for bovine protein C.
Authors: Long G L , Belagaje R M , MacGillivray R T
Published: May 2006

cDNA coding for protein C has been cloned from a bovine liver library in plasmid vector pBR322 and its sequence has been determined. Two overlapping clones code for the entire light and heavy chains of the mature protein, as well as a previously unreported connecting dipeptide (Lys-Arg) and a 39-amino acid leader peptide region. Identification and characterization of the clones establishes the liver as a site of protein C biosynthesis. A contiguous coding region reveals that a one-chain precursor protein is made that upon limited proteolysis yields both the mature light and heavy chains. The codon for aspartic acid is found at light chain amino acid position 71, showing that the beta-hydroxyaspartic acid that exists in this position of the mature protein is the result of post-translational modification of an aspartic acid residue. Amino acid sequence homology in the amino-terminal region of the light chain with other vitamin K-dependent coagulation factors is continued into the leader peptide region.
Localization of the gangliosides GD2 and GD3 in adhesion plaques and on the surface of human melanoma cells.
Authors: Cheresh D A , Harper J R , Schulz G , Reisfeld R A
Published: May 2006

The predominant gangliosides produced by two cultured human melanoma cell lines are GD3 and/or GD2. These gangliosides were found to be cell associated and present in substratum-attached material after cell removal by EDTA. Monoclonal antibodies directed to GD2 and GD3 specified the cell-surface distribution of these gangliosides and localized them in focal adhesion plaques at the interface of cells and their substratum. These attachment sites did not represent indiscriminant membrane fragments remaining after removal of cells with EDTA, because neither melanoma-associated proteoglycan nor class I histocompatibility antigens were detected by their respective antibodies. Our data suggest that the disialogangliosides GD2 and GD3 may be involved in the interaction between human melanoma cells and solid substrata.
Suppression of the first stage of phorbol 12-tetradecanoate 13-acetate-effected tumor promotion in mouse skin by nontoxic inhibition of DNA synthesis.
Authors: Kinzel V , Loehrke H , Goerttler K , Fürstenberger G , Marks F
Published: May 2006

In order to evaluate the significance of epidermal cell proliferation for the first stage of skin tumor promotion, the effect of hydroxyurea (HU), an inhibitor of DNA synthesis, on tumor formation was studied. Mice initiated with 7,12-dimethylbenz[a]anthracene received a single dose of phorbol 12-myristate 13-acetate (PMA) in stage I of promotion, followed by twice weekly application of the irritant skin mitogen phorbol 12-retinoate 13-acetate in stage II. A single dose of HU given intraperitoneally at different times before or after treatment with PMA was found to interfere with tumor formation, exhibiting an almost complete inhibition if administered 18 hr after PMA--i.e., at the time of maximal DNA synthesis. The inhibition of tumor formation by HU in the two-stage promotion experiment did not prevent a subsequent promotion of cells by repetitive PMA treatment. This indicates that the inhibitory effect of HU was due neither to cytotoxicity (killing of initiated cells) nor to an interference with initiation. The data indicate that epidermal DNA synthesis is obligatory for PMA-induced first-stage promotion. The causal relationship between both events remains to be established.
Pilus genes of Neisseria gonorrheae: chromosomal organization and DNA sequence.
Authors: Meyer T F , Billyard E , Haas R , Storzbach S , So M
Published: May 2006

We have mapped two regions of the Neisseria gonorrheae genome, pilE1 and pilE2, which are involved in pilus expression. When the cells are in the piliated P+ state, these two loci carry sequences necessary for pilin production. A silent locus, pilS1, also maps near pilE1 and pilE2. pilS1 contains structural gene information but lacks pilus promoter sequences. The pilus gene sequences in pilE1 and pilE2 are identical in strain MS11.
Human placental syncytiotrophoblastic Mr 75,000 polypeptide defined by antibodies to a synthetic peptide based on a cloned human endogenous retroviral DNA sequence.
Authors: Suni J , Närvänen A , Wahlström T , Aho M , Pakkanen R , Vaheri A , Copeland T , Cohen M , Oroszlan S
Published: May 2006

Antibodies to a synthetic undecapeptide (NH2-Cys-Glu-Asn-Pro-Ser-Gln-Phe-Tyr-Glu-Arg-Leu-COOH), the sequence (except cysteine) of which was deduced from a previously reported cloned human retroviral gag-gene-related DNA sequence erv-1, were raised in rabbits. In immunohistochemical staining these antibodies reacted with normal human first-trimester placentas and with blighted ova and benign and malignant trophoblastic tumors (hydatidiform and destructive moles, choriocarcinomas) but not with any other normal embryonic or adult tissues tested. In all tissues the reactivity was mainly confined to cells with trophoblastic morphology. In immunoblotting the antibody detected an Mr 75,000 polypeptide in syncytiotrophoblasts isolated from first-trimester placentas and in three different lines of cultured choriocarcinoma cells. The undecapeptide blocked the reactivity of the antibody.
H2-uptake activity of the MoFe protein component of Azotobacter vinelandii nitrogenase.
Authors: Wang Z C , Watt G D
Published: May 2006

The MoFe protein from Azotobacter vinelandii catalyzes the reduction of methylene blue and other oxidants by H2 under anaerobic conditions. H2 uptake followed manometrically or by 3H2 transfer from the gas to aqueous phase occurs concomitantly with methylene blue disappearance monitored optically or coulometrically. The stoichiometry was found to be 1:1 methylene blue/H2. MoFe protein oxidized by transfer of approximately 4 e- seems to be the redox state of the protein most active in the catalytic step, although both the S2O4(2-)-reduced and 6-e- oxidized state have been shown to react, but at a much lower rate. The presence of H2 in the atmosphere above the MoFe protein offers increased protection against O2 inactivation.
Glycosylated ovine prolactin.
Authors: Lewis U J , Singh R N , Lewis L J , Seavey B K , Sinha Y N
Published: May 2006

A modification of prolactin, in which the asparagine at position 31 carries a carbohydrate unit, was isolated from ovine pituitary glands. Sequence and amino acid analyses identified the point of linkage of the carbohydrate. Glucosamine was found in acid hydrolysates, an indication that the carbohydrate is attached through N-acetylglucosamine. The glycosylated prolactin binds to concanavalin A and lentil lectin and is eluted with methyl alpha-D-mannopyranoside. During gel electrophoresis in sodium dodecyl sulfate, the glycosylated hormone migrates as a Mr 25,000 protein; prolactin has a Mr of 23,000. The modified prolactin had a potency of 20 international units/mg, approximately equal to 60% the potency of a reference prolactin preparation when measured by the pigeon crop sac assay. In a radioimmunoassay, the glycosylated form had only 34% the immunoreactivity of ovine prolactin.
Chimeric influenza virus hemagglutinin containing either the NH2 terminus or the COOH terminus of G protein of vesicular stomatitis virus is defective in transport to the cell surface.
Authors: McQueen N L , Nayak D P , Jones L V , Compans R W
Published: May 2006

Chimeric cDNA clones of influenza virus hemagglutinin (HA) were constructed in which the DNA encoding either the NH2 terminus or the COOH terminus of HA was replaced with that of a vesicular stomatitis virus G protein. The chimeric cDNAs (GHA or HAG) were expressed in CV1 cells using the simian virus 40 late replacement promoter. Both chimeric proteins are synthesized, glycosylated, and transported to the rough endoplasmic reticulum. These results show that the NH2-terminal sequences of vesicular stomatitis virus G protein can provide a signal function for translocation and the COOH-terminal sequences can provide the anchor function for the influenza virus HA, when substituted for similar sequences. However, the chimeric glycoproteins were not transported to the Golgi complex or the plasma membrane. The implication of these results in translocation, sorting, and transport processes is discussed.
Association of cytokeratin p39 with DNA in intact Novikoff hepatoma cells.
Authors: Ward W S , Schmidt W N , Schmidt C A , Hnilica L S
Published: May 2006

The relationship between the rat tumor-associated cytokeratin p39 (Mr 39,000) and cellular DNA has been studied in intact cells. Using a DNA-protein crosslinking technique, incubation with cis-dichlorodiammineplatinum, we present evidence for the association of p39 with DNA in intact Novikoff ascites hepatoma cells. The cells were treated with cis-dichlorodiammineplatinum and solubilized in NaDodSO4-containing buffer, and the DNA was pelleted by high-speed centrifugation. By immunotransfer analysis, the cytokeratin was found in the DNA pellet of the crosslinked samples while absent from the controls. This result was further substantiated by CsCl density-gradient centrifugation. Collectively, these results suggest a cytokeratin-DNA association at the filament binding sites on or near the nuclear lamina.
Role of carbon monoxide dehydrogenase in the autotrophic pathway used by acetogenic bacteria.
Authors: Pezacka E , Wood H G
Published: May 2006

Anaerobic acetogenic bacteria utilize a pathway of autotrophic growth that differs from any previously described. One part of the pathway involves the reduction of CO2 to formate and its subsequent conversion to the methyl moiety of methyltetrahydrofolate. The second part involves the formation of a one-carbon intermediate from CO, CO2 and H2, or the carboxyl of pyruvate and combination of the intermediate with CoA and methyltetrahydrofolate mediated by a corrinoid enzyme to yield acetyl-CoA. Our studies have been concerned with this latter portion of the pathway and we have proposed that a one-carbon intermediate is formed via carbon monoxide dehydrogenase. It remained possible, however, that the function of the CO dehydrogenase is to reduce the cobalt of the corrinoid enzyme to Co+, which is required for it to act as a methyl acceptor, and that the dehydrogenase is not involved directly in the formation of a C1 intermediate. All the enzymes required for the synthesis of acetyl-CoA from CO and methyltetrahydrofolate or from methyltetrahydrofolate and the carboxyl of pyruvate have now been purified. With these purified enzymes, it has been possible to show that CO dehydrogenase is essential for acetyl-CoA synthesis with CO as the substrate under conditions in which the cobalt of the corrinoid is reduced by other means. In addition, using pyruvate ferredoxin oxidoreductase, it has been shown that a 14C1-CO dehydrogenase complex is formed from [1-14C]pyruvate. Furthermore, [1-14C]acetyl-CoA was synthesized using the 14C1-CO dehydrogenase complex. Thus the evidence appears conclusive that CO dehydrogenase has a direct role in the formation of the carboxyl of acetyl-CoA.
Invariant temporal order of replication of the four actin gene loci during the naturally synchronous mitotic cycles of Physarum polycephalum.
Authors: Pierron G , Durica D S , Sauer H W
Published: May 2006

The chronological sequence of replication for the four unlinked actin gene loci of Physarum has been established. Southern hybridization analysis of density-labeled, bromodeoxyuridine-substituted DNA isolated from defined periods of S phase demonstrates that three actin loci (ardB, ardC, ardD) are duplicated early, corresponding to the first 10% of the genome. The fourth locus (ardA) replicates later, between 80 and 100 min into S phase and after 75% of DNA synthesis is completed. Gene-dosage determinations, based on the quantitation of hybridization signals from DNAs isolated from various times during S phase, confirm the results obtained with bromodeoxyuridine-substituted DNA and increase the temporal resolution. The chronological order of replication in the macroplasmodium appears constant through two consecutive cell cycles and after prolonged growth in suspension culture. The precise chronology of DNA synthesis at the gene level extends to the coordinate replication of allele pairs.
Brain- and liver cell-derived factors are required for growth of human endothelial cells in serum-free culture.
Authors: Hoshi H , McKeehan W L
Published: May 2006

A test of the mitogenic activity of greater than 40 purified and crude sources of hormones and growth factors revealed that epidermal growth factor, high density lipoprotein, an extract of bovine pituitary, hypothalamus, or whole brain, and the medium conditioned by differentiated human hepatoma cells were mitogenic for cultured endothelial cells derived from human umbilical vein. The four active agents combined with an improved nutrient medium and a collagen- or fibronectin-coated culture surface supported the growth of the endothelial cell population at a rate of 0.70-0.80 generations per day at both low and high cell densities in serum-free medium. The brain-derived activity exhibited properties reported by Maciag et al. [Maciag, T., Hoover, A. & Weinstein, R. (1982) J. Biol. Chem. 257, 5333-5336] and Gordon et al. [Gordon, P. B., Sussman, I. I. & Hatcher, V. B. (1983) In Vitro 19, 661-671]. The liver cell-derived activity was a specific product of differentiated hepatoma cells. The medium from HeLa cells, relatively undifferentiated rat liver cell lines, and human fibroblasts was inactive. Purified plasma proteins of liver origin could not substitute for the hepatoma cell-conditioned medium. The hepatoma cell-derived activity was non-dialyzable, heat-labile, stable between pH 4 to 11, inactivated by trypsin and mercaptoethanol treatment, and stable after treatment with 6 M urea and phenylmethylsulfonyl fluoride. The results provide a simplified model for elucidation of the endocrinology of human endothelial cell growth, function, and aging. We suggest an endocrine role of both the nervous system and liver in the regeneration of endothelial cells.
Purification and chemical characterization of the receptor for interleukin 2 from activated human T lymphocytes and from a human T-cell lymphoma cell line.
Authors: Urdal D L , March C J , Gillis S , Larsen A , Dower S K
Published: May 2006

The cell surface receptor for interleukin 2 plays a central role in the biology of this T-cell growth factor. A combination of affinity chromatography, high-performance liquid chromatography, and NH2-terminal protein sequencing was used to purify and chemically characterize the interleukin 2 receptor both from phytohemagglutinin-activated T cells and from the human T-cell lymphoma cell line HuT-102. The receptor isolated from HuT-102 cells was purified 16,000-fold to homogeneity as evidenced by (i) a final specific activity close to the theoretical specific activity of 18,182 fmol of receptor per microgram of protein, (ii) a single band on polyacrylamide gel electrophoresis with an Mr of 55,000, and (iii) a unique, unambiguous NH2-terminal protein sequence. The receptor purified from phytohemagglutinin-activated T lymphocytes had an Mr of 60,000 but it had the same NH2-terminal protein sequence.
Isolated rat hepatocyte couplets: a primary secretory unit for electrophysiologic studies of bile secretory function.
Authors: Graf J , Gautam A , Boyer J L
Published: May 2006

Hepatocyte couplets were isolated by collagenase perfusion from rat liver. Between adjacent cells, the bile canaliculus forms a closed space into which secretion occurs. As in intact liver, Mg2+-ATPase is localized at the canalicular lumen, the organic anion fluorescein is excreted, and secretion is modified by osmotic gradients. By passing a microelectrode through one cell into the canalicular vacuole, a transepithelial potential profile was obtained. In 27 cell couplets the steady-state intracellular (-26.3 +/- 5.3 mV) and intracanalicular (-5.9 +/- 3.3 mV) potentials were recorded at 37 degrees C with reference to the external medium. Input resistances were determined within the cell (86 +/- 23 M omega) and in the bile canalicular lumen (32 +/- 17 M omega) by passing current pulses through the microelectrode. These data define electrical driving forces for ion transport across the sinusoidal, canalicular, and paracellular barriers and indicate ion permeation across a leaky paracellular junctional pathway. These findings indicate that the isolated hepatocyte couplet is an effective model for electrophysiologic studies of bile secretory function.
Identification and partial purification of the cardiac sodium-calcium exchange protein.
Authors: Hale C C , Slaughter R S , Ahrens D C , Reeves J P
Published: May 2006

Cardiac sarcolemmal vesicles were solubilized in 2% sodium cholate/0.5 M NaCl/soybean phospholipids (25 mg/ml), and reconstituted following the procedure of Miyamoto and Racker [Miyamoto, H. & Racker, E. (1980) J. Biol. Chem. 255, 2656-2658]. Initial rates of Na-Ca exchange in the reconstituted proteoliposomes were 17-fold higher than in the native vesicles. Total recovery of exchange activity often exceeded 100%, indicating that the exchange system had been activated by the reconstitution procedure. Examination of native and reconstituted vesicles by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed two protein bands that were substantially enriched in the reconstituted system: one at 48 kDa and a diffuse band centered at 82 kDa. A cholate extract of the vesicles was applied to a Sephacryl S-300 column and the various eluted fractions were reconstituted, assayed for Na-Ca exchange and examined by polyacrylamide gel electrophoresis. The activity profile, after correcting for loss of activity on the column, showed a good correlation with the presence of the diffuse 82-kDa band. A cholate extract of the vesicles was treated with Pronase (1 mg/ml) for 10 min at 37 degrees C and reconstituted using a procedure similar to that described by Wakabayashi and Goshima [Wakabayashi, S. & Goshima, K. (1982) Biochim. Biophys. Acta 693, 125-133]. The resulting proteoliposomes catalyzed Na-Ca exchange with a specific activity 30- to 100-fold greater than that of native vesicles. Upon examination by polyacrylamide gel electrophoresis, these proteoliposomes exhibited a single major band at 82 kDa, with several minor bands at lower molecular weights that migrated identically to the components of Pronase. The results suggest that the 82-kDa band represents the cardiac Na-Ca exchange protein.
Stimulatory GTP regulatory unit Ns and the catalytic unit of adenylate cyclase are tightly associated: mechanistic consequences.
Authors: Arad H , Rosenbusch J P , Levitzki A
Published: May 2006

Turkey erythrocyte membranes were solubilized in the mild detergent octylpenta(oxyethylene) [CH3(CH2)7-(OCH2CH2)5OH], which possesses a high critical micelle concentration (approximately equal to 6 mM) and forms small, dynamic micelles. Both the native enzyme Ns(GDP) X C and the p[NH]ppG-preactivated species N's X p[NH]ppG X C' were found to possess the same molecular mass of 215,000 +/- 17,000 daltons. Both enzyme species migrate as a tight complex between Ns and C on both gel permeation columns and on DEAE-Sephacel columns in detergent. The two functional units, Ns and C, remain associated even in dilute detergent solutions and throughout a 300- to 400-fold purification in octylpoly(oxyethylene). These results strongly support the view that Ns and C do not come apart during the process of enzyme activation by the beta-adrenergic receptor. Furthermore, these results strongly support our previous assertion that the beta-adrenergic receptor activation of adenylate cyclase is by a simple "collision coupling" between the receptor and NsC. These results are not compatible with shuttle mechanisms that postulate that Ns physically migrates from the receptor R to the catalytic unit C and back during the activation cycle, as suggested by Citri and Schramm [Citri, Y. & Schramm, M. (1980) Nature (London) 287, 297-300] and by De Lean et al. [De Lean, A., Stadel, J. M. & Lefkowitz, R. J. (1980) J. Biol. Chem. 255, 5108-5117].
Triplication of a four-gene set during evolution of the goat beta-globin locus produced three genes now expressed differentially during development.
Authors: Townes T M , Fitzgerald M C , Lingrel J B
Published: May 2006

Distinct hemoglobins are synthesized in goats at different stages of development, similar to humans. Embryonic hemoglobins (zeta 2 epsilon 2 and alpha 2 epsilon 2) are synthesized initially and are followed sequentially by fetal (alpha 2 beta F2), preadult (alpha 2 beta C2), and adult (alpha 2 beta A2) hemoglobins. To help understand the basis of these switches, the genes of the beta-globin locus have been cloned and their linkage arrangement has been determined by the isolation of lambda phage carrying overlapping inserts of genomic goat DNA. The locus extends over 120 kilobase pairs and consists of 12 genes arranged in the following order: epsilon I-epsilon II-psi beta X-beta C-epsilon III-epsilon IV-psi beta Z-beta A-epsilon V-epsilon VI-psi beta Y-beta F. Comparison of the nucleotide sequence of the 12 genes shows that the locus is organized into three homologous four-gene sets that presumably evolved by the triplication of an ancestral set of four genes (epsilon-epsilon-psi beta-beta). Interestingly, the three genes (beta C, beta A, and beta F) located at the ends of the four-gene sets are expressed at different stages of development. Therefore, the goat beta F-, beta C-, and beta A-globin genes appear to have evolved by a mechanism that includes the triplication of 40-50 kilobase pairs of DNA and the recruitment of newly formed genes for expression in fetal, preadult, and adult life.
Heat shock regulatory gene htpR influences rates of protein degradation and expression of the lon gene in Escherichia coli.
Authors: Goff S A , Casson L P , Goldberg A L
Published: May 2006

Upon a shift to high temperature, Escherichia coli increase their rate of protein degradation and also the expression of a set of "heat shock" genes. Nonsense mutants of htpR (also called hin), suppressed by a temperature-sensitive suppressor, show lower expression of heat shock genes at 30 degrees C and fail to respond to a shift to 42 degrees C. These mutants were found to have a lower capacity to degrade abnormal or incomplete proteins than that of wild-type cells. This reduction in proteolysis equals or exceeds that in lon mutants, which encode a defective ATP-dependent protease, protease La, and is particularly large in htpR lon double mutants. The activity of protease La was higher in wild-type cells than in htpR mutants grown at 30 degrees C and increased upon shift to 42 degrees C only in the wild type. To determine whether htpR influences transcription of the lon gene, a lon-lacZ operon fusion was utilized. Introduction of the htpR mutation reduced transcription from the lon promoter at 30 degrees C and 37 degrees C. This defect was corrected by a plasmid (pFN97) carrying the wild-type htpR allele. Induction of the heat shock response with ethanol had little or no effect in htpR mutants but stimulated lon transcription 2-3 fold in wild-type cells and htpR cells carrying pFN97. Thus, lon appears to be a heat shock gene, and increased synthesis of protease La under stressful conditions may help to prevent the accumulation of damaged cellular protein.
Chimeric human antibody molecules: mouse antigen-binding domains with human constant region domains.
Authors: Morrison S L , Johnson M J , Herzenberg L A , Oi V T
Published: May 2006

We have created mouse-human antibody molecules of defined antigen-binding specificity by taking the variable region genes of a mouse antibody-producing myeloma cell line with known antigen-binding specificity and joining them to human immunoglobulin constant region genes using recombinant DNA techniques. Chimeric genes were constructed that utilized the rearranged and expressed antigen-binding variable region exons from the myeloma cell line S107, which produces an IgA (kappa) anti-phosphocholine antibody. The heavy chain variable region exon was joined to human IgG1 or IgG2 heavy chain constant region genes, and the light chain variable region exon from the same myeloma was joined to the human kappa light chain gene. These genes were transfected into mouse myeloma cell lines, generating transformed cells that produce chimeric mouse-human IgG (kappa) or IgG (kappa) anti-phosphocholine antibodies. The transformed cell lines remained tumorigenic in mice and the chimeric molecules were present in the ascitic fluids and sera of tumor-bearing mice.
Pharmacomechanical coupling in smooth muscle may involve phosphatidylinositol metabolism.
Authors: Baron C B , Cunningham M , Strauss J F , Coburn R F
Published: May 2006

Cholinergic contraction of canine trachealis muscle, a contraction that primarily utilizes membrane potential-independent mechanisms for activating contractile proteins (pharmacomechanical coupling), is associated with a decline in the phosphatidylinositol pool, an increase in the phosphatidic acid and diacylglycerol pools, and an increased incorporation of 32PO4 into phosphatidylinositol. We found that these changes occur during development of the contraction and during maintenance of tension and are independent of membrane depolarization or increases in cytosolic Ca2+ concentration. These findings suggest that phosphatidylinositol turnover may be part of a receptor transduction process controlling receptor-operated Ca2+ channels or other membrane potential-independent mechanisms involved in pharmacomechanical coupling in smooth muscle.
Nonrandom binding of the carcinogen N-hydroxy-2-acetylaminofluorene to repetitive sequences of rat liver DNA in vivo.
Authors: Gupta R C
Published: May 2006

We have examined the distribution of individual adducts in repetitive DNA sequences of rat liver in vivo after a single dose of the carcinogen N-hydroxy-2-acetyl-aminofluorene. Repetitive fragments [82, 125, 179, 225, and 370 base pairs (bp)] were isolated by digestion of hepatic DNA with HindIII restriction endonuclease (EC 3.1.23.21) and gel electrophoresis. As assayed by 32P postlabeling, no qualitative differences were observed between the DNA-bound metabolites in repetitive sequences and total DNA, but preferential binding to these sequences occurred. After 1 day of treatment, the amounts of N-hydroxy-2-acetylaminofluorene-induced adducts were found to be 13.8, 2.0, and 3.0 times higher in 179-, 225-, and 370-bp repeats, respectively, than in total DNA, while 82- and 125-bp repeats showed no differences. The relative distribution of individual adducts varied among the various sequences. After 9 days, all five sequences showed 1.3-1.7 times higher binding as compared to total DNA. In contrast, a random binding was observed when DNA reacted in vitro with an active metabolite, N-acetoxy-2-acetylaminofluorene. Taken together, these results suggest that the enrichment and differential excision of adducts in the repetitive DNA sequences may be a function of the nuclear organization of DNA. This application of the 32P assay constitutes a means to study the DNA damage and excision repair in vivo in chromatin structural components, including transcribed and nontranscribed multiple-copy genes, in a much more sensitive and precise way than has been hitherto possible.
RNA structure analysis using methidiumpropyl-EDTA.Fe(II): a base-pair-specific RNA structure probe.
Authors: Vary C P , Vournakis J N
Published: May 2006

Methidiumpropyl-EDTA.Fe(II) [MPE.Fe(II)] in the presence of dithiothreitol, is shown to cleave phenylalanine-accepting tRNA (tRNAPhe) in a structure-specific fashion. Molar ratios of MPE.Fe(II) to tRNAPhe of less than 1 preferentially cleave phosphodiester bonds known to occur in double-stranded regions of the tRNAPhe molecule. Microdensitometric analysis of autoradiograms of MPE.Fe(II) cleavage products following gel electrophoresis reveals a correspondence between preferred sites of MPE.Fe(II) cleavage and sites in tRNAPhe most sensitive to cobra venom ribonuclease, a double-strand-specific endoribonuclease. Conversely, sites of cleavage by the single-strand-specific S1 nuclease correspond to those nucleotides that are least susceptible to MPE.Fe(II) hydrolysis. Sensitive helical regions in tRNAPhe include the dihydrouracil and the "T psi C" stems, which cannot be detected by cobra venom ribonuclease because of steric constraints. Phosphodiester bonds within the T psi C and dihydrouracil loop regions, which are not detected by S1 nuclease under rigorously controlled digestion conditions, are revealed by inference from their relative insensitivity to MPE.Fe(II). These results demonstrate the utility of MPE.Fe(II) as a general small molecular weight probe of RNA structure, having a greater accessibility to base-paired regions than do the more bulky enzymic probes.
Cloning and sequencing of the genes encoding the alpha and beta subunits of C-phycocyanin from the cyanobacterium Agmenellum quadruplicatum.
Authors: Pilot T J , Fox J L
Published: May 2006

Synthetic oligonucleotide probes were used to identify a cloned DNA fragment from the cyanobacterium Agmenellum quadruplicatum that contains the genes for the alpha and beta subunits of C-phycocyanin. The coding region for the alpha-subunit gene begins 108 base pairs downstream from the 3' end of the beta-subunit structural gene. The sequences of the coding regions for both genes have been determined as well as 379 base pairs of 5' flanking region, 204 base pairs of 3' flanking region, and the 108 base pairs between the two genes. The site of transcriptional initiation is located approximately equal to 325 base pairs upstream from the beta-subunit gene, and an open reading frame 114 base pairs long is found within this region. The significance of this additional open reading frame is not yet known. The derived amino acid sequences for both C-phycocyanin subunits were compared with other known C-phycocyanin sequences for homology. Homologies between the A. quadruplicatum alpha subunit and alpha subunits from other species were approximately equal to 70%, as were homologies between the A. quadruplicatum beta subunit and other beta subunits. Homologies between the various alpha and beta subunits were 21%-27%. Codon usage for both the C-phycocyanin alpha- and beta-subunit genes shows asymmetries for many amino acids that correspond closely to those seen in highly expressed Escherichia coli genes.
Mechanisms of glycosylation and sulfation in the Golgi apparatus: evidence for nucleotide sugar/nucleoside monophosphate and nucleotide sulfate/nucleoside monophosphate antiports in the Golgi apparatus membrane.
Authors: Capasso J M , Hirschberg C B
Published: May 2006

The mechanism of translocation in vitro of sugar nucleotides and adenosine 3'-phosphate 5'-phosphosulfate (PAPS) into the lumen of rat liver Golgi apparatus vesicles has been studied. It has been previously shown that the Golgi apparatus membrane has specific carrier proteins for PAPS and sugar nucleotides. We now report that translocation of the above nucleotide derivatives across Golgi membranes occurs via a coupled equimolar exchange with the corresponding nucleoside monophosphates. An initial incubation of Golgi vesicles with GDP-fucose radiolabeled in the guanidine ring resulted in accumulation within the lumen of radiolabeled GMP. Exit of GMP from these vesicles was specifically dependent on the entry of (additional) GDP-fucose into the vesicles (GDP-mannose and other sugar nucleotides had no effect). GDP-fucose-stimulated exit of GMP was temperature dependent, was blocked by inhibitors of GDP-fucose transport, such as 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, and appeared to be equimolar with GDP-fucose entry. Preliminary evidence for specific, equimolar exchange of CMP-N-acetylneuraminic acid with CMP, PAPS with 3'-AMP, and UDP-galactose and UDP-N-acetylglucosamine with UMP was also obtained. These results strongly suggest the existence of different antiport proteins within the Golgi membrane that mediate the 1:1 exchange of sugar nucleotides or PAPS with the corresponding nucleoside monophosphate. Such proteins may have a regulatory role in glycosylation and sulfation reactions within the Golgi apparatus.
Escherichia coli translational initiation factor IF3: a unique case of translational regulation.
Authors: Gold L , Stormo G , Saunders R
Published: May 2006

The Escherichia coli translational initiation factor IF3 is encoded by an mRNA that has an unusual ribosome binding site. We have explored a mechanism that may account for the translation of IF3 and that provides regulation of the quantity of IF3 relative to ribosomes.
A correlation between proton pumping and the bacteriorhodopsin photocycle.
Authors: Li Q , Govindjee R , Ebrey T G
Published: May 2006

In an attempt to establish a relationship between proton pumping and the photocycle intermediates of bacteriorhodopsin, we have studied the effects of pH and temperature on flash-induced proton pumping and the photointermediates O640 and M412. The relative quantum yield of flash-induced proton pumping is both pH and temperature dependent. It is high in the acid pH range and at low temperatures but decreases in the basic pH range and at high temperatures. The decay of M412 is biphasic. The amplitude of the slowly decaying component (Ms) was found to be pH dependent with a pK similar to that of the delta pH. The pH dependence of the fast-decaying component (Mf) is opposite to that of Ms and delta pH. Like that of Ms, the amplitude of O640 is high in the acid pH range, but unlike the amplitude of Ms, it declines very rapidly at pHs greater than 6.5; the amplitude of O640 becomes zero around pH 8. The temperature dependence of the amplitude of Ms was found to be similar to that of delta pH, being high at low temperatures and low in the high-temperature range. The temperature dependence of Mf and O640 is opposite to that of delta pH and Ms. Whereas, O640 and Ms seem to be likely candidates (from the pH dependence) for coupling between proton pumping and the photocycle, Ms is more likely because (i) the relative quantum yield of both Ms and delta pH seem not to go to zero at higher pHs and (ii) Ms and delta pH have similar temperature dependence.
Structural stability in the 4-zinc human insulin hexamer.
Authors: Smith G D , Swenson D C , Dodson E J , Dodson G G , Reynolds C D
Published: May 2006

X-ray studies on human insulins prepared by semisynthetic and biosynthetic methods have recently been undertaken. Human insulin differs from porcine insulin only at the COOH terminus of the B-chain. The present study reports the crystal structure of 4-zinc human insulin, which is used clinically as a slow-acting preparation. The structure has been refined, using 1.85-A resolution data, to a residual of 0.173. The unit cell is rhombohedral, space group R3, with hexagonal cell constants a = 80.953 and c = 37.636 A, and it is nearly isomorphous with that of 4-zinc porcine insulin. As a result of a conformational change of the first eight residues of the B-chain of molecule 1 from an extended conformation observed in the 2-zinc structure to an alpha-helical one, the coordination around one of the zinc ions on the 3-fold axis has changed, an additional zinc ion in a general position is bound by the hexamer, and additional hydrogen-bonded interactions help stabilize dimer and hexamer formation. Unlike the surface of the 2-zinc insulin hexamer, which possesses a shallow depression containing a zinc ion and its coordinating water molecules, the 4-zinc human insulin hexamer contains a zinc and chloride ion at the bottom of an 8-A tunnel produced by three parallel alpha-helices. These alpha-helices shield the zinc ion from the environment, decreasing the rate of dissociation of the hexamer, and provide an explanation for the slow-acting aspect of the 4-zinc crystalline form.