bluuebunny/crossref_metadata_2025 huggingface dataset:
Continuous production of erythropoietin by an established human renal carcinoma cell line: development of the cell line.
Authors: Sherwood J B , Shouval D
Published: May 2006

Establishment of a stable, transformed human renal carcinoma cell line that produces erythropoietin in vitro and has maintained this function continuously since 1981 and for greater than 150 passages in monolayer culture was accomplished by transplantation of human renal clear cell carcinoma tissue from a patient with erythrocytosis into an immunosuppressed athymic mouse. In addition to its immunocrossreactivity with native human urinary erythropoietin, the tumor erythropoietin demonstrates biological activity in the in vitro mouse erythroid colony-forming unit assay and in tumor-bearing nude mice. The cloned renal carcinoma cell line has an abnormal human karyotype and has ultrastructural features characteristic of human renal clear cell carcinoma. This cell line provides a reproducible model system for the production of an erythropoietin-like material and for the study of its synthesis and secretion.
In vitro secondary activation (memory effect) of avian vitellogenin II gene in isolated liver nuclei.
Authors: Jost J P , Moncharmont B , Jiricny J , Saluz H , Hertner T
Published: May 2006

The vitellogenin II gene is specifically reactivated in vitro (secondary stimulation, memory effect) in purified liver nuclei that had ceased to express the gene in vivo a month after the roosters had received a single injection of estradiol (primary stimulation). The in vitro reactivation depends on the addition to the nuclei of nuclear and cytoplasmic extracts from estradiol-stimulated livers, polyamines (0.1-1.0 mM), and calmodulin (0.1 mM). Under identical incubation conditions the vitellogenin gene could not be reactivated in oviduct, embryonic, and immature chicken liver nuclei. Two other genes, those for ovalbumin and lysozyme, which are regulated by estradiol in the oviduct, could not be activated in the liver nuclei. The correct initiation of vitellogenin gene transcription in the liver nuclei was tested by primer extension studies. Addition of the antiestrogen tamoxifen (0.1 microM) to the system decreased vitellogenin mRNA synthesis by about 45% without affecting total RNA synthesis. Addition of quercetin (0.1 mM) and trans-flupenthixol (0.2 mM), inhibitors of nuclear protein kinase II and calmodulin-dependent kinase, respectively, inhibited the synthesis of vitellogenin mRNA by about 55% without affecting total RNA synthesis. The inhibitory effects of the antiestrogen and the kinase inhibitors were not additive, suggesting that both classes of inhibitor act on the same target or related targets. Depleting the estradiol receptors from the cell and nuclear extracts by means of estradiol-receptor antibodies covalently bound to Matrex beads reduced the stimulation of the vitellogenin gene by 40%. We conclude that in addition to the estradiol receptor and phosphorylation of nuclear protein(s) there are additional factors responsible for the in vitro secondary activation of the avian vitellogenin II gene.
Structural studies of the vacuolar membrane ATPase from Neurospora crassa and comparison with the tonoplast membrane ATPase from Zea mays.
Authors: Bowman E J , Mandala S , Taiz L , Bowman B J
Published: May 2006

The H+-translocating ATPase located on vacuolar membranes of Neurospora crassa was partially purified by solubilization in two detergents, Triton X-100 and N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, followed by centrifugation on sucrose density gradients. Two polypeptides of Mr approximately equal to 70,000 and approximately equal to 62,000 consistently migrated with activity, along with several minor bands of lower molecular weight. Radioactively labeled inhibitors of ATPase activity, N-[14C]ethylmaleimide and 7-chloro-4-nitro[14C]benzo-2-oxa-1,3-diazole, labeled the Mr approximately equal to 70,000 polypeptide; this labeling was reduced in the presence of ATP. N,N'-[14C]dicyclohexylcarbodiimide labeled a polypeptide of Mr approximately equal to 15,000. Estimation of the functional size of the vacuolar membrane ATPase by radiation inactivation gave a value of Mr 5.2 X 10(5), 10-15% larger than the mitochondrial ATPase. The Neurospora vacuolar ATPase showed no crossreactivity with antiserum to plasma membrane or mitochondrial ATPase but strongly crossreacted with antiserum against a polypeptide of Mr approximately equal to 70,000 associated with the tonoplast ATPase of corn coleoptiles. These results suggest that fungal and plant vacuolar ATPases may be large multisubunit complexes, somewhat similar to, but immunologically distinct from, known F0F1 ATPases.
An arachidonate metabolite is involved in the conversion from alpha 1- to beta-adrenergic glycogenolysis in isolated rat liver cells.
Authors: Ishac E J , Kunos G
Published: May 2006

In vitro incubation of isolated rat liver cells in a serum-free buffer leads to the suppression of the glycogenolytic effect of phenylephrine and the simultaneous emergence of a glycogenolytic response to isoproterenol within 4 hr. This time-dependent conversion of the adrenergic receptor response from alpha 1 to beta type is prevented by the presence in the incubation medium of 0.5% fatty-acid-free, but not regular, bovine serum albumin. A 20-min exposure of freshly isolated liver cells to arachidonic acid (10 micrograms/ml), but not to stearic or palmitic acid, causes an acute shift in the receptor response from alpha 1 to mixed alpha 1/beta type, similar in direction to that seen after prolonged incubation of the cells. This effect of arachidonic acid is prevented by 0.2 microM ibuprofen but not by the same concentration of nordihydroguaiaretic acid. Ibuprofen (1 microM) or indomethacin (1 microM) also inhibits the time-dependent shift in the receptor response. Actinomycin D inhibits the change in receptor response that is caused by prolonged incubation but not the change that is caused by exogenous arachidonic acid. It is proposed that the time-dependent conversion from alpha 1- to beta-adrenergic receptor-mediated glycogenolysis in isolated rat liver cells is related to a parallel increase in the phospholipase-mediated release of arachidonic acid and the subsequent formation of a key cyclooxygenase metabolite. A protein factor appears to be involved in the regulation of the release of arachidonic acid but not in the action of its metabolite. A possible mechanism by which this metabolite may regulate inverse changes in the coupling of alpha 1- and beta-receptors to postreceptor pathways is discussed.
Lateral diffusion of phospholipids in the plasma membrane of soybean protoplasts: Evidence for membrane lipid domains
Authors: Metcalf Thomas N. , Wang John L. , Schindler Melvin
Published: May 2006

Fluorescent lipid and phospholipid probes were incorporated at 4°C into soybean protoplasts prepared from cultured soybean (SB-1) cells. Fluorescence microscopy showed that the plasma membrane as well as the nucleus were labeled. Fluorescence redistribution after photobleaching (FRAP) analysis was performed on these cells at 18°C to monitor the lateral mobility of the incorporated probes. After labeling at low concentrations (40 μg/ml) of phosphatidyl-
N
-(4-nitrobenzo-2-oxa-1,3-diazolyl)ethanolamine (NBD-PtdEtn), a single mobile component was observed with a diffusion coefficient (
D
) of ≈3 × 10
-9
cm
2
/sec. After labeling at higher probe concentrations (≥100 μg/ml), two diffusing species were observed, with diffusion coefficients of ≈3 × 10
-9
cm
2
/sec (“fast”) and ≈5 × 10
-10
cm
2
/sec (“slow”). Similar results were observed with fluorescent derivatives of phosphatidylcholine and fatty acids. In contrast to these results, parallel analysis of 3T3 fibroblasts, using the same probes and conditions, yielded only a single diffusion component. These results suggest that the soybean plasma membrane may contain two distinct lipid domains in terms of lipid mobility. Consistent with this idea, experiments with soybean protoplasts yielded a single diffusion component under the following conditions: (
i
) labeling with NBD-PtdEtn (100 μg/ml), FRAP analysis at 37°C (
D
= 1.1 × 10
-8
cm
2
/sec); (
ii
) labeling with NBD-PtdEtn (100 μg/ml), FRAP analysis at 18°C in the presence of 2 mM EGTA (
D
= 4.2 × 10
-9
cm
2
/sec); (
iii
) labeling with 5-(
N
-dodecanoyl)aminofluorescein (a short-chain lipid probe), FRAP analysis at 18°C or 37°C (
D
= 2.5 × 10
-8
cm
2
/sec). These results suggest that the plasma membrane of soybean cells may contain stable immiscible domains of fluid and gel-like lipids.
A mutation that prevents glucosylation of the lipid-linked oligosaccharide precursor leads to underglycosylation of secreted yeast invertase.
Authors: Ballou L , Gopal P , Krummel B , Tammi M , Ballou C E
Published: May 2006

A mutant of Saccharomyces cerevisiae with the genotype mnn1 mnn2 mnn9 gls1 synthesizes mannoproteins with oligosaccharides having the composition Glc3Man10Glc-NAc2- owing to the mnn9 defect, which prevents synthesis of the outer chain, the mnn1 defect, which prevents branching of the core, and the gls1 mutation, which prevents deglucosylation of the resultant glycoprotein as a consequence of a defective glucosidase-I [Tsai, P.-K., Ballou, L., Esmon, B., Schekman, R. & Ballou, C. E. (1984) Proc. Natl. Acad. Sci. USA 81, 6340-6343]. (The mnn2 defect is not expressed in presence of the mnn9 mutation.) This strain spontaneously forms new colonies in which gls1 is suppressed owing to a defect in synthesis of dolichol phosphoglucose, the glucosylation substrate. The new mutant, designated mnn1 mnn2 mnn9 gls1 dpg1, synthesizes and secretes invertase (EC 3.2.1.26) that has a higher mobility on native gel electrophoresis than that made by the parent strain, the consequence of a reduction in both the size and the number of carbohydrate chains. The mannoprotein chains have the mnn1 mnn9 structure (Man10Glc-NAc2-), and the invertase is resolved by gel electrophoresis in sodium dodecyl sulfate into two major and two minor bands that represent homologs with about 4-7 carbohydrate units, in contrast to about 8-11 chains in the parent strain. Thus, the inability to glucosylate the lipid-linked precursor reduces the efficiency of glycosylation of the protein chains. The genetic defect is in synthesis of the glucose donor dolichol phosphoglucose, but the mutation is nonallelic with the reported alg5-1 mutation, which has a similar phenotype [Runge, K. W., Huffaker, T. C. & Robbins, P. W. (1984) J. Biol. Chem. 259, 412-417].
Selection and amplification of heterologous genes encoding adenosine deaminase in mammalian cells.
Authors: Kaufman R J , Murtha P , Ingolia D E , Yeung C Y , Kellems R E
Published: May 2006

We demonstrate that an adenosine deaminase (ADA) cDNA gene can function as a dominant selectable and amplifiable marker for gene transfer experiments in mammalian cells. Cells that incorporate the gene can be selected by growth in the presence of low concentrations of the ADA inhibitor 2'-deoxycoformycin with cytotoxic concentrations of adenosine or its analogue 9-beta-D-xylofuranosyl adenine. The DNA copy number of the transfected ADA minigene in the isolated transformants of Chinese hamster ovary cells can be amplified greater than 100-fold by growth in ADA selection media and increasing concentrations of 2'-deoxycoformycin. This selection scheme may allow for the introduction and subsequent amplification of heterologous DNA in a variety of mammalian cells.
Gene for lysine tRNA1 may be a progenitor of the highly repetitive and transcribable sequences present in the salmon genome.
Authors: Matsumoto K , Murakami K , Okada N
Published: May 2006

When salmon total DNA was transcribed in a HeLa cell extract, a discrete 6S RNA was found to be synthesized by RNA polymerase III. We isolated several phage clones containing the 6S RNA gene from a salmon genomic library and determined the sequences of two representative clones. The 5' part of the gene showed remarkable sequence homology with the lysine tRNA1 molecule. This homology extended to secondary structures, and the numbers of nucleotides in the stem and loop structures in the 6S RNA were the same as those in lysine tRNA1. Further, the pseudouridylic acid residues synthesized by HeLa pseudouridylate synthase(s) were determined to be at uridine-27 and uridine-55, which are the positions of these modified nucleosides in lysine tRNA1. These results strongly suggest that the lysine tRNA1 gene is a progenitor of the highly repetitive and transcribable sequences in the salmon genome.
Alpha 4, the major regulatory protein of herpes simplex virus type 1, is stably and specifically associated with promoter-regulatory domains of alpha genes and of selected other viral genes.
Authors: Kristie T M , Roizman B
Published: May 2006

Herpes simplex virus type 1 genes form at least five groups (alpha, beta 1, beta 2, gamma 1, and gamma 2) whose expression is coordinately regulated and sequentially ordered in a cascade fashion. Previous studies have shown that functional alpha 4 gene product is essential for the transition from alpha to beta protein synthesis and have suggested that alpha 4 gene expression is autoregulatory. However, the mechanism by which alpha 4 regulates gene expression remained unknown. We report that labeled DNA fragments containing promoter-regulatory domains of three alpha (alpha 0, alpha 4, and alpha 27) and a gamma 2 gene form stable complexes with proteins from infected-cell lysates as detected by a gel electrophoresis binding assay. The protein(s) exhibits sequence specificity since autologous DNA fragments but not heterologous DNA fragments, synthetic polydeoxynucleotide chains, or salmon sperm DNA competitively displace the DNA probe from the complexes. Murine monoclonal antibody to alpha 4 protein added before or after DNA-protein complex formation further retarded the electrophoretic mobility of the complexes whereas monoclonal antibody to alpha 0, alpha 27, or to a viral glycoprotein had no effect. Complexes consisting of the promoter-regulatory domain of the beta-class thymidine kinase gene and infected cell proteins were low in abundance and could be detected only in the presence of antibody to alpha 4 protein. The alpha 4 protein, therefore, forms stable complexes with promoter-regulatory domains of alpha genes and of selected other herpes simplex virus type 1 genes either alone or in combination with other proteins.
Exocrine secretion granules contain peptide amidation activity.
Authors: von Zastrow M , Tritton T R , Castle J D
Published: May 2006

Exocrine secretion granules from the rat parotid gland contain a carboxyl-terminal peptide alpha-amidation enzyme resembling closely an enzyme from the pituitary (peptidyl-glycine alpha-amidating monooxygenase) that functions in post-translational processing of secretory polypeptides within neural and endocrine secretion granules. alpha-Amidation is a characteristic (often essential) chemical feature of a variety of biologically active regulatory peptides in animals. The parotid and pituitary activities exhibit very similar ascorbate and copper requirements, pH dependence, and kinetic properties. Further, like the pituitary enzyme(s), the parotid activity is found predominantly in secretion granule content and is discharged by exocytosis. These results establish the presence of a novel enzyme in exocrine secretion granules and suggest a potential role of the L-ascorbic acid contained in parotid granules. Two additional findings--the detection of similar levels of amidation activity in purified secretion granule fractions from other exocrine glands and the observation, in parotid granule fractions, of a B-type carboxypeptidase activity similar to that involved in post-translational processing in other systems--form a rational basis for considering whether exocrine secretion granules (like their neural and endocrine counterparts) serve as post-translational processing sites. The identity and functional role of the modified polypeptides remain to be determined.
Rearrangement of chicken immunoglobulin genes is not an ongoing process in the embryonic bursa of Fabricius.
Authors: Weill J C , Reynaud C A , Lassila O , Pink J R
Published: May 2006

We report a molecular analysis of the chicken Ig loci in single bursal follicles from 3- to 7-week-old chickens. Each follicle contained between 10(5) and 3 X 10(5) cells. The Ig gene rearrangement patterns obtained were compared to the pattern observed with the corresponding total bursal DNA. The results obtained for the light chain locus imply that a very small number (two on average) of rearrangement events takes place in each follicle. For the heavy chain locus similar results were obtained, each follicle showing a more restricted pattern than the total bursa. These data favor a model in which each follicle is colonized by a very few prebursal stem cells that are committed to a particular Ig gene rearrangement at the very beginning of the development of the embryonic bursa. The role of the bursa as the organ in which such a committed stem cell population for the B-cell lineage arises is discussed.
Prostaglandin E2, a seminal constituent, facilitates the replication of acquired immune deficiency syndrome virus in vitro.
Authors: Kuno S , Ueno R , Hayaishi O , Nakashima H , Harada S , Yamamoto N
Published: May 2006

Acquired immune deficiency syndrome (AIDS)-associated virus is thought to be transmitted effectively through semen during sexual activities from male to male or from male to female. Prostaglandin (PG) E2 is one of the immunosuppressive compounds present in high concentrations in human semen. We, therefore, investigated direct effects of PGE2 and other PGs on AIDS-associated virus infection and replication in vitro. First, type III human T-lymphotropic virus (HTLV-III) was used to infect a T-cell line (MT-4) in culture. PGE2 (10 nM to 10 microM) added to the culture medium enhanced the production of infectious virus in a dose-dependent fashion. In the presence of 5 microM PGE2, 2.5-fold more virus were released from the infected MT-4 cells as compared to untreated control cells on day 3 after infection. Second, when we used an HTLV-III continuous-producer cell line (Molt-4/HTLV-III), PGE2 and PGD2 added to the culture medium increased the number of viruses released from Molt-4/HTLV-III cells. Other PGs such as PGF2 alpha and 13,14-dihydro-15-keto PGE2 did not affect the replication of HTLV-III in this system. These results indicate that some PGs including seminal PGs enhance the AIDS-associated virus replication in vitro. We propose that PGE2 in human semen might directly facilitate the infection of AIDS-associated virus and cause the efficient transmission of the virus during sexual activities.
Stable expression of selectable genes introduced into human hematopoietic stem cells by electric field-mediated DNA transfer.
Authors: Toneguzzo F , Keating A
Published: May 2006

The successful introduction of DNA into human bone marrow cells by electric field-mediated transfer was initially demonstrated by the detection of transient chloramphenicol acetyltransferase (acetyl-CoA:chloramphenicol O3-acetyltransferase, EC 2,3.1.28) activity in marrow cell extracts. To determine whether DNA was transferred into hematopoietic stem cells, human nucleated marrow cells were subjected to electroporation in the presence of a plasmid construct containing the bacterial genes conferring resistance to the neomycin analogue G418 (neo) and to mycophenolic acid (gpt). The growth of granulocyte/macrophage colonies in selective media, followed by hybridization analyses of resistant cells, established that DNA was transferred into human granulopoietic progenitor cells and was stably maintained and expressed in their differentiated progeny. Electroporation, therefore, offers the opportunity to transfer genes effectively into human hematopoietic stem cells and avoids some of the disadvantages associated with other methods of gene transfer.
Isolation and characterization of the gene encoding Drosophila DNA topoisomerase II.
Authors: Nolan J M , Lee M P , Wyckoff E , Hsieh T S
Published: May 2006

We have isolated the gene coding for the Drosophila type II DNA topoisomerase by immunochemically screening a Drosophila cDNA library constructed with a phage lambda expression vector, lambda gt11. The identity of the cloned gene is confirmed by the analysis of an antigenic fusion protein produced in Escherichia coli and by the in vitro translation of its RNA. The gene is 5.1 kilobases in length, the expected size for a gene encoding topoisomerase II (Mr 170,000), and it is divided into five exons. By in situ hybridization to the polytene chromosomes from salivary glands, we have mapped it to chromosome 2L at 37D.
Site-directed mutagenesis and the role of the oxyanion hole in subtilisin.
Authors: Bryan P , Pantoliano M W , Quill S G , Hsiao H Y , Poulos T
Published: May 2006

Oligonucleotide-directed mutagenesis was used to investigate the nature of transition state stabilization in the catalytic mechanism of the serine protease, subtilisin BPN'. The gene for this extracellular enzyme from Bacillus amyloliquefaciens has been cloned and expressed in Bacillus subtilis. In the transition state complex, the carbonyl group of the peptide bond to be hydrolyzed is believed to adopt a tetrahedral configuration rather than the ground-state planar configuration. Crystallographic studies suggest that stabilization of this activated complex is accomplished in part through the donation of a hydrogen bond from the amide side group of Asn-155 to the carbonyl oxygen of the peptide substrate. To specifically test this hypothesis, leucine was introduced at position 155. Leucine is isosteric with asparagine but is incapable of donating a hydrogen bond to the tetrahedral intermediate. The Leu-155 variant was found to have an unaltered Km but a greatly reduced catalytic rate constant, kcat, (factor of 200-300 smaller) when assayed with a peptide substrate. These kinetic results are consistent with the Asn-155 mediating stabilization of the activated complex and lend further experimental support for the transition-state stabilization hypothesis of enzyme catalysis.
Membrane vesicles from multidrug-resistant human cancer cells contain a specific 150- to 170-kDa protein detected by photoaffinity labeling.
Authors: Cornwell M M , Safa A R , Felsted R L , Gottesman M M , Pastan I
Published: May 2006

Multiple drug resistance of tumor cells is a common problem in cancer therapy. We have demonstrated that membrane vesicles from highly multidrug-resistant human KB carcinoma cell lines exhibit increased specific and saturable binding of vinblastine. To identify the molecules that bind vinblastine, membrane vesicles from multidrug-resistant cells were exposed to two analogs of vinblastine, N-(p-azido-[3,5-3H]benzoyl)-N'-(beta-aminoethyl)vindesine and N-(p-azido-[3-125I]salicyl)-N'-(beta-aminoethyl)vindesine, that could be photoactivated. Our studies show the specific labeling of a 150- to 170-kDa protein in membrane vesicles from two independently selected multidrug-resistant KB cell lines, which was not seen in drug-sensitive parental or revertant cell lines. The labeling of the high molecular weight protein was inhibited in a dose-dependent manner by vinblastine with half-maximal inhibition at about 1 microM. Photolabeling was also inhibited by 100 microM vincristine or 100 microM verapamil but not by 100 microM colchicine or 100 microM dexamethasone. The data suggest that the 150- to 170-kDa protein may play an important role in the multidrug-resistance phenotype.
A unique serotonin receptor in choroid plexus is linked to phosphatidylinositol turnover.
Authors: Conn P J , Sanders-Bush E , Hoffman B J , Hartig P R
Published: May 2006

A novel serotonergic binding site, the 5-HT1C site, has been characterized recently in choroid plexus and several brain regions. The biochemical and physiological roles of this site have not been previously described. In this report we show that serotonin (5-hydroxytryptamine, 5-HT) stimulates phosphatidylinositol turnover in rat choroid plexus. The pharmacology of serotonin-stimulated phosphatidylinositol hydrolysis in choroid plexus was compared to the pharmacology in cerebral cortex, where this response is mediated by the serotonin 5-HT2 receptor. Serotonin increased phosphatidylinositol turnover in choroid plexus by 6-fold and in cerebral cortex by 2.5-fold. Serotonin was greater than 10-fold more potent in choroid plexus (EC50 = 46 nM) than in cerebral cortex (EC50 = 540 nM). The serotonin antagonists ketanserin, mianserin, and spiperone inhibited the response in the two tissues with different potencies. In cerebral cortex all three exhibited nanomolar affinities consistent with their potencies at the 5-HT2 site. In choroid plexus, however, the rank order (mianserin greater than ketanserin much greater than spiperone) and absolute potencies were consistent with binding to the 5-HT1C site. These data suggest that the 5-HT1C site in choroid plexus is a functional receptor that utilizes phosphatidylinositol turnover as its biochemical effector system.
Translational regulation is responsible for growth-rate-dependent and stringent control of the synthesis of ribosomal proteins L11 and L1 in Escherichia coli.
Authors: Cole J R , Nomura M
Published: May 2006

The physiological importance of translational regulation in controlling the synthesis of ribosomal proteins from the L11 ribosomal protein operon was determined for the classical regulatory phenomena of growth rate dependence and stringent control. Translational regulation of the L11 operon by ribosomal protein L1, the L11 operon-specific translational repressor protein, was abolished by introducing a chromosomal mutation that causes an alteration of the site where L1 interacts with L11 operon mRNA. It was found that abolishing translational regulation of the L11 operon also abolished growth-rate-dependent regulation and stringent control of the L11 operon ribosomal proteins without affecting the normal regulation of ribosomal proteins from other operons that are not regulated by L1. These results show that both growth-rate-dependent control and stringent control of ribosomal protein synthesis in the L11 operon are a direct result of translational regulation.
Purification to apparent homogeneity of a mu-type opioid receptor from rat brain.
Authors: Cho T M , Hasegawa J , Ge B L , Loh H H
Published: May 2006

A mu-opioid-specific receptor was purified to apparent homogeneity from rat brain membranes by 6-succinylmorphine affinity chromatography, gel filtration, wheat germ agglutinin affinity chromatography, and isoelectric focusing. The purified receptor had a molecular weight of 58,000 as determined by NaDodSO4/polyacrylamide gel electrophoresis and was judged to be homogeneous by the following criteria: (i) a single band was detected by autoradiography after NaDodSO4/polyacrylamide gel electrophoresis of 125I-labeled receptor and (ii) the purified preparation had a specific opioid-binding activity of 17,720 pmol/mg of protein, close to the theoretical value. In addition, the Mr 58,000 value agrees closely with that determined by covalently labeling purified receptor with bromoacetyl-[3H]dihydromorphine or with 125I-labeled beta-endorphin and dimethyl suberimidate.
Secretion of beta-lactamase into the periplasm of Escherichia coli: evidence for a distinct release step associated with a conformational change.
Authors: Minsky A , Summers R G , Knowles J R
Published: May 2006

The secretion of beta-lactamase (EC 3.5.2.6) into the periplasm of Escherichia coli has been followed by pulse-chase labeling at 15 degrees C. Though the periplasmic fraction contains only the mature form of the enzyme, the spheroplast fraction contains the completed precursor and a hitherto undocumented processed form. When whole spheroplasts are treated with trypsin, the processed form in this fraction is completely digested. This is in contrast to the native mature enzyme localized in the periplasm, which is trypsin resistant. The beta-lactamase is evidently processed after translocation to a trypsin-sensitive form that is transiently bound to the periplasmic face of the inner membrane. The release of this processed form into the periplasm occurs concomitantly with a conformational change that results in the soluble, catalytically active, trypsin-resistant structure.
Genetic definition of the translational operator of the threonine-tRNA ligase gene in Escherichia coli.
Authors: Springer M , Graffe M , Butler J S , Grunberg-Manago M
Published: May 2006

The Escherichia coli gene thrS that codes for threonine-tRNA ligase (tRNAThr ligase, formerly threonine-tRNA synthetase, EC 6.1.1.3) has previously been shown to be negatively autoregulated at the level of translation. Here we describe the use of several thrS-lac gene fusions to isolate cis-acting regulatory mutations that increase the translation but not the transcription of the thrS gene. These mutations lead to a total loss of control of repression and derepression of thrS. DNA sequence analysis locates the mutations between 10 and 40 base pairs upstream of the translation initiation codon of thrS and more than 100 base pairs downstream of the transcription initiation site. The mRNA region where these mutations are located shares primary and secondary structure homologies with specific parts of several isoacceptor tRNAThr species. These findings suggest that the ligase regulates its translation by binding to its mRNA at a place that shares some homology with its natural substrate.
Two types of human mast cells that have distinct neutral protease compositions.
Authors: Irani A A , Schechter N M , Craig S S , DeBlois G , Schwartz L B
Published: May 2006

Two human mast cell types were identified by immunohistochemical techniques in skin, lung, and small intestine. One type contains the neutral proteases, tryptase and chymotryptic proteinase, and is termed the TC mast cell. The second type contains only tryptase and is termed the T mast cell. Both types are fixed better by Carnoy's fluid than by formalin. The percentage of mast cells accounted for by the T type was 12 in skin; 98 in mucosa and 13 in submucosa of small intestine; and 77 in bronchial/bronchiolar subepithelium, about 97 in bronchial/bronchiolar epithelium, and 93 in alveoli of lung. Dispersed lung cells contained 90% T mast cells. The mean area of TC mast cells (76 micron2) was slightly larger than that of T mast cells (66 micron2); however, there was such extensive overlap that individual mast cells belonging to different types could not be distinguished on the basis of size. The recognition of human mast cell types with distinct protease compositions suggests a higher level of complexity of human mast cell-mediated reactions than heretofore appreciated.
Coordinate developmental regulation of high and low molecular weight mRNAs for rat insulin-like growth factor II.
Authors: Graham D E , Rechler M M , Brown A L , Frunzio R , Romanus J A , Bruni C B , Whitfield H J , Nissley S P , Seelig S , Berry S
Published: May 2006

Insulin-like growth factor II (IGF-II) is a mitogenic polypeptide that is thought to play a role in fetal growth and development. To study the hormonal and developmental regulation of IGF-II gene expression, we have isolated a cDNA clone for rat IGF-II (rIGF-II) from a 12S [1.2-kilobase-pair (kbp)] fraction of mRNA from a rat liver cell line (BRL-3A) that directs the cell-free synthesis of pre-pro-rIGF-II. In the present study, the rIGF-II probe was used to determine the size of IGF-II RNA. Surprisingly, in BRL-3A cells and in neonatal liver, the probe hybridized under stringent conditions 10-20 times more strongly to a larger (4 kbp) RNA than to 1.2-kbp RNA. The 4-kbp RNA is almost exclusively cytoplasmic and is colinear with a 551-base fragment of the rIGF-II cDNA insert containing coding and 3' noncoding regions. The 4-kbp and 1.2-kbp RNA species are regulated coordinately with developmental age, being high in liver from neonatal rats but not detectable in liver from older animals, suggesting that both IGF-II mRNA species arise from a single primary transcript by alternative RNA processing. Although oligodeoxynucleotide hybridization and S1 nuclease protection experiments suggest that the 4-kbp RNA contains an intact protein-coding region, fractions enriched in 4-kbp RNA do not direct the translation of pre-pro-rIGF-II in vitro. This may indicate that the 4-kbp RNA specifies an altered protein product that has not yet been recognized, or alternatively that it contains a normal protein-coding region but requires further RNA processing to be activated for translation.
Differentiation stage and lineage-specific inhibitor from the stroma of mouse bone marrow that restricts lymphoma cell growth.
Authors: Zipori D , Tamir M , Toledo J , Oren T
Published: May 2006

We have examined the mechanism by which stromal cells from the microenvironment of the bone marrow restricted the in vitro growth of certain hemopoietic tumors. A series of leukemia cell lines was used to monitor biological activities of stromal cell lines representing five distinct subtypes. Only an endothelial-like clone derived from mouse stroma (MBA-2.1) was consistently found to produce a cell-surface-associated glycoprotein that selectively inhibited the growth of plasmacytomas. The factor, designated leukemia cell inhibitory activity (LCIA), was not detected in anchorage-dependent cells of nonhemopoietic origin. Tumors of the lymphoid lineage and plasmacytomas in particular were the most sensitive to LCIA. Myeloid, macrophage, and erythroleukemia tumors were resistant to the factor, as were normal hemopoietic target cells including pluripotent stem cells, myeloid progenitor cells, and mitogen-stimulated spleen cells. Fractionation of trypsin-released proteins from MBA-2.1 cells by gel filtration and affinity binding to concanavalin A-Sepharose revealed two types of inhibitors; one was the specific leukemia cell inhibitor (i.e., LCIA); the other, present at a lower titer, was non-target-cell specific. The high sensitivity of plasmacytomas to LCIA versus the resistance of normal stem cells may be utilized for selective elimination of plasma cell tumors from bone marrow inocula.
Modified low density lipoproteins suppress production of a platelet-derived growth factor-like protein by cultured endothelial cells.
Authors: Fox P L , DiCorleto P E
Published: May 2006

Cultured endothelial cells (EC) produce a platelet-derived growth factor-like protein (PDGF-c) that stimulates the growth of cultured cells of mesenchymal origin. We have examined the effect of native plasma low density lipoprotein (LDL) and chemically modified LDL on production of PDGF-c by EC. Acetyl-LDL, but not native LDL, suppressed the production of PDGF-c by bovine aortic EC. Half-maximal inhibition was observed at a concentration of 25-75 micrograms of cholesterol per ml, and maximal inhibition (0-25% of control) at 150 micrograms of cholesterol per ml. EC treated with acetyl-LDL showed no morphological damage, there was no change in cell number, and the effect on production of PDGF-c was substantially reversed upon removal of the acetyl-LDL. The observed inhibition of PDGF-c production was specific, since total cellular and secreted protein synthesis were unaffected by acetyl-LDL. Acetyl-LDL suppressed PDGF-c production in both bovine aortic and human umbilical vein EC, but not in rat heart EC. This cell specificity correlated with the presence of scavenger receptors as measured by degradation of 125I-labeled acetyl-LDL and uptake of fluorescently labeled acetyl-LDL. Dimethylpropanediamine-LDL, a cationic modified lipoprotein, also inhibited PDGF-c production. The inhibition by both types of modified LDL was accompanied by significant intracellular cholesterol accumulation, suggesting a role for EC lipid composition in the regulation of production of PDGF-c.
Interaction of cAMP with the cell-surface receptor induces cell-type-specific mRNA accumulation in Dictyostelium discoideum.
Authors: Oyama M , Blumberg D D
Published: May 2006

The accumulation of many postaggregative mRNA species in Dictyostelium discoideum is dependent upon the continuous presence of elevated levels of cAMP. We have analyzed the cyclic nucleotide specificity of this requirement and show that it is similar to that of the cell-surface receptor and distinct from the specificity displayed by the cAMP-dependent protein kinase. The same specificity is displayed for the accumulation of two classes of prespore mRNAs (class I, early; class II, late) and a prestalk mRNA and for the shutoff of a growth-phase mRNA. Under conditions in which cAMP phosphodiesterase activity is competitively inhibited, half-maximal accumulation of prestalk mRNA can be obtained at cAMP concentrations of 320-520 nM, whereas a higher concentration, 1-2 microM, is required for half-maximal accumulation of the prespore mRNAs and shutoff of the growth-phase mRNA. These effects of cAMP and its analogues on gene expression have been obtained under conditions in which cAMP-mediated activation of adenylate cyclase is completely inhibited. We conclude that cAMP acts to stimulate postaggregative gene expression by interacting at the cell-surface receptor.
Transposition of the maize controlling element “Activator” in tobacco
Authors: Baker Barbara , Schell Jeff , Lörz Horst , Fedoroff Nina
Published: May 2006

Transposition of the maize autonomous controlling element Activator (
Ac
) and a nonautonomous derivative, Dissociation (
Ds
), was investigated in tobacco cells. Tobacco protoplasts were transformed with Ti-plasmid vectors that contained
Ac
or
Ds
flanked by short maize
wx
gene sequences. The structures of the elements and surrounding
wx
and T-DNA sequences were investigated in nine
Ac
and five
Ds
tobacco transformants by digestion with restriction enzymes, Southern blotting, and hybridization using specific probes. In four of the nine
Ac
transformed lines,
Ac
had excised from its original position in the T-DNA and inserted at new sites in the tobacco genome.
Ds
did not excise from its original T-DNA position in any of the transformants examined. Two
Ac
fragments and cellular flanking sequences were cloned from a line of tobacco in which
Ac
had transposed. Fragments, comprised of sequences flanking the newly integrated
Ac
elements, were used as hybridization probes to normal tobacco DNA and to the tobacco DNA from which they were isolated. The
Ac
copies were integrated into repetitive tobacco DNA sequences. Two tobacco fragments containing empty
Wx
donor sites were cloned from the DNA of the same
Ac
transformant and sequenced. Both sequences are among the types of excision products observed to result from
Ac
-catalyzed excision events in maize. Our results indicate that the maize controlling element
Ac
is capable of self-catalyzed transposition in tobacco.
Three distinct signals can induce class II gene expression in a murine pre-B-cell line.
Authors: Polla B S , Poljak A , Geier S G , Nathenson S G , Ohara J , Paul W E , Glimcher L H
Published: May 2006

Expression of class II genes of the major histocompatibility complex (MHC) has been studied in an Abelson-murine-leukemia-virus-transformed pre-B-cell line, R8, and its class II molecule (Ia)-negative variant, R8205. These variant cells contained barely detectable levels of RNA specific for all class II genes, including the nonpolymorphic invariant chain gene (Ii), and did not express cell surface Ia. Fusion of this murine Ia-negative cell line to the human Ia-positive Raji cell produced an interspecies hybridoma that expressed the murine Ia. These data are further evidence for the existence of a trans-acting factor(s) that can regulate class II gene expression. Furthermore, the T-cell-derived lymphokine B-cell-stimulatory factor 1 (BSF-1) induced expression of class II genes in the R8205 cells. Exposure of R8205 cells to an antibody that has been shown to mimic BSF-1 activity on normal B cells also resulted in expression of class II genes. These data demonstrate that three distinct signals--a lymphokine, an alloantibody binding to membrane structures, and an interspecies trans-acting factor--can induce expression of class II genes.
Modes of transmission and evidence for viral latency from studies of human T-cell lymphotrophic virus type I in Japanese migrant populations in Hawaii.
Authors: Blattner W A , Nomura A , Clark J W , Ho G Y , Nakao Y , Gallo R , Robert-Guroff M
Published: May 2006

Human T-cell lymphotrophic virus type I (HTLV-I) seroprevalence was 20% among Hawaiian Japanese migrants (issei) and their offspring (nisei) from Okinawa compared to 35% in similarly aged men who were lifetime residents of Okinawa. A control group of migrants from a nonendemic area of Japan, Niigata, had low rates of HTLV-I antibodies, suggesting that Hawaii per se is not an endemic area for HTLV-I. Factors that were significantly associated with seropositivity in the Okinawa migrant groups were years of residence in Japan before migration (issei) and age for offspring of Okinawa migrants (nisei). Antibody titer was highest in Okinawa lifetime residents, intermediate in migrants (issei), and significantly lower in offspring of Okinawa migrants (nisei), with increasing titer observed with advancing age in the offspring of the migrant group. Based on these data, infection within the household occurring early in life appears to be a major route of HTLV-I transmission and may help to explain the curious geographic clustering of this virus in certain locales. As yet to be defined cofactors, including sexual transmission and/or environmental exposures (e.g., particularly before age 20), also may contribute to HTLV-I seropositivity. The pattern of rising seroprevalence and titer with age in the offspring of migrants who resided all of their lives in Hawaii raises the possibility that HTLV-I infection acquired early in life may become dormant and reexpressed with reactivation of latently infected T cells. The importance of this model in the process of viral leukemogenesis is supported by recent reports of adult T-cell leukemia in offspring (nisei) of Okinawa migrants.
The Gy mutation: another cause of X-linked hypophosphatemia in mouse.
Authors: Lyon M F , Scriver C R , Baker L R , Tenenhouse H S , Kronick J , Mandla S
Published: May 2006

An X-linked dominant mutation (gyro, gene symbol Gy) in the laboratory mouse causes hypophosphatemia, rickets/osteomalacia, circling behavior, inner ear abnormalities, and sterility in males and a milder phenotype in females. Gy maps closely (crossover value 0.4-0.8%) to another X-linked gene (Hyp) that also causes hypophosphatemia in the mouse. Gy and Hyp genes have similar quantitative expression in serum phosphorus values, renal excretion of phosphate, and impairment of Na+/phosphate cotransport by renal brush-border membrane vesicles. These findings indicate that independent translation products of two X-linked genes serve phosphate transport in mouse kidney and thereby control phosphate content of extracellular fluid. The Gy translation product, unlike the Hyp product, is also expressed in the inner ear. These findings have implications for our understanding of the human counterpart known as "X-linked hypophosphatemia."
Hydrogen peroxide-induced injury of cells and its prevention by inhibitors of poly(ADP-ribose) polymerase.
Authors: Schraufstatter I U , Hyslop P A , Hinshaw D B , Spragg R G , Sklar L A , Cochrane C G
Published: May 2006

H2O2, in concentrations achieved in the proximity of stimulated leukocytes, induces injury and lysis of target cells. This may be an important aspect of inflammatory injury of tissues. Cell lysis in two target cells, the murine macrophage-like tumor cell line P388D1 and human peripheral lymphocytes, was found to be associated with activation of poly(ADP-ribose) polymerase (EC 2.4.2.30), a nuclear enzyme. This enzyme is activated under various conditions of DNA damage. Poly(ADP-ribose) polymerase utilizes nicotinamide adenine dinucleotide (NAD) as substrate and has been previously shown to consume NAD during exposure of cells to oxidants that was associated with inhibition of glycolysis, a decrease in cellular ATP, and cell death. In the current studies, inhibition of poly(ADP-ribose) polymerase by 3-aminobenzamide, nicotinamide, or theophylline in cells exposed to lethal concentrations of H2O2 prevented the sequence of events that eventually led to cell lysis--i.e., the decrease in NAD, followed by depletion of ATP, influx of extracellular Ca2+, actin polymerization and, finally, cell death. DNA damage, the initial stimulus for poly(ADP-ribose) polymerase activation, occurred despite the inhibition of this enzyme. Cells exposed to oxidant in the presence of the poly(ADP-ribose) polymerase inhibitor 3-aminobenzamide failed to demonstrate repair of DNA strand breaks.
Escherichia coli mutS-encoded protein binds to mismatched DNA base pairs.
Authors: Su S S , Modrich P
Published: May 2006

The Escherichia coli mutS gene product is involved in mismatch correction in this organism. We have purified a biologically active form of the 97,000 Mr protein to near homogeneity from an overproducing strain. Enzymatic and chemical protection ("footprinting") experiments have demonstrated that mutS-encoded protein specifically binds to DNA regions containing a single base-pair mismatch. The protein displayed variable affinity for the limited set of mismatches tested (G-T greater than G-A approximately equal to A-C greater than T-C).
Mutants of Salmonella typhimurium that cannot survive within the macrophage are avirulent.
Authors: Fields P I , Swanson R V , Haidaris C G , Heffron F
Published: May 2006

Salmonella typhimurium is a facultative intracellular pathogen capable of surviving within phagocytic cells of the reticuloendothelial system. To identify the genes important for intracellular survival, 9516 independent Tn10 insertional mutations were isolated in a virulent strain of S. typhimurium. By using an in vitro assay for survival within macrophages, 83 Tn10 mutants have been identified that have a diminished capacity for intracellular survival (designated MS or macrophage survival mutants). All of the MS mutants are less virulent than the parent strain in vivo, demonstrating that, for Salmonella, survival within the macrophage is essential for virulence. Thirty-seven of the MS mutants have been characterized as to their phenotype, including several mutations that confer sensitivity to specific microbiocidal mechanisms of the macrophage.
Suppression of tumorigenicity with continued expression of the c-Ha-ras oncogene in EJ bladder carcinoma-human fibroblast hybrid cells.
Authors: Geiser A G , Der C J , Marshall C J , Stanbridge E J
Published: May 2006

A human tumor cell line (EJ) expressing an activated c-Ha-ras oncogene was fused with a normal human fibroblast cell line. This fusion resulted in hybrids that behaved as transformed cells in culture but failed to form tumors in nude (athymic) mice. After repeated cell passage, two tumorigenic segregants of the hybrids arose in culture. The levels of expression of activated c-Ha-ras mRNA and its protein product, p21, were similar in the EJ cell line, the nontumorigenic hybrids, and the tumorigenic segregants. DNA transfections of the hybrids were performed with activated c-Ha-ras plasmid constructs, and transfectants expressing a 2-fold level of c-Ha-ras relative to the hybrid cells were found to maintain the nontumorigenic phenotype. We suggest that expression of the active c-Ha-ras oncogene is insufficient for the malignant transformation of these human cells.
Antigen presentation by a B-cell line transfected with cloned immunoglobulin heavy- and light-chain genes specific for a defined hapten.
Authors: Watanabe M , Wegmann D R , Ochi A , Hozumi N
Published: May 2006

The rearranged genes encoding immunoglobulin heavy (mu) and light (kappa) chains specific for the hapten 2,4,6-trinitrophenyl (Tnp) were introduced into a B-lymphoma line that bears surface IgG with an unknown specificity and expresses surface Ia molecules. A transformant expressing surface IgM specific for Tnp was obtained. The transformant was found to present Tnp-proteins to antigen (protein)-specific T cells far more efficiently than the parenteral B-lymphoma line. This artificial system, utilizing recombinant DNA technology and gene transfer, provides several approaches for the study of T-cell-B-cell interactions.