bluuebunny/crossref_metadata_2025 huggingface dataset:
Specific regulation of c-myc oncogene expression in a murine B-cell lymphoma.
Authors: McCormack J E , Pepe V H , Kent R B , Dean M , Marshak-Rothstein A , Sonenshein G E
Published: May 2006

The c-myc oncogene has been implicated in a wide spectrum of B-cell neoplasias. In normal cells, the level of expression of the c-myc gene correlates with growth status. In the present study, we examined the effect of receptor-mediated inhibition of growth on c-myc expression in a B-cell lymphoma. The murine lymphoma line WEHI 231 has been characterized as an early B cell; it bears surface-bound IgM and has unrearranged c-myc genes. Following treatment of a WEHI 231 culture with anti-mouse Ig antiserum, the cells undergo one round of division and further proliferation is inhibited. We observed that this treatment specifically affected cytoplasmic levels of c-myc mRNA. An initial early increase is followed by a precipitous drop such that by 4 hr (after exposure) the amount of c-myc mRNA is below control values by a factor of approximately equal to 10. The drop in c-myc precedes cessation of DNA synthesis. During the 2- to 4-hr period, c-myc mRNA had a maximal half-life of between 20 and 30 min. In contrast, even 24 hr after anti-Ig exposure, the amounts of most major mRNAs, including mu heavy chain and actin, were not significantly altered. These results indicate that expression of an unrearranged c-myc gene can be selectively responsive to receptor-mediated regulatory events.
Molecular model of ribosome frameshifting.
Authors: Weiss R B
Published: May 2006

Normal tRNAs can cause two- and four-base translocation errors by mistranslating certain noncognate codons. Several cases have been reported that reveal the identities of the frameshift-provoking tRNAs--i.e., the shifty tRNAs--and the mRNA sequence encompassing the site of frameshifting--i.e., the shifty codons. Here, a striking uniformity between the anticodon loops of the shifty tRNAs and their shifty codons is described. Stable "offset" anti-codon X codon pairs are postulated for each of the shifty tRNA-shifty codon combinations. This offset anticodon X codon pair leads to two- and four-base translocations when viewed in terms of the reciprocating ratchet mechanism of translocation.
Expression of β-galactosidase controlled by a nitrogenase promoter in stem nodules of
Aeschynomene scabra
Authors: Legocki Roman P. , Yun Allen C. , Szalay Aladar A.
Published: May 2006

A 365-base-pair (bp) DNA fragment, containing the promoter region of the nitrogenase reductase (
nifH
) gene from stem
Rhizobium
BTAi1, has been isolated and sequenced. The transcription initiation sites were localized at positions 152 (major initiation) and 114 (minor initiation) nucleotides upstream of the translation initiation codon. The 200-bp nucleotide sequence upstream of the
nifH
structural gene shows substantial homology to the corresponding
nifH
regions of cowpea
Rhizobium
(100%),
Parasponia Rhizobium
(89%), and
Rhizobium japonicum
(88%). The
nifH
promoter region of stem
Rhizobium
BTAi1 was fused to the
lacZ
gene of
Escherichia coli
. The fusion and a 1.6-kilobase DNA specifying neomycin phosphotransferase were inserted into a 3,4-kilobase fragment of stem
Rhizobium
chromosome, and the resulting construct was placed on a mobilizable vector, pREV1000. Stem
Rhizobium
transconjugants resistant to kanamycin were found to contain the
nifH
promoter region-
lacZ
fusion linked to the neomycin phosphotransferase gene at the site of chromosomal homology. Analysis of the DNA from stable transconjugants showed integration of a single copy of these sequences into the chromosome by a double-reciprocal crossover event. The transconjugants formed nitrogen-fixing nodules, indicating that the insertion occurred in a “nonessential” region of the stem
Rhizobium
chromosome. Transconjugant strain BTAi1000 grows on β-galactosidase indicator plates under aerobic conditions as white colonies, whereas under microaerobic conditions (97% N
2
/3% O
2
), which derepress nitrogenase, the colonies turn blue within 15-24 hr. β-Galactosidase activity in derepressed cultures of BTAi1000 showed a 200-fold increase in comparison to the wild-type strain, whereas stem nodules formed by BTAi1000 exhibited 15- to 20-fold higher β-galactosidase values than wild-type nodules. Nitrogenase promoter-dependent expression of β-galactosidase in stem nodules was inhibited by fixed nitrogen, suggesting that the
nifH
promoter-
lacZ
fusion is controlled coordinately in
trans
with the native
nif
region.
Somatic mutation of the T15 heavy chain gives rise to an antibody with autoantibody specificity.
Authors: Diamond B , Scharff M D
Published: May 2006

The S107 IgA kappa-chain myeloma cell line makes an antiphosphocholine antibody of the T15 idiotype. A somatic mutant of this line, U4, makes an immunoglobulin with a single amino acid substitution of an alanine for a glutamic acid at residue 35. This single amino acid change results in a loss of phosphocholine binding activity. However, the U4 immunoglobulin has acquired reactivity with a variety of phosphorylated macromolecules, including double-stranded DNA, protamine, and cardiolipin. Thus, a single amino acid change in the T15 heavy chain can transform an antibacterial antibody into an antibody that resembles the autoantibodies seen in mice and man with autoimmune disease.
Placental lactogen administration reverses the effect of low-protein diet on maternal and fetal serum somatomedin levels in the pregnant rat.
Authors: Pilistine S J , Moses A C , Munro H N
Published: May 2006

Female rats were studied on day 20 of pregnancy after being fed either a 5% lactalbumin (low protein) diet or a 20% lactalbumin (adequate) diet for the last 2 weeks of pregnancy. Rats on the lower intake of protein showed decreased serum levels of rat placental lactogen and reduced numbers of lactogenic receptors in the maternal liver. These changes were accompanied by much reduced serum levels of somatomedins IGF I(insulin-like growth factor) and II (multiplication-stimulating activity, MSA). Infusion of human placental lactogen or human growth hormone into the rats on the low-protein intake during the last 2 weeks of pregnancy partially restored the maternal serum levels of both somatomedins, but only human placental lactogen increased the number of lactogenic receptors on liver cell membranes. It was concluded that protein deficiency may reduce secretion of somatomedins by the liver (or other tissues) of the pregnant rat indirectly through reduction in output of rat placental lactogen by the placenta. In the same experiments, the effect of maternal protein deficiency on fetal development and serum somatomedin levels was examined. Protein deficiency resulted in smaller fetuses and placentas and lower fetal serum levels of IGF I and MSA. Unlike the response in maternal serum levels, the concentration of MSA in the fetal serum increased during infusion of hPL or hGH but the concentration of IGF I did not. This suggests that placental lactogen enters the fetal circulation and affects tissues producing MSA but not those making IGF I. Despite the restoration of MSA levels, fetal and placental weights did not increase when the rats on the protein-deficient diets were treated with human placental lactogen or growth hormone.
Hippocampal denervation causes rapid forgetting of olfactory information in rats.
Authors: Stäubli U , Ivy G , Lynch G
Published: May 2006

Rats were trained on a succession of two-odor discriminations for a water reward in a modified radial maze. A different odor pair was used each day. After three or four pairs, rats would learn to choose the correct odor in only 3-5 trials. Animals were then subjected to electrolytic lesions in the lateral entorhinal cortex, which is innervated by the lateral olfactory tract, or in the dorsal entorhinal cortex, which is not a target of the olfactory system. Lesions of the first type did not interfere with performance, provided a short interval (30 sec to 2 min) was used between trials. However, the rats were severely impaired when trials were separated by 3-10 min. Dorsal lesions had no effect on olfactory discrimination irrespective of length of delay. In additional experiments, the rats were trained for 10 trials with short inter-trial intervals and then tested 1 hr later with the significance of the cues reversed. Animals with dorsal lesions continued to respond to the formerly correct odor while those with lateral entorhinal damage immediately reversed their response choices. These results provide evidence that lesions to the hippocampal system produce a rapid forgetting syndrome in rats comparable with that reported for humans with temporal lobe damage or dysfunction.
A theorem about Hamiltonian systems
Authors: Case K. M.
Published: May 2006

A simple theorem in Hamiltonian mechanics is pointed out. One consequence is a generalization of the classical result that symmetries are generated by Poisson brackets of conserved functionals. General applications are discussed. Special emphasis is given to the Kadomtsev-Petviashvili equation.
Mercuric ion-resistance operons of plasmid R100 and transposon Tn501: the beginning of the operon including the regulatory region and the first two structural genes.
Authors: Misra T K , Brown N L , Fritzinger D C , Pridmore R D , Barnes W M , Haberstroh L , Silver S
Published: May 2006

The mercuric ion-resistance operons of plasmid R100 (originally from Shigella) and transposon Tn501 (originally from a plasmid isolated in Pseudomonas) have been compared by DNA sequence analysis. The sequences for the first 1340 base pairs of Tn501 are given with the best alignment with the comparable 1319 base pairs of R100. The homology between the two sequences starts at base 58 after the end of the insertion sequence IS-1 of R100. The sequences include the transcriptional regulatory region, and the homology is particularly strong in regions just upstream from potential transcriptional initiation sites. The trans-acting regulatory gene merR consists of 180 base pairs in both cases and codes for a highly basic polypeptide of 60 amino acids, which is also rich in serine. The Tn501 and R100 merR genes differ in 25 of the 180 base positions, and the resulting polypeptides differ in seven amino acids. The regulatory region before the major transcription initiation site contains potential -35 and -10 sequences and dyad symmetrical sequences, which may be the merR binding sites for transcriptional regulation. The first structural gene, merT, encodes a highly hydrophobic polypeptide of 116 amino acids. The R100 and Tn501 merT genes differ in 17% of their positions, leading to 14 (12%) amino acid changes. This region had previously been shown to encode a protein governing membrane transport of mercuric ions. The second structural gene, merC, would give a 91 amino acid polypeptide with a hydrophobic amino-terminal segment. The Tn501 and R100 merC genes differ at 37 base positions, leading to 10 amino acid changes.
Demonstration of two operator elements in gal: in vitro repressor binding studies.
Authors: Majumdar A , Adhya S
Published: May 2006

Genetic and DNA base sequence analyses of cis-dominant mutations that derepress the gal operon of Escherichia coli suggested the existence of two operator loci needed for gal repression. One (OE) is located immediately upstream to the two overlapping gal promoters and the other (OI) is inside the first structural gene. We have investigated the ability of wild-type and mutant OE and OI DNA sequences to bind to gal repressor. The repressor has been purified from cells containing a multicopy plasmid in which the repressor gene is brought under the control of phage lambda PL promoter. The DNA-repressor interactions are detected by the change in electrophoretic mobility of labeled DNA that accompanies its complex formation with repressor protein. The purified repressor shows concentration-dependent binding to both O+E and O+I but not to OEc and OIc sequences. These results authenticate the proposed operator role of the two homologous gal DNA control elements and thereby establish that the negative control of the gal operon requires repressor binding at both OE and OI, which are separated by greater than 90 base pairs.
Time-dependent conversion of alpha 1- to beta-adrenoceptor-mediated glycogenolysis in isolated rat liver cells: role of membrane phospholipase A2.
Authors: Kunos G , Hirata F , Ishac E J , Tchakarov L
Published: May 2006

Incubation of isolated rat liver cells in a serum-free buffer leads to the reduction of the glycogenolytic effect of phenylephrine and the simultaneous emergence of a glycogenolytic response to isoproterenol within 4 hr. This conversion of the adrenergic activation of phosphorylase from an alpha 1- to a beta-adrenoceptor-mediated response is associated with no change in the glycogenolytic response to the calcium-linked activator vasopressin, and a reduction of the glycogenolytic response to the cAMP-linked activator glucagon. In vitro incubation of hepatocytes does not influence the density of affinity of [3H]prazosin-labeled alpha 1-receptors and [3H]CGP-12177-labeled beta-receptors. In cells preincubated for 4 hr, a further 30-min incubation with 50 nM lipomodulin, an endogenous inhibitor of membrane phospholipase A2 (EC 3.1.1.4), reverses the adrenergic activation of phosphorylase from a beta- to an alpha 1-receptor-mediated event, whereas in freshly isolated cells lipomodulin does not affect the predominant alpha-receptor response. Conversely, exposure of freshly isolated cells to a monoclonal antibody to lipomodulin in the presence of 10 microM phenylephrine, or to melittin, an activator of phospholipase A2, at 2 micrograms/ml, results in the suppression of the effect of phenylephrine and the emergence of a response to isoproterenol within 30 min. It is proposed that coupling of hepatic alpha 1- and beta-adrenoceptors to postreceptor pathways is regulated in an inverse reciprocal manner by changes in membrane phospholipase A2 activity.
Developing dorsal root ganglion neurons require trophic support from their central processes: evidence for a role of retrogradely transported nerve growth factor from the central nervous system to the periphery.
Authors: Yip H K , Johnson E M
Published: May 2006

Injury to the peripheral processes produces a profound cell loss (40-50%) in the dorsal root ganglion of newborn rats. Although division of central processes produces little or no cellular change in sensory ganglion of adult animals, no information has been available on the effect of dorsal root section in developing dorsal root ganglion. We show that 6 days after dorsal rhizotomy on newborn rats, there is a 50% decrease in neuronal number in L5 dorsal root ganglion. A combined central and peripheral lesion of the sensory process results in a greater decrease in neuronal number (70%). Both of these effects can be prevented by the concomitant treatment with nerve growth factor. We also demonstrate that 125I-Ia-labeled nerve growth factor is retrogradely transported with high selectivity from the spinal cord to the dorsal root ganglion via the dorsal roots. The results indicate that trophic support for developing sensory neurons is provided through the central processes. This is presumably due to the uptake and retrograde transport of a trophic factor by the terminals of the central processes. The data suggest that nerve growth factor may be the trophic factor.
Antithrombin III Toyama: replacement of arginine-47 by cysteine in hereditary abnormal antithrombin III that lacks heparin-binding ability.
Authors: Koide T , Odani S , Takahashi K , Ono T , Sakuragawa N
Published: May 2006

Structural analyses of a hereditary abnormal antithrombin III, antithrombin III Toyama, which has normal progressive antithrombin activity but no heparin cofactor activity, have been carried out to elucidate the molecular abnormality causing recurrent thrombophlebitis of a patient and to identify an amino acid residue essential for the binding with heparin. Abnormal antithrombin III was reduced, S-pyridylethylated, and treated with cyanogen bromide. Eleven fragments were isolated by the combination of Sephadex G-50 gel filtration and reversed-phase HPLC and compared with those from normal antithrombin III. One large fragment (CN-III) that appeared to have a different amino acid composition from that of the corresponding fragment from normal antithrombin III was digested with trypsin, and the digests were separated by HPLC. The abnormal peptide was identified by comparing the peptide map with that from normal antithrombin III. Amino acid sequence analysis of the abnormal peptide indicated that the arginine-47 of normal antithrombin III had been replaced by cysteine in antithrombin III Toyama. One base mutation, C leads to T, in the 5' terminal position of the arginine-47 genetic codon (CGT) is probably responsible for this substitution. These results also suggest that arginine-47 is an essential amino acid residue for the binding with heparin.
Trifluoperazine inhibits insulin action on glucose metabolism in fat cells without affecting inhibition of lipolysis.
Authors: Shechter Y
Published: May 2006

One of the specific inhibitors of calmodulin action, trifluoperazine, blocked the stimulating action of insulin on 2-deoxyglucose uptake and glucose metabolism. The inhibitory effect of insulin on lipolysis was not altered by the drug. The active (insulin-stimulated) state and the basal state of lipogenesis were inhibited half-maximally at 80 and 550 microM trifluoperazine, respectively. 2-Deoxyglucose uptake was inhibited half-maximally at a trifluoperazine concentration of 70 microM. Other less potent calmodulin inhibitors also inhibited glucose metabolism in fat cells but in a nonspecific manner. The inhibition was noncompetitive and was not altered in Ca2+- free medium. The stimulating activity of wheat germ agglutinin and of sodium vanadate were also inhibited by trifluoperazine. The dose-dependent inhibitions were indistinguishable whether the active (stimulated) state was produced by insulin, wheat germ agglutinin, or vanadate. The data indicate that a late event in the sequence that ultimately leads to enhanced glucose transport activity in fat cells is specifically inhibited by trifluoperazine. The possible involvement of calmodulin or another related Ca2+-dependent regulatory protein in the exocytic (fusion) reaction that recruits glucose-transport activity from storage sites to the plasma membranes is discussed.
Microinjected pBR322 stimulates cellular DNA synthesis in Swiss 3T3 cells.
Authors: Hyland J K , Hirschhorn R R , Avignolo C , Mercer W E , Ohta M , Galanti N , Jonak G J , Baserga R
Published: May 2006

When pBR322 is manually microinjected into the nuclei of quiescent Swiss 3T3 cells it stimulates the incorporation of [3H]thymidine into DNA. The evidence clearly shows that this increased incorporation that is detected by in situ autoradiography in microinjected cells represents cellular DNA synthesis and not DNA repair or plasmid replication. The effect is due to pBR322 and not due to impurities, mechanical perturbances due to the microinjection technique, or aspecific effects. This stimulation is striking in Swiss 3T3 cells. Some NIH 3T3 cells show a slight stimulation, but hamster cells, derived from baby hamster kidney (BHK) cells, are not stimulated when microinjected with pBR322. The preliminary evidence seems to indicate that the integrity of the pBR322 genome is important for the stimulation of cellular DNA synthesis in quiescent Swiss 3T3 cells. These results, although of a preliminary nature, are of interest because they indicate that a prokaryotic genome may alter the cell cycle of mammalian cells. From a practical point of view the stimulatory effect of microinjected pBR322 on cellular DNA synthesis has a more immediate interest, because pBR322 is the vector most commonly used for molecular cloning and 3T3 cells are very frequently used for gene transfer experiments.
Long-term cultivation and differentiation of human erythroleukemia cells in a protein-free chemically defined medium.
Authors: Okabe T , Fujisawa M , Takaku F
Published: May 2006

To examine whether a human erythroleukemia cell line, K-562, can proliferate and differentiate without protein supplements, long-term cultivation of the cells was carried out in a protein-free chemically defined medium. By the use of stepwise decreases in the fetal bovine serum concentration, continuous growth of K-562 cells was established in a protein-free F-10 medium. The cells have been propagated in this medium for 3 years. The population-doubling time of the cells is about 30 hr. Growth was not stimulated by the addition of insulin, epidermal growth factor, fibroblast growth factor, multiplication-stimulating activity, transferrin, platelet-derived growth factor, or dexamethasone. Addition of serum stimulated the cell growth slightly and increased the saturation density. The saturation density of the cells could be increased to that seen with serum-supplemented cultures by changing the serum-free medium daily. The cells synthesized significant amounts of hemoglobin in the presence of hemin without serum supplementation. The results suggest that the human erythroleukemia cells grown in a protein-free medium do not require serum components for their growth or hemin-induced hemoglobin synthesis and provide an excellent model for better understanding of the growth and differentiation of human leukemia cells.
Calcium-dependent, cyclic nucleotide-independent steroidogenesis in the bovine placenta.
Authors: Shemesh M , Hansel W , Strauss J F
Published: May 2006

Dispersed bovine placental cells (fetal cotyledon and maternal caruncle) were shown to synthesize progesterone. To determine if their steroidogenic activity could be modulated by a cyclic nucleotide-mediated process, we added luteinizing hormone, 8-bromoadenosine 3',5'-monophosphate, 8-bromoguanosine 3',5'-monophosphate, adenosine, or cholera toxin to dispersed cells from placentomes of 100-283 days gestational age and examined progesterone synthesis during 3-to 16-hr incubation periods. Net progesterone production, defined as the amount of progesterone released in excess of the zero-time cellular progesterone content, was determined by using a specific RIA. None of these agents significantly affected progesterone synthesis. In contrast, the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (MIX; 0.2-0.5 mM) caused a marked increase in progesterone formation. In time course studies it was found that MIX produced a 5-fold increase in progesterone production in 16 hr, with steroid production increasing linearly during this time. MIX also increased the conversion of exogenous pregnenolone to progesterone by placental cells. In view of the failure of cyclic nucleotide analogues and activators of adenylate cyclase to stimulate steroidogenesis, it was necessary to consider other modes of action of MIX. Since MIX is known to affect intracellular calcium translocation, we examined the effects of the calcium ionophore A23187 on progesterone formation. This drug enhanced progesterone formation and augmented the stimulatory effects of MIX. The stimulatory action of A23187 was not affected by cyclic nucleotide analogues. Our data suggest that progesterone synthesis in the bovine placentome is calcium dependent and cyclic nucleotide independent.
Phorbol 12-myristate 13-acetate, ionomycin or ouabain, and raised extracellular magnesium induce proliferation of chicken heart mesenchymal cells.
Authors: Balk S D , Morisi A , Gunther H S
Published: May 2006

Cultured chicken heart mesenchymal cells are proliferatively quiescent at low densities in medium containing plasma at 10%. Mitogenic hormones like epidermal growth factor and insulin-like growth factors cause these cells to proliferate very actively, as does infection with avian sarcoma viruses, erythroblastosis virus, or myelocytomatosis virus. We have found that the combination of phorbol 12-myristate 13-acetate (PMA), ionomycin or ouabain, and raised extracellular magnesium, likewise, causes these cells to proliferate very actively. Although these agents have no significant effect when acting singly, the combination of PMA at 100 ng/ml and 0.5 microM ionomycin induces a 6-fold increase in cell number at 4 days, and the combination of PMA, ionomycin, and 5.6 mM magnesium induces 12-fold multiplication. Likewise, PMA plus 1 microM ouabain induces 3-fold multiplication, whereas the combination of PMA, ouabain, and magnesium induces 6-fold multiplication. The tumor promoter PMA, like diacylglycerol released by breakdown of plasma membrane phosphatidylinositol diphosphate, is known to activate the serine- and threonine-specific intracellular enzyme kinase C. The divalent cation ionophore ionomycin is known to carry calcium into cells down an electrochemical gradient, and the Na+,K+-ATPase inhibitor ouabain appears to elevate intracellular calcium by means of a sodium-mediated exchange mechanism. Magnesium, like calcium, is known to enter cells passively down an electrochemical gradient and to be involved in the regulation of many key intracellular reactions. Our findings with PMA, ionotropes, and magnesium support a hypothesis that diacylglycerol-mediated activation of kinase C plus cellular divalent cation influx and/or mobilization, caused by the action of mitogenic hormones or the protein products of onc genes, are key events in the initiation of cell replication.
In vivo metabolic effects of hyperglycemia in murine radiation-induced fibrosarcoma: a 31P NMR investigation.
Authors: Evelhoch J L , Sapareto S A , Jick D E , Ackerman J J
Published: May 2006

The hyperglycemia-induced in vivo metabolic changes produced in subcutaneous murine RIF-1 tumors, grown on female C3H/Anf mice, were examined with 31P surface-coil NMR. Serum glucose levels were elevated 4-fold by bolus intraperitoneal injection of 0.3 ml of an aqueous 50% glucose solution. Tumor pH was calculated from the chemical shift of Pi and relative phosphocreatine and ATP concentrations were determined by Simpson's rule integration of the peak areas. Tumor pH decreased by ca. 0.45 unit over 2 hr while phosphocreatine concentrations decreased by ca. 50% over the same time period (n = 9). Initial tumor pH correlated inversely with the initial peak intensity ratio of Pi:ATP (r = -0.77). In a significant number of tumors (n = 4), two pH populations were observed. In these tumors, one population was unaffected by hyperglycemia and the other showed a decrease in pH. In the other tumors (n = 5), the pH distribution broadened as the pH decreased. In these tumors, the observed decreased in phosphocreatine concentration correlated with that calculated from the effect of measured tumor pH on the intracellular creatine kinase equilibrium (n = 18, r = 0.91). This correlation and consideration of the Pi distribution in the tumor suggest that the pH measured by 31P NMR is weighted heavily by intracellular pH for the RIF-1 tumor. The presence of two distinct tumor pH populations or a broadened pH distribution likely reflects variations in tumor microcellular environment. Control experiments showed negligible changes in tumor pH and high energy phosphate concentrations after bolus intraperitoneal injection of 0.3 ml of isotonic saline. In addition, negligible changes in leg muscle pH and high energy phosphate concentrations were observed after glucose injection into mice with or without tumors. These results indicate that hyperglycemia induced by intraperitoneal glucose injection is effective in lowering the tumor pH of the murine RIF-1 tumor.
Enzymes processing somatostatin precursors: an Arg-Lys esteropeptidase from the rat brain cortex converting somatostatin-28 into somatostatin-14.
Authors: Gluschankof P , Morel A , Gomez S , Nicolas P , Fahy C , Cohen P
Published: May 2006

The post-translational proteolytic conversion of somatostatin-14 precursors was studied to characterize the enzyme system responsible for the production of the tetradecapeptide either from its 15-kDa precursor protein or from its COOH-terminal fragment, somatostatin-28. A synthetic undecapeptide Pro-Arg-Glu-Arg-Lys-Ala-Gly-Ala-Lys-Asn-Tyr(NH2), homologous to the amino acid sequence of the octacosapeptide at the putative Arg-Lys cleavage locus, was used as substrate, after 125I labeling on the COOH-terminal tyrosine residue. A 90-kDa proteolytic activity was detected in rat brain cortex extracts after molecular sieve fractionation followed by ion exchange chromatography. The protease released the peptide 125I-Ala-Gly-Ala-Lys-Asn-Tyr(NH2) from the synthetic undecapeptide substrate and converted somatostatin-28 into somatostatin-14 under similar conditions (pH 7.0). Under these experimental conditions, the product tetradecapeptide was not further degraded by the enzyme. In contrast, the purified 15-kDa hypothalamic precursor remained unaffected when exposed to the proteolytic enzyme under identical conditions. It is concluded that this Arg-Lys esteropeptidase from the brain cortex may be involved in the in vivo processing of the somatostatin-28 fragment of prosomatostatin into somatostatin-14, the former species being an obligatory intermediate in a two-step proteolytic mechanism leading to somatostatin-14.
Genomic structure and possible retroviral origin of the chicken CR1 repetitive DNA sequence family.
Authors: Stumph W E , Hodgson C P , Tsai M J , O'Malley B W
Published: May 2006

We have analyzed the sequence and structure of three CR1 family repetitive elements found in the region adjoining the 3' end of a chicken calmodulin gene. Members of this family are approximately equal to 300 base pairs long and are dispersed throughout the chicken genome. The present data, when taken together with that from four CR1s sequenced previously, reveal that the CR1 family has an overall structure possessing several features associated with the long terminal repeats of avian retroviruses. This finding implies that a retroviral mechanism may be responsible for the dispersion of CR1 sequences throughout the chicken genome. The seven different CR1 repeats that have been analyzed exist at defined locations in the chicken genome relative to nearby structural genes. A directional polarity has been assigned to the CR1 family based upon limited sequence homology to mammalian Alu-type sequences. Interestingly, whether present in 5' - or 3'-flanking DNA, the CR1 sequences have an inverse orientation such that they all "point toward" the nearby structural genes. This is consistent with the previously proposed concept that chicken CR1 sequences may be involved in defining the boundaries of active chromosomal domains of gene expression.
High-affinity binding of the regulatory subunit (RII) of cAMP-dependent protein kinase to microtubule-associated and other cellular proteins.
Authors: Lohmann S M , DeCamilli P , Einig I , Walter U
Published: May 2006

Interaction of the regulatory subunit of the type II cAMP-dependent protein kinase (RII) with tissue-specific cellular binding proteins has been demonstrated by two independent methods. Complexes of RII and its binding proteins were isolated on a cAMP analog-Sepharose affinity column, eluted from the column, and analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Alternatively, nitrocellulose blots made from polyacrylamide gels containing samples of tissue extracts or affinity column eluates were treated with sequential overlays of RII, monospecific antibody, and radioiodinated protein A. In bovine cerebrum, specific high-affinity interactions between RII and several binding proteins, including major proteins of 300, 80, and 68 kDa, were recognized by the two methods. The 300-kDa and 68-kDa proteins were identified as microtubule-associated protein 2 (300 kDa) and a protein of lower molecular weight (68 kDa) that copurifies with it. The additional major binding protein of 80 kDa requires further characterization. In addition, several binding proteins distinct from those observed in bovine cerebrum were found in bovine heart. Many of the RII binding proteins from brain and heart served to differing extents as substrates for the purified catalytic subunit of cAMP-dependent protein kinase. One hypothesis of the significance of the protein kinase regulatory subunit interaction with cellular binding proteins is that this may control the protein kinase holoenzyme localization and, thereby, define the substrate targets most accessible for phosphorylation by the activated protein kinase catalytic subunit. Alternatively, RII binding to a variety of cellular proteins might regulate their function--i.e., RII could be a regulator for multiple proteins in addition to the catalytic subunit of the cAMP-dependent protein kinase.
Interactions between human tumor cells and fibroblasts stimulate hyaluronate synthesis.
Authors: Knudson W , Biswas C , Toole B P
Published: May 2006

Several types of tumors contain high concentrations of hyaluronate, yet isolated tumor cells in culture often produce little glycosaminoglycan. To explore the possibility that interactions between tumor cells and host fibroblasts stimulate hyaluronate synthesis, human tumor cells were grown separately from and in coculture with normal human fibroblasts. Stimulation was observed with each of the three types of tumor cells used: LX-1 lung carcinoma, DAN pancreatic carcinoma, and TRIG melanoma. The interaction between LX-1 cells and fibroblasts was studied in detail. Under serum-free conditions, cocultures of LX-1 and fibroblasts synthesized 3-fold more hyaluronate than the sum of that produced by LX-1 and fibroblast cultures grown separately. This stimulation was linear over 72 hr and hyaluronate represented 80% of the glycosaminoglycan synthesized. Maximum stimulation occurred at a ratio of fibroblasts to LX-1 cells of 1-2:1. Quantitation of unlabeled glycosaminoglycans by HPLC analysis of disaccharides generated by digestion with chondroitin ABC and AC lyases (EC 4.2.2.4 and 4.2.2.5) demonstrated that net accumulation of hyaluronate increased 2-fold and that hyaluronate represented 80% of total chondroitinase-sensitive glycosaminoglycan produced by the cocultures. The disaccharide patterns obtained showed that accumulations of chondroitin-4- and chondroitin-6-sulfates were stimulated proportionately to that of hyaluronate in these cocultures. Similar levels of stimulation due to coculture were obtained in serum-containing and serum-free media. Stimulation was not effected by addition of LX-1-conditioned medium to fibroblast cultures or by culturing LX-1 and fibroblasts under conditions where they shared the same medium but were physically separated. Cell contact between LX-1 and fibroblasts thus appears to be necessary for the stimulation of hyaluronate synthesis.
Activation of a translocated c-myc gene: role of structural alterations in the upstream region.
Authors: Wiman K G , Clarkson B , Hayday A C , Saito H , Tonegawa S , Hayward W S
Published: May 2006

The translocated c-myc gene in AW-Ramos, a Burkitt lymphoma cell line carrying the 8;14 translocation, is expressed at 2- to 5-fold higher levels than c-myc in lymphoblastoid cell lines. The translocation event has joined c-myc to the IgM switch region. As a consequence, a recently identified immunoglobulin transcriptional enhancer element is not linked to the translocated c-myc gene. Chromosomal recombination occurs approximately equal to 340 nucleotides upstream of the c-myc 5' cap site, leaving all three c-myc exons intact. The nucleotide sequences of the two coding exons in the translocated c-myc gene are identical to those of the normal c-myc gene. Nucleotide sequence analyses of the first, noncoding c-myc exon and of the region between this exon and the chromosomal recombination point reveal two single-base differences from normal c-myc. Our data indicate that altered expression rather than an altered gene product is responsible for c-myc activation in AW-Ramos cells and that this is a result of either loss of regulatory sequences located greater than 340 nucleotides upstream of c-myc or disruption of normal c-myc regulation by one or both base substitutions. Alternatively, unidentified enhancer-like sequences in the Ig locus may alter the expression of c-myc.
Spontaneous deletions and duplications of sequences in the genome of cowpox virus.
Authors: Pickup D J , Ink B S , Parsons B L , Hu W , Joklik W K
Published: May 2006

Examination of the genomes of 10 white-pock variants of cowpox virus strain Brighton red (CPV-BR) revealed that 9 of them had lost 32 to 38 kilobase pairs (kbp) from their right-hand ends and that the deleted sequences had been replaced by inverted copies of regions from 21 to 50 kbp long from the left-hand end of the genome. These variants thus possess inverted terminal repeats (ITRs) from 21 to 50 kbp long; all are longer than the ITRs of CPV-BR (10 kbp). The 10th variant is a simple deletion mutant that has lost the sequences between 32 and 12 kbp from the right-hand end of the genome. The limits of the inner ends of the observed deletions (between 32 and 38 kbp from the right-hand end of the CPV-BR genome) appear to be defined by the location of the nearest essential gene on the one hand and the location of the gene that encodes "pock redness" on the other. The genomes of the deletion/duplication white-pock variants appear to have been generated either by single crossover recombinational events between two CPV-BR genomes aligned in opposite directions or by the nonreciprocal transfer of genetic information. The sites where such recombination/transfer occurred were sequenced in four variants. In all of them, the sequences adjacent to such sites show no sequence homology or any other unusual structural feature. The analogous sites at the internal ends of the two ITRs of CPV-BR also were sequenced and also show no unusual features. It is likely that the ITRs of CPV-BR and of its white-pock variants, and probably those of other orthopox-virus genomes, arise as a result of nonhomologous recombination or by random nonreciprocal transfer of genetic information.
Monocytes are required to trigger Ca2+ uptake in the proliferative response of human t lymphocytes to staphylococcus aureus protein A.
Authors: Lederman H M , Lee J W , Cheung R K , Grinstein S , Gelfand E W
Published: May 2006

We have used the T-cell mitogen Staphylococcus aureus protein A (SpA) to study the role of monocytes in the early events of T-lymphocyte activation. The mitogenic response of human peripheral blood mononuclear cells (PBM) was compared to the response of populations enriched for T cells by E-rosetting (PBM-E+). In response to SpA, the [3H]thymidine uptake of PBM-E+ was reduced by 80% compared to PBM. The reduced response of PBM-E+ was completely restored by the addition of irradiated PBM-E- or the monocyte-like human cell line U-937 but not by addition of irradiated PBM-E+. A direct interaction of SpA with monocytes is important since proliferative responses could be generated by preincubation of U-937 with SpA followed by washing and subsequent addition to PBM-E+; incubation of PBM-E+ with SpA followed by washing and subsequent addition of U-937 did not result in a proliferative response. To further delineate the role of the monocyte, we examined the ability of soluble SpA, U-937, or U-937 preincubated with SpA to trigger Ca2+ flux into T lymphocytes, an early step in initiation of the proliferative response. SpA-pretreated U-937, but neither SpA nor monocytes alone, triggered Ca2+ movement into the T lymphocytes. This defines a new role for the monocyte in the early events of T-lymphocyte activation.
Phosphorylation of the nicotinic acetylcholine receptor by an endogenous tyrosine-specific protein kinase.
Authors: Huganir R L , Miles K , Greengard P
Published: May 2006

Postsynaptic membranes from the electric organ of Torpedo californica, rich in the nicotinic acetylcholine receptor, were shown to contain an endogenous tyrosine protein kinase. This endogenous kinase phosphorylated three major proteins with molecular masses corresponding to 50 kDa, 60 kDa, and 65 kDa. The phosphorylation of these three proteins occurred exclusively on tyrosine residues under the experimental conditions used and was abolished by 0.1% Nonidet P-40 and stimulated by Mn2+. The 50-kDa, and 60-kDa, and 65-kDa phosphoproteins were demonstrated to be the beta, gamma, and delta subunits, respectively, of the nicotinic acetylcholine receptor by purification of the phosphorylated receptor using affinity chromatography. The endogenous tyrosine kinase specifically phosphorylated the beta, gamma, and delta subunits rapidly to a final stoichiometry of approximately equal to 0.5 mol of phosphate per mol of sub-unit. Two-dimensional phosphopeptide mapping of the phosphorylated beta, gamma, and delta subunits, after limit proteolysis with trypsin or thermolysin, indicated that each subunit was phosphorylated on a single site. Locations are proposed for the amino acid residues phosphorylated on the receptor by the tyrosine-specific protein kinase and by two other protein kinases (cAMP-dependent protein kinase and protein kinase C) which phosphorylate the receptor.
Complete nucleotide sequence of Torpedo marmorata mRNA coding for the 43,000-dalton nu 2 protein: muscle-specific creatine kinase.
Authors: Giraudat J , Devillers-Thiery A , Perriard J C , Changeux J P
Published: May 2006

DNA sequences coding for the muscle-specific subunit of creatine kinase have been isolated from cDNA libraries constructed from Torpedo marmorata electric organ. Clones were screened by differential in situ hybridization and hybrid-selected translation. The in vitro translation product of the selected mRNA was immunoprecipitated by anti-chicken creatine kinase antibodies and comigrated with Torpedo muscle creatine kinase on two-dimensional gels at the same position as the cytosolic 43,000-dalton protein referred to as nu 2. The cDNA inserts hybridized to a mRNA species present in adult Torpedo muscle but not in brain. The complete sequence of the mRNA was determined on one of the clones except for the 78 nucleotides of the mRNA 5' terminal sequence, which were identified by the primer extension method. The amino acid sequence of muscle-specific creatine kinase from T. marmorata was deduced and analyzed. It includes the known sequence of a peptide from the active site of rabbit muscle-specific creatine kinase.
A 12-base-pair DNA motif that is repeated several times in metallothionein gene promoters confers metal regulation to a heterologous gene.
Authors: Stuart G W , Searle P F , Chen H Y , Brinster R L , Palmiter R D
Published: May 2006

To define DNA sequences involved in mouse metallothionein-I (MT-I) gene promoter function and metal regulation, we fused the 5' flanking sequences of the MT-I gene to the coding sequences of a viral thymidine kinase (TK) gene. A series of 5' deletion, 3' deletion, linker-scanning, and internal deletion mutants of the MT-I promoter was constructed and assayed by microinjection into mouse eggs. The results indicate that at least two related promoter elements can confer some metal regulation independently. Those mutations that had the most severe effect on regulation impinge on a 12-base-pair conserved sequence that is repeated several times within the mouse MT-I and other MT promoters. To test the regulatory function of this sequence, it was synthesized as a pair of complementary oligonucleotides and inserted into the promoter of the TK gene. A single insertion of this sequence conferred limited metal regulation onto the TK promoter, whereas a construct with two separate inserts was regulated as efficiently as the MT-I promoter.
An immunological method for detecting gene expression in yeast colonies.
Authors: Lyons S , Nelson N
Published: May 2006

A method for detection of cloned, expressed genes in yeast colonies has been developed. The 70-kilodalton (kDa) mitochondrial outer membrane protein of yeast was used as a model protein. Transformation of a strain deficient in the gene for the 70-kDa protein was performed, and transformed colonies were detected with the antibody decoration technique. This technique is based upon gentle lysis of yeast colonies that have been grown on nitrocellulose filters such that the yeast proteins remain bound in discrete spots after lysis. The lysis is carried out by alkaline conditions in the presence of 2-mercaptoethanol and sodium dodecyl sulfate. After lysis, empty sites on the nitrocellulose filter are blocked to eliminate nonspecific binding of proteins by either 0.5% bovine serum albumin or 0.05% Tween 20. Decoration with antibody is visualized by using 125I-labeled protein A or peroxidase-conjugated second antibody. Antigens amounting to less than 0.1% of the total protein in the cell can be readily detected by the assay. The sensitivity of the assay enables detection of 1 positive colony per plate containing about 1000 colonies.
Isolation of the heterogeneous nuclear RNA-ribonucleoprotein complex (hnRNP): a unique supramolecular assembly.
Authors: Choi Y D , Dreyfuss G
Published: May 2006

The packaging of heterogeneous nuclear RNA (hnRNA), the fate of hnRNA in the nucleus, and the conversion of hnRNA to mRNA are believed to occur as the hnRNA transcript is associated with specific proteins to form a ribonucleoprotein complex termed the hnRNP complex. The identity and organization of the protein constituents of the hnRNP have been a matter of considerable controversy. We report here the isolation of the hnRNP complex from vertebrate cell nuclei, employing immunoprecipitation with monoclonal antibodies against the major proteins that are in contact with hnRNA in HeLa cells. Rapid immunoprecipitation from HeLa nucleoplasm with two different monoclonal antibodies to the hnRNP C proteins (41 and 43 kDa) isolates a similar complex that contains proteins and hnRNA of up to approximately equal to 10 kilobases. The major steady-state [35S]methionine-labeled proteins of the isolated complex are of 34 kDa, 36 kDa (A1 and A2), 37 kDa, 38 kDa (B1 and B2), 41 kDa, 43 kDa (C1 and C2), and doublets at 68 kDa and at 120 kDa. Additional proteins from 45 kDa to very high molecular mass are also seen. The major proteins of the complex appear identical by NaDodSO4/polyacrylamide gel electrophoresis to genuine hnRNP proteins--those which become crosslinked by UV light to the hnRNA in vivo. Immunoprecipitation with a different, noncrossreacting monoclonal antibody to the 120-kDa protein isolates an apparently identical complex of proteins that are present at a similar relative stoichiometry. Similar hnRNP complexes are found in rodent and avian cells. Nuclease digestions indicate that RNA plays a role in maintaining the integrity of the structure and that intact RNA of approximately equal to 125 nucleotides is sufficient to hold the complex of proteins together. The coimmunoprecipitation of the hnRNA and of all of the proteins through antibodies against different genuine hnRNP proteins and from divergent species strongly suggests that the hnRNP complex is a unitary structure of consistent, defined, and conserved components.