bluuebunny/crossref_metadata_2025 huggingface dataset:
Proline metabolism in N2-fixing root nodules: energy transfer and regulation of purine synthesis.
Authors: Kohl D H , Schubert K R , Carter M B , Hagedorn C H , Shearer G
Published: May 2006

N2-fixing root nodules of soybean (Glycine max L. Merr.) convert atmospheric N2 to ammonia(um) in an energy-intensive enzymatic reaction. These nodules synthesize large quantities of purines because nitrogen fixed by bacteria contained within this tissue is transferred to the shoots in the form of ureides, which are degradation products of purines. In animal systems, it has been proposed that proline biosynthesis by pyrroline-5-carboxylate reductase (P5CR) is used to generate the NADP+ required for the synthesis of the purine precursor ribose 5-phosphate. We have examined the levels, properties, and location of P5CR and proline dehydrogenase (ProDH) in soybean nodules. Nodule P5CR was found in the plant cytosol. Its activity was substantially higher than that reported for other animal and plant tissues and is 4-fold higher than in pea (Pisum sativum) nodules (which export amides). The Km for NADPH was lower by a factor of 25 than the Km for NADH, while the Vmax with NADPH was one-third of that with NADH. P5CR activity was diminished by NADP+ but not by proline. These characteristics are consistent with a role for P5CR in supporting nodule purine biosynthesis rather than in producing proline for incorporation into protein. ProDH activity was divided between the bacteroids and plant cytosol, but less than 2% was in the mitochondria-rich fractions. The specific activity of ProDH in soybean nodule bacteroids was comparable to that in rat liver mitochondria. In addition, we propose that some of the proline synthesized in the plant cytosol by P5CR is catabolized within the bacteroids by ProDH and that this represents a novel mechanism for transferring energy from the plant to its endosymbiont.
The predicted DBL oncogene product defines a distinct class of transforming proteins.
Authors: Eva A , Vecchio G , Rao C D , Tronick S R , Aaronson S A
Published: May 2006

The DBL transforming gene was originally identified by transfection of NIH 3T3 cells with DNA from a human B-cell lymphoma. This gene was found to have arisen as a result of recombination of the 3' portion of the DBL protooncogene coding sequences with an unrelated segment of human DNA. It encodes a cytoplasmic protein that is equally distributed between cytosol and crude membrane fractions. To further characterize this transforming gene, a biologically active cDNA clone of the DBL transforming gene mRNA was isolated. Analysis of the sequence of the DBL oncogene cDNA revealed a long open reading frame that encodes a hybrid protein whose first 50 amino acids (at least) derive from a complete exon of a different locus. No significant homology with known oncogenes or any known protein sequences was demonstrated. The computer analysis of the predicted DBL protein indicated it is highly hydrophilic with no hydrophobic domains characteristic of a membrane-spanning region or signal peptide. Thus, the DBL oncoprotein is distinct among known transforming gene products.
Neural networks counting chimes.
Authors: Amit D J
Published: May 2006

It is shown that the ideas that led to neural networks capable of recalling associatively and asynchronously temporal sequences of patterns can be extended to produce a neural network that automatically counts the cardinal number in a sequence of identical external stimuli. The network is explicitly constructed, analyzed, and simulated. Such a network may account for the cognitive effect of the automatic counting of chimes to tell the hour. A more general implication is that different electrophysiological responses to identical stimuli, at certain stages of cortical processing, do not necessarily imply synaptic modification, a la Hebb. Such differences may arise from the fact that consecutive identical inputs find the network in different stages of an active temporal sequence of cognitive states. These types of networks are then situated within a program for the study of cognition, which assigns the detection of meaning as the primary role of attractor neural networks rather than computation, in contrast to the parallel distributed processing attitude to the connectionist project. This interpretation is free of homunculus, as well as from the criticism raised against the cognitive model of symbol manipulation. Computation is then identified as the syntax of temporal sequences of quasi-attractors.
Binding of antiestrogens exposes an occult antigenic determinant in the human estrogen receptor.
Authors: Martin P M , Berthois Y , Jensen E V
Published: May 2006

Treatment of human breast cancer cytosol with tamoxifen (Tam) or 4-monohydroxytamoxifen (MHT) enhances the immunoreactivity of the estrogen receptor toward monoclonal antibody H222 but not monoclonal antibodies D547 or D75. This effect is evident from an increase in the apparent receptor content measured by the Abbott enzyme immunoassay, which uses peroxidase-labeled H222 as the chromogenic marker, and in the rate and size of the sedimentation peak of the immune complex of the receptor with radiolabeled H222. In contrast, MHT shows no effect in reversed immunoassay systems that use peroxidase-labeled D547 or D75 as chromogenic markers, nor does it affect the sedimentation peak of the complex of D547 with the receptor. MHT can exert its action on receptor bound to immobilized antibody. These results indicate that reaction with antiestrogens causes a change, probably conformational, in the receptor protein that exposes an occult antigenic determinant recognized uniquely by H222. Since this can occur in cytosol previously treated with excess estradiol in the cold, it appears to result from an interaction of antiestrogens with a region of the receptor distinct from the estrogen-binding site, suggesting that agonist and antagonist actions may involve different parts of the receptor molecule.
The mutY gene: a mutator locus in Escherichia coli that generates G.C----T.A transversions.
Authors: Nghiem Y , Cabrera M , Cupples C G , Miller J H
Published: May 2006

We have used a strain with an altered lacZ gene, which reverts to wild type via only certain transversions, to detect transversion-specific mutators in Escherichia coli. Detection relied on a papillation technique that uses a combination of beta-galactosides to reveal blue Lac+ papillae. One class of mutators is specific for the G.C----T.A transversion as determined by the reversion pattern of a set of lacZ mutations and by the distribution of forward nonsense mutations in the lacI gene. The locus responsible for the mutator phenotype is designated mutY and maps near 64 min on the genetic map of E. coli. The mutY locus may act in a similar but reciprocal fashion to the previously characterized mutT locus, which results in A.T----C.G transversions.
Lysophosphatidylcholine: a chemotactic factor for human monocytes and its potential role in atherogenesis.
Authors: Quinn M T , Parthasarathy S , Steinberg D
Published: May 2006

Native low density lipoprotein (LDL) does not affect monocyte/macrophage motility. On the other hand, oxidatively modified LDL inhibits the motility of resident peritoneal macrophages yet acts as a chemotactic factor for circulating human monocytes. We now show that lysophosphatidylcholine (lyso-PtdCho), which is generated by a phospholipase A2 activity during LDL oxidation, is a potent chemotactic factor for monocytes. It is not chemotactic for neutrophils or for resident macrophages. Platelet-activating factor, after treatment with phospholipase A2, becomes chemotactic for monocytes, whereas the intact factor is not. Synthetic 1-palmitoyl-lyso-PtdCho showed chemotactic activity comparable to that of the lyso-PtdCho fraction derived from oxidized LDL. The results suggest that lyso-PtdCho in oxidized LDL may favor recruitment of monocytes into the arterial wall during the early stages of atherogenesis. Generation of lyso-PtdCho, either from LDL itself or from membrane phospholipids of damaged cells, could play a more general role in inflammatory processes throughout the body.
Platelet-derived growth factor gene expression in human atherosclerotic plaques and normal artery wall.
Authors: Barrett T B , Benditt E P
Published: May 2006

We previously demonstrated that the B chain of platelet-derived growth factor (PDGF-B) is transcribed in human atherosclerotic plaques, indicating that production of growth factors within plaques could occur during atherogenesis. However, since atherosclerotic plaques are composed of several cell types and three of these--macrophages, endothelial cells, and smooth muscle cells--can express the PDGF genes, the cell type responsible for PDGF gene expression was not clear. In the present study we explore further the expression of PDGF-A and -B and identify transcriptionally active cell types. We assayed PDGF-A and -B mRNA levels in dissected fractions of carotid atherosclerotic plaques and normal artery and then sequentially rehybridized these blots with three cDNA probes that recognize cell type-specific markers: fms for macrophages, von Willebrand factor for endothelial cells, and smooth muscle alpha-actin for smooth muscle cells. In plaques, PDGF-A expression correlated with smooth muscle actin; PDGF-B expression correlated strongly with fms. PDGF-A expression correlated with smooth muscle actin. In normal vessel wall, PDGF-A expression was high in the media and again correlated with smooth muscle actin, whereas PDGF-B expression was high in the adventitia. Since transcripts from both PDGF genes are found in normal artery where cell turnover is very low, we suggest that PDGF gene expression does not necessarily function to produce smooth muscle cell proliferation. We propose that these genes may have an important nonmitogenic, maintenance function in normal arterial tissue and in the atherosclerotic plaque.
Membrane depolarization and carbamoylcholine stimulate phosphatidylinositol turnover in intact nerve terminals.
Authors: Audigier S M , Wang J K , Greengard P
Published: May 2006

Synaptosomes, purified from rat cerebral cortex, were prelabeled with [3H]inositol to study phosphatidylinositol turnover in nerve terminals. Labeled synaptosomes were either depolarized with 40 mM K+ or exposed to carbamoylcholine (carbachol). K+ depolarization increased the level of inositol phosphates in a time-dependent manner. The inositol trisphosphate concentration increased rapidly and transiently, reaching maximum (250% of control) in less than 3 sec and returning to near basal levels by 30 sec. The inositol bisphosphate level also increased rapidly, but its elevated level (220% of control) was sustained during continued depolarization. The elevated level of inositol bisphosphate was reversed upon repolarization of the synaptosomes. The level of inositol monophosphate increased slowly to 120-130% of control. These effects of K+ depolarization depended on the presence of Ca2+ in the incubation medium. Carbachol stimulated the turnover of phosphatidylinositol in a dose- and time-dependent manner. The level of inositol trisphosphate increased only slightly (120-130% of control) during carbachol stimulation. The level of inositol bisphosphate increased to 210% of control, and this maximal response was seen from 15 to 60 min. Accumulation of inositol monophosphate (250% of control) was larger than that of inositol bisphosphate, but its time course was slower. Atropine and pirenzepine inhibited the carbachol effect with high affinities of 0.8 nM and 16 nM, respectively, indicating that the effect of carbachol was mediated by activation of a M1 muscarinic receptor. Incubation of synaptosomes in Ca2+-free buffer reduced the response to carbachol by 30%, and addition of EGTA abolished it. These data show that both Ca2+ influx and M1 muscarinic receptor activation stimulate phospholipase C activity in synaptosomes, suggesting that phosphatidylinositol turnover may be involved in regulating neurotransmitter release from nerve terminals.
Cell volume regulation by the thin descending limb of Henle's loop.
Authors: Lopes A G , Amzel L M , Markakis D , Guggino W B
Published: May 2006

Thin descending limb cells from Henle's loop (from the inner strip of the outer medulla of long loops) were studied with optical and video techniques to identify the mechanisms of ion transport and cell volume regulation. Increasing the K+ concentration in the basolateral solution from 5 to 90 mM caused the cells to swell. This K+-induced swelling was inhibited by exposure of the basolateral membrane to 9 mM Ba2+ and was abolished by removing Cl- from the perfusion solutions. Decreasing the perfusion osmolality caused an increase in cell volume followed by a return to the preexposure volume. The latter regulatory decrease in hypoosmolality was slowed by basolateral Ba2+ and the removal of HCO-3 from the solutions. Further slowing occurred when both HCO-3 and Cl- were removed. Exposure of cells to ouabain abolished volume regulation. These data suggest that the basolateral cell membrane of the thin descending limb has a Cl- -dependent K+ permeability, which is important in cell volume regulation. The cells also possess Cl- and HCO-3 transport pathways that participate in volume regulation. Finally, volume regulation is dependent upon the operation of the Na/K pump.
Calcium influx and the Ca2+-calmodulin complex are involved in interferon-gamma-induced expression of HLA class II molecules on HL-60 cells.
Authors: Koide Y , Ina Y , Nezu N , Yoshida T O
Published: May 2006

Interferon gamma (IFN-gamma) induces HLA-DR and -DQ molecules and causes an accumulation of transcripts in HL-60 cells. Experiments were, therefore, designed to investigate the intracellular signaling molecules regulating the appearance of HLA class II molecules. The expression of HLA class II (DR and DQ) molecules induced by IFN-gamma was blocked by a calmodulin antagonist, W7, but not by a protein kinase C inhibitor, H7. Furthermore, a direct activator of protein kinase C, phorbol 12-myristate 13-acetate, was unable to induce HLA class II (DR) molecule expression. These results suggest that IFN-gamma induces HLA class II molecules on HL-60 cells by way of a calcium-calmodulin pathway and not by way of a protein kinase C pathway. Calmodulin is activated by a transient rise in the cytosolic free calcium. In fact, IFN-gamma evoked a calcium influx into HL-60 cells, whereas depletion of Ca2+ from culture medium resulted in a failure of IFN-gamma to induce DR expression. Furthermore, the calcium ionophore A23187 by itself induced DR molecule expression. These results suggest that IFN-gamma stimulates calcium influx by a so-called receptor-mediated calcium channel and activates the calmodulin branch of the calcium messenger system, resulting in the induction of DR molecules on the surface of HL-60 cells.
Prolactin and glucocorticoid hormones synergistically induce expression of transfected rat beta-casein gene promoter constructs in a mammary epithelial cell line.
Authors: Doppler W , Groner B , Ball R K
Published: May 2006

We have detected hormone response elements in the promoter region of the rat beta-casein gene that confer the synergistic action of prolactin and glucocorticoid hormones upon transcription of chimeric gene constructs. A 2800-base-pair (bp) rat beta-casein gene fragment containing 2300 bp of 5' flanking sequence was placed in front of a chloramphenicol acetyltransferase (CAT) reporter gene and stably transfected into the mouse mammary epithelial cell line HC11. Addition of prolactin or dexamethasone alone was sufficient for a modest induction of the fusion gene. The simultaneous presence of both hormones produced a strongly synergistic effect, which did not require the presence of insulin. Induction of the beta-casein-CAT gene was only observed in stably transfected confluent cell cultures. Analysis of a 5' deletion series of the beta-casein-CAT gene construct revealed a stepwise loss of hormone inducibility; 285 bp of 5' flanking sequence was sufficient to mediate the synergistic action of lactogenic hormones on expression. The response was reduced by half when compared with the construct containing 2300 bp of the 5' flanking region. Synergistic inducibility further decreased in deletion mutants between -285 and -265 and was completely abolished between -180 and -170. Thus, the 5' flanking region between -285 and -170 contains cis-acting sequences, which are required for mediating the effect of prolactin and dexamethasone.
Physiological localization of an agonist-sensitive pool of Ca2+ in parotid acinar cells.
Authors: Foskett J K , Gunter-Smith P J , Melvin J E , Turner R J
Published: May 2006

Muscarinic stimulation of fluid secretion by mammalian salivary acinar cells is associated with a rise in the level of intracellular free calcium ([Ca2+]i) and activation of a calcium-sensitive potassium (K+) conductance in the basolateral membrane. To test in the intact cell whether the rise of [Ca2+]i precedes activation of the K+ conductance (as expected if Ca2+ is the intracellular messenger mediating this response), [Ca2+]i and membrane voltage were measured simultaneously in carbachol-stimulated rat parotid acinar cells by using fura-2 and an intracellular microelectrode. Unexpectedly, the cells hyperpolarize (indicating activation of the K+ conductance) before fura-2 detectable [Ca2+]i begins to rise. This occurs even in Ca2+-depleted medium where intracellular stores are the only source of mobilized Ca2+. Nevertheless, when the increase in [Ca2+]i was eliminated by loading cells with the Ca2+ chelator bis(2-amino-5-methylphenoxy)ethane-N,N,N',N'-tetraacetate (Me2BAPTA) and stimulating in Ca2+-depleted medium, membrane hyperpolarization was also eliminated, indicating that a rise of [Ca2+] is required for the agonist-induced voltage response. Stimulation of Me2BAPTA-loaded cells in Ca2+-containing medium dramatically accentuates the temporal dissociation between the activation of the K+ conductance and the rise of [Ca2+]i. The data are consistent with the hypothesis that muscarinic stimulation results in a rapid localized increase in [Ca2+]i at the acinar basolateral membrane followed by a somewhat delayed increase in total [Ca2+]i. The localized increase cannot be detected by fura-2 but is sufficient to open the Ca2+-sensitive K+ channels located in the basolateral membrane. We concluded that a receptor-mobilized intracellular store of Ca2+ is localized at or near the basolateral membrane.
Circadian regulation of bioluminescence in Gonyaulax involves translational control.
Authors: Morse D , Milos P M , Roux E , Hastings J W
Published: May 2006

A 10-fold circadian variation in the amount of luciferin binding protein (LBP) in the marine dinoflagellate Gonyaulax polyedra is reported. This protein binds and stabilizes luciferin, the bioluminescence substrate. In early night phase, when bioluminescence is increasing and LBP levels are rising in the cell, pulse labeling experiments show that LBP is being rapidly synthesized in vivo. At other times, the rate of LBP synthesis is at least 50 times lower, while the rate of synthesis of most other proteins remains the same. The LBP mRNA levels, as determined by in vitro translations and by RNA (Northern) hybridizations, do not vary over the daily cycle, indicating that circadian control of bioluminescence in this species is mediated by translation.
Cytolysis by tumor necrosis factor is preceded by a rapid and specific dissolution of microfilaments.
Authors: Scanlon M , Laster S M , Wood J G , Gooding L R
Published: May 2006

Tumor necrosis factor (TNF) is cytotoxic to certain transformed cells, whereas normal cells are resistant to its effects. The resistance of normal cells can often be overcome by treatment with inhibitors of transcription or translation such as actinomycin D or cycloheximide (CHI), suggesting that normal cells produce a protein(s) that protects them from TNF-induced cytolysis. In this report, we examine the mechanism of cytolysis in a 3T3-like mouse cell line, C3HA, which was sensitized to TNF by treatment with CHI. We found that an early change in TNF/CHI-treated cells was a significant loss of stress fibers in perinuclear areas of the cytoplasm. The disruption of microfilaments, which was observed within 15 min of treatment, was not seen in untreated cells or in cells treated with either TNF or CHI alone. The dissolution of microfilaments spread peripherally over time and preceded other TNF/CHI-induced effects such as cytoplasmic "boiling," decrease in cell volume, and lysis of the plasma membrane. The breakdown of stress fibers occurred without a change in microtubules or intermediate filaments. Cytochalasin E, which disrupts microfilaments, induced cytolysis of TNF-treated cells even in the absence of CHI; however, demecolcine, which depolymerizes microtubules, did not sensitize cells to TNF. We propose that the TNF-induced cytolysis of certain cell types is preceded by a selective disruption of the microfilament lattice.
Identification of murine complement receptor type 2.
Authors: Fingeroth J D , Benedict M A , Levy D N , Strominger J L
Published: May 2006

A rabbit antiserum reactive with the human complement component C3d/Epstein-Barr virus receptor (complement receptor type 2, CR2) immunoprecipitates a Mr 155,000 murine B-cell surface antigen. The apparent molecular weight and cellular distribution of this murine antigen are similar to those of human CR2. Cells expressing the murine protein bind sheep erythrocytes coated with antibody and murine C1-C3d but do not bind Epstein-Barr virus at all. The monospecific antiserum to human CR2 together with goat F(ab')2 anti-rabbit IgG blocks attachment of the C3d-coated erythrocytes to receptor-bearing murine B lymphocytes. To further characterize murine CR2, a lambda gt11 library from the murine late pre-B-cell line 70Z/3 was screened with human CR2 cDNA. A partial cDNA clone of 3.5 kilobases with 79% amino acid sequence identity to human CR2 in the unique intracytoplasmic region and 63% identity to the sixth human CR2 repeat was obtained. Blot hybridization with the murine cDNA clone identified an RNA species of approximately 4.7 kilobases, similar in size to human CR2 mRNA, from a murine B-cell line but not from a murine T-cell line.
Common acute lymphoblastic leukemia antigen (CALLA) is active neutral endopeptidase 24.11 ("enkephalinase"): direct evidence by cDNA transfection analysis.
Authors: Shipp M A , Vijayaraghavan J , Schmidt E V , Masteller E L , D'Adamio L , Hersh L B , Reinherz E L
Published: May 2006

The common acute lymphoblastic leukemia antigen (CALLA) is a 749-amino acid type II integral membrane protein expressed by most acute lymphoblastic leukemias, certain other lymphoid malignancies with an immature phenotype, and normal lymphoid progenitors. A computer search against the most recent GenBank release (no. 56) indicates that human CALLA cDNA encodes a protein nearly identical to the rat and rabbit neutral endopeptidase 24.11 ("enkephalinase;" EC 3.4.24.11). This zinc metalloendopeptidase, which has been shown to inactivate a variety of peptide hormones including enkephalin, chemotactic peptide, substance P, neurotensin, oxytocin, bradykinin, and angiotensins I and II, had not been identified in lymphoid cells. To determine whether CALLA cDNA derived from human acute lymphoblastic leukemia cells (Nalm-6 cell line) encodes functional neutral endopeptidase activity, we generated CALLA+ stable transfectants in the CALLA- murine myeloma cell line J558 and analyzed them for enzymatic activity in a fluorometric assay based upon cleavage of the substrate glutaryl-Ala-Ala-Phe 4-methoxy-2-naphthylamide at the Ala-Phe bond. Total lysates as well as whole-cell suspensions of the Nalm-6 line and of the CALLA+ transfectants, but not of the CALLA- J558 cells, possessed neutral endopeptidase activity. This enzymatic activity was associated with the cellular membrane fraction and was abrogated by the specific neutral endopeptidase inhibitor phosphoramidon. The unequivocal identification of CALLA as a functional neutral endopeptidase provides insight into its potential role in both normal and malignant lymphoid function.
Neuropeptide K potently stimulates salivary gland secretion and potentiates substance P-induced salivation.
Authors: Takeda Y , Krause J E
Published: May 2006

Neuropeptide K (NPK) is an N-terminally extended derivative of neurokinin A (NKA) that can be a final product in the posttranslational processing of beta-preprotachykinin. A rat salivation bioassay was used to demonstrate potent effects of NPK at low doses, while effects due to NKA were much weaker at higher doses. The rank order of potency of beta-preprotachykinin-derived peptides on salivation responses was NPK greater than substance P greater than NKA much greater than beta-preprotachykinin-(72-96)-peptide. The time course of the NPK response was longer than that observed with substance P. The responses elicited by NPK were blocked by the tachykinin antagonist [D-Pro2,D-Trp7,9]substance P but not by atropine. In peptide coinfusion studies, NPK strikingly potentiated the salivation responses elicited by substance P. NPK in vitro displayed a 100 times lower potency than substance P in displacing 3H-labeled substance P binding in submandibular gland membranes, a tissue rich in SP-P type (NK-1) receptors. The possible cellular mechanisms by which NPK stimulates salivary gland secretion are discussed. We conclude that NPK and substance P may be cotransmitters derived by posttranslational processing of beta-preprotachykinin.
Chiral synthesis of D- and L-myo-inositol 1,4,5-trisphosphate.
Authors: Tegge W , Ballou C E
Published: May 2006

Chiral inositols (D-chiro-inositol from D-pinitol and L-chiro-inositol from L-quebrachitol) were converted to the 3,4-di-O-benzyl ethers, which were selectively benzoylated to yield the 1,2,5-tri-O-benzoyl-3,4-di-O-benzyl-chiro-inositols. The free hydroxyl group in each derivative was inverted by way of the trifluoromethane sulfonate ester to provide D- and L-1,2,4-tri-O-benzoyl-5,6-di-O-benzyl-myo-inositol. Hydrogenolysis to remove the benzyl ether groups gave the enantiomeric 1,2,4-tri-O-benzoyl-myo-inositols, which were phosphorylated by a dibenzylphosphite triester method. After hydrogenolysis and saponification of the derivatives, the D- and L-myo-inositol 1,4,5-trisphosphates were isolated as the crystalline cyclohexylammonium salts in gram quantity.
Granulocyte-macrophage colony-stimulating factor and human neutrophils: role of guanine nucleotide regulatory proteins.
Authors: Gomez-Cambronero J , Yamazaki M , Metwally F , Molski T F , Bonak V A , Huang C K , Becker E L , Sha'afi R I
Published: May 2006

The addition of granulocyte-macrophage colony-stimulating factor (GM-CSF) to human neutrophils causes a rapid increase in the basal and fMet-Leu-Phe-stimulated Na+ influx and an increase in intracellular pH. The increase can be seen as early as 5 min after the addition of GM-CSF. Changes produced by GM-CSF are totally inhibited by amiloride and are significantly reduced in pertussis toxin-treated cells. The stimulation of the Na+/H+ exchange mechanism by GM-CSF inhibits further stimulation of this system with either fMet-Leu-Phe or phorbol 12-myristate 13-acetate. In addition, membrane preparations isolated from GM-CSF-treated neutrophils have higher basal and stimulated GTPase activities. The basal and the fMet-Leu-Phe- or platelet-activating factor-stimulated GTPase activities are reduced in pertussis toxin-treated cells. Cells pretreated with GM-CSF accumulate more radioactive phosphate than control cells, and this increase is diminished by pertussis toxin treatment. In addition, GM-CSF causes a rapid increase in the tyrosine phosphorylation levels of five proteins with molecular masses of 118 kDa, 92 kDa, 78 kDa, 54 kDa, and 40 kDa. These results clearly show that GM-CSF, on its own, can initiate several changes and that these changes are mediated in part by the pertussis toxin-sensitive guanine nucleotide regulatory protein.
Isolation and characterization of a mammalian gene encoding a high-affinity cAMP phosphodiesterase.
Authors: Colicelli J , Birchmeier C , Michaeli T , O'Neill K , Riggs M , Wigler M
Published: May 2006

A rat brain cDNA library has been constructed in a Saccharomyces cerevisiae expression vector and used to isolate genes that can function in yeast to suppress the phenotypic effects of RAS2val19, a mutant form of the RAS2 gene analogous to an oncogenic mutant of the human HRAS gene. One cDNA, DPD, was cloned and its genetic and biochemical properties were characterized. A DPD product would share 80% amino acid sequence identity with the Drosophila melanogaster dunce-encoded protein over an extended region. We have shown that the DPD protein is a high-affinity cAMP-specific phosphodiesterase.
Mechanism of regulation of the 422(aP2) gene by cAMP during preadipocyte differentiation.
Authors: Yang V W , Christy R J , Cook J S , Kelly T J , Lane M D
Published: May 2006

During differentiation of 3T3-L1 preadipocytes into adipocytes, expression of the gene encoding adipocyte 422(aP2) protein is activated. We have shown that the first 248 base pairs of the 422(aP2) gene promoter (which lacks a consensus cAMP response element) are sufficient to confer inducibility of a reporter gene by cAMP in preadipocytes. We now demonstrate by deletion analysis that this DNA segment contains overlapping negative and positive regulatory elements. The positive regulatory element contains a consensus activator protein 1 (AP-1) binding sequence. The effect of the negative regulatory element is observed in preadipocytes but not in fully differentiated adipocytes, suggesting that it is an important component of the regulatory mechanism governing expression of the 422(aP2) gene during differentiation. cAMP activates the 422(aP2) promoter in confluent preadipocytes but not in proliferating preadipocytes or fully differentiated adipocytes. The stimulatory effect of cAMP is abolished by deletions that enter the negative element, suggesting that cAMP increases expression by relieving the inhibitory effect of the negative regulatory element.
Enhanced meiotic recombination on the smallest chromosome of Saccharomyces cerevisiae.
Authors: Kaback D B , Steensma H Y , de Jonge P
Published: May 2006

Chromosome I is the smallest chromosome in Saccharomyces cerevisiae and contains a DNA molecule that is only 250 kilobases (kb). Approximately 75% of this DNA molecule has been cloned. A restriction map for the entire DNA molecule from chromosome I was determined and most of its genetically mapped genes were located on this physical map. Based on the average rate of recombination (centimorgans/kb) found for other S. cerevisiae chromosomes, the outermost markers on the genetic map of chromosome I were expected to be close to the ends of the DNA molecule. While the rightmost genetic marker was 3 kb from the end, the leftmost marker, CDC24, was located near the middle of the left arm, suggesting that the genetic map would be much longer. To extend the genetic map, a copy of the S. cerevisiae URA3 gene was integrated in the outermost cloned region located 32 kb centromere distal to CDC24, and the genetic map distance between these two genes was determined. The new marker substantially increased the genetic map length of chromosome I. In addition, we determined the relationship between physical and genetic map distance along most of the length of the chromosome. Consistent with the longer genetic map, the average rate of recombination between markers on chromosome I was greater than 50% higher than the average found on other yeast chromosomes. Owing to its small size, it had been estimated that approximately 5% of the chromosome I homologues failed to undergo meiotic recombination. New measurements of the zero-crossover class indicated that the enhanced rate of recombination ensures at least one genetic exchange between virtually every pair of chromosome I homologues.
Insertional mutation in a transgenic mouse allelic with Purkinje cell degeneration.
Authors: Krulewski T F , Neumann P E , Gordon J W
Published: May 2006

Purkinje cell degeneration (pcd) is an autosomal recessive mutation which maps to chromosome 13 in the mouse. The pcd mutation causes loss of cerebellar Purkinje cells, retinal photoreceptor cells, and olfactory bulb mitral cells, as well as abnormalities of spermatogenesis. pcd is among a number of autosomal recessive mutations in mice and humans that affect neurologic function and male fertility. The cloning of one or more of these loci would contribute significantly to our understanding of the genetic control of development and maintenance of affected cell types. We report here identification of a transgenic mouse line with an insertional mutation that is allelic with pcd and manifests histopathologic features indistinguishable from those of the spontaneous mutation. The creation of an allele of pcd by transgene insertion should make possible the cloning of the pcd locus.
Induction of diapause in Drosophila melanogaster: photoperiodic regulation and the impact of arrhythmic clock mutations on time measurement.
Authors: Saunders D S , Henrich V C , Gilbert L I
Published: May 2006

The fruit fly Drosophila melanogaster displays an ovarian diapause that is regulated by photoperiod. Newly eclosed female flies (Canton-S wild type) exposed to short days (less than 14 hr of light per day) at 12 degrees C (or 10 degrees C) enter a fairly shallow reproductive diapause. Females exposed to long days (16 hr of light per day) at the same low temperature undergo ovarian maturation. The short day induced diapause continues for 6-7 weeks under a 10:14 light/dark cycle at 12 degrees C but is terminated rapidly after a transfer to higher temperature (18 or 25 degrees C) or to long days (18:6 light/dark cycle). Females from three strains homozygous for alleles of the period (per) locus, reportedly arrhythmic for behavioral circadian rhythms, and females that possessed two overlapping deletions of per were also capable of discriminating between long and short days, although, when compared with the wild-type flies, the critical day length was shifted to shorter values by approximately 2 hr. It is concluded that the period locus is not causally involved in photoperiod time measurement.
Loss of heterozygosity for the short arm of chromosome 1 in human neuroblastomas: correlation with N-myc amplification.
Authors: Fong C T , Dracopoli N C , White P S , Merrill P T , Griffith R C , Housman D E , Brodeur G M
Published: May 2006

Partial monosomy of the short arm of chromosome 1 is the most consistent cytogenetic abnormality found in human neuroblastomas, but its overall frequency and significance are unclear. Using a panel of chromosome-1-specific DNA probes that identify restriction fragment length polymorphisms, we demonstrate that 13 of 47 human neuroblastomas (28%) have somatic loss of heterozygosity (LOH) at one or more loci on the distal short arm of chromosome 1. the chromosomal region that shows LOH most consistently is between 1p36.1 and 1p36.3; loss of a gene or genes in this region may be critical for the development or progression of neuroblastomas. The region of LOH in human neuroblastoma may resemble that described for pheochromocytoma, medullary thyroid carcinoma, and melanoma, which are also tumors of neural-crest origin. Although LOH for distal chromosome 1p can occur in early stages of neuroblastoma, the loss usually occurs in tumors of advanced clinical stages. LOH for the short arm of chromosome 1 correlates significantly with N-myc amplification, suggesting that these two genetic events are related. Indeed, these two lesions appear to characterize a genetically distinct subset of particularly aggressive neuroblastomas.
Potential antiinflammatory effects of interleukin 4: suppression of human monocyte tumor necrosis factor alpha, interleukin 1, and prostaglandin E2.
Authors: Hart P H , Vitti G F , Burgess D R , Whitty G A , Piccoli D S , Hamilton J A
Published: May 2006

Stimulated human monocytes/macrophages are a source of mediators such as tumor necrosis factor alpha (TNF-alpha), interleukin 1 (IL-1), and prostaglandin E2 (PGE2), which can modulate inflammatory and immune reactions. Therefore, the ability to control the production of such mediators by monocytes/macrophages may have therapeutic benefits, and it has been proposed that glucocorticoids may act in this way. Purified human monocytes, when stimulated in vitro with lipopolysaccharide (LPS) or with LPS and gamma interferon (IFN-gamma), produce TNF-alpha, IL-1, and PGE2. Cotreatment of stimulated cells with the purified human lymphokine, interleukin 4 (IL-4 greater than or equal to 0.1-0.5 unit/ml; 12-60 pM) dramatically blocked the increased levels of these three mediators; for TNF-alpha and IL-1, the inhibition was manifest at the level of mRNA. Thus, IL-4 can suppress some parameters of monocyte activation and, as for B cells, have opposite effects to IFN-gamma. The effects of IL-4 on human monocytes are similar to those obtained with the glucocorticoid dexamethasone (0.1 microM).
Construction and use of a replication-competent human immunodeficiency virus (HIV-1) that expresses the chloramphenicol acetyltransferase enzyme.
Authors: Terwilliger E F , Godin B , Sodroski J G , Haseltine W A
Published: May 2006

The construction and properties of an infectious human immunodeficiency virus (HIV) that expresses the bacterial gene chloramphenicol acetyltransferase are described. This virus can be used in vitro to screen for drugs that inhibit HIV infection. The marked virus may also be used to trace the routes of infection from the site of inoculation in animal experiments.
Active transport of gamma-aminobutyric acid and glycine into synaptic vesicles.
Authors: Kish P E , Fischer-Bovenkerk C , Ueda T
Published: May 2006

Although gamma-aminobutyric acid (GABA) and glycine are recognized as major amino acid inhibitory neurotransmitters in the central nervous system, their storage is poorly understood. In this study we have characterized vesicular GABA and glycine uptakes in the cerebrum and spinal cord, respectively. We present evidence that GABA and glycine are each taken up into isolated synaptic vesicles in an ATP-dependent manner and that the uptake is driven by an electrochemical proton gradient. Uptake for both amino acids exhibited kinetics with low affinity (Km in the millimolar range) similar to vesicular glutamate uptake. The ATP-dependent GABA uptake was not inhibited by the putative amino acid neurotransmitters glycine, taurine, glutamate, or aspartate or by GABA analogs, agonists, and antagonists. Similarly, ATP-dependent glycine uptake was hardly affected by GABA, taurine, glutamate, or aspartate or by glycine analogs or antagonists. The GABA uptake was not affected by chloride, which is in contrast to the uptake of the excitatory neurotransmitter glutamate, whereas the glycine uptake was slightly stimulated by low concentrations of chloride. Tissue distribution studies indicate that the vesicular uptake systems for GABA, glycine, and glutamate are distributed in different proportions in the cerebrum and spinal cord. These results suggest that the vesicular uptake systems for GABA, glycine, and glutamate are distinct from each other.
Partial diversion of a mutant proinsulin (B10 aspartic acid) from the regulated to the constitutive secretory pathway in transfected AtT-20 cells.
Authors: Gross D J , Halban P A , Kahn C R , Weir G C , Villa-Komaroff L
Published: May 2006

A patient with type II diabetes associated with hyperproinsulinemia has been shown to have a point mutation in one insulin gene allele, resulting in replacement of histidine with aspartic acid at position 10 of the B-chain. To investigate the basis of the proinsulin processing defect, we introduced an identical mutation in the rat insulin II gene and expressed both the normal and the mutant genes in the AtT-20 pituitary corticotroph cell line. Cells expressing the mutant gene showed increased secretion of proinsulin relative to insulin and rapid release of newly synthesized proinsulin. Moreover, the mutant cell lines did not store the prohormone nor did they release it upon stimulation with secretagogues. These data indicate that a significant fraction of the mutant prohormone is released via the constitutive secretory pathway rather than the regulated pathway, thereby bypassing granule-related processing and regulated release.
Single adduct mutagenesis: strong effect of the position of a single acetylaminofluorene adduct within a mutation hot spot.
Authors: Burnouf D , Koehl P , Fuchs R P
Published: May 2006

2-Acetylaminofluorene (AAF), a potent rat liver carcinogen that binds covalently to the C-8 position of guanine residues in DNA, is an effective frameshift mutagen. The mutations are distributed nonrandomly, in that most are located at a few specific DNA sequences (i.e., mutation hot spots). Among these hot spots, the Nar I sequence (GGCGCC) is especially susceptible to the induction of -2 frameshift mutations (GGCGCC----GGCC). Due to the nature of the Nar I sequence, G1G2CG3CC, three different molecular events, each involving the deletion of two contiguous base pairs (i.e., G2C, CG3, G3C), can give rise to the observed end point (GGCC). To compare the potential role of each of the three possible guanine-AAF adducts in the Nar I site to induce the -2 frameshift mutation, we constructed double-stranded plasmid molecules containing a single-AAF adduct bound to one of the three guanine positions. Using these plasmids, we found that only the adduct in the G3 position induces the -2 frameshift mutation. This strong effect of the position of the -AAF adduct within the Nar I site is discussed in relation to the possible involvement of an unusual DNA conformation in the mutagenic processing.
Computer-simulation methods in human linkage analysis.
Authors: Ott J
Published: May 2006

In human linkage analysis, many statistical problems without analytical solution could be solved by ad hoc Monte Carlo procedures were efficient computer-simulation methods available for members of family pedigrees. In this paper, a general method is described for randomly generating genotypes at one or more marker loci, given observed phenotypes at loci linked among themselves and with the markers. The method is based on a well-known expansion of the multivariate probability of genotypes, given phenotypes, into a product of conditional univariate probabilities that may be viewed as corresponding to conditionally independent univariate random variables. This representation allows a recursive evaluation of the univariate probabilities that can be implemented in a surprisingly simple manner by carrying out successive "risk calculations" with respect to marker genotypes, given observed phenotypes and marker genotypes already generated. Potential applications to various unresolved problems are discussed. The method is applied to 28 published families analyzed for genetic linkage between hereditary motor and sensory neuropathy I and the Duffy (FY) blood group locus and confirms heterogeneity of hereditary motor and sensory neuropathy I. An implementation of the simulation methods developed in the LINKAGE program package will be available later in 1989.
Cross-linking of interleukin 4 to surface molecules on murine T and B lymphocytes.
Authors: Fernandez-Botran R , Uhr J W , Vitetta E S
Published: May 2006

Interleukin 4 (IL-4) is a T-cell derived lymphokine with multiple activities on a variety of cells. An intriguing feature of the different IL-4-mediated activities is their requirements for markedly different concentrations of IL-4 even on the same cell population. To gain some insight into the phenomenon, we have analyzed the structure of IL-4-binding proteins on T and B cells by cross-linking different concentrations of 125I-labeled IL-4 (125I-IL-4) to cells. Cross-linking of 125I-IL-4 at relatively low concentrations in the presence of the cross-linking agent 3,3'-dithiobis(propionic acid hydroxysuccinimide ester) resulted in the detection of the previously described 60- to 75-kDa protein. At higher 125I-IL-4 concentrations, however, an additional molecule of 105 kDa was observed. Cross-linking of 125I-IL-4 to both molecules was specific; it was inhibited by the presence of a 50-fold excess of either unlabeled IL-4 or monoclonal anti-IL-4 antibody. Furthermore, considerable size heterogeneity of the IL-4-binding proteins was evident in different cell populations. These results suggest that IL-4 receptors might have a more complex structure than originally reported and/or that high concentrations of IL-4 might induce interactions of the previously described IL-4 receptor (60-75 kDa) with a 105-kDa molecule. Hence, it is possible that IL-4 might generate a different signal at high concentrations through interaction of its receptor with another membrane molecule.
Inositolphospholipid-linked glutamate receptors mediate cerebellar parallel-fiber-Purkinje-cell synaptic transmission.
Authors: Blackstone C D , Supattapone S , Snyder S H
Published: May 2006

In slices of adult rat cerebellum inositolphospholipid turnover is stimulated markedly by glutamate and its rigid analogues quisqualate and ibotenate. The drug and amino acid specificity of the response reflects a quisqualate-preferring excitatory amino acid receptor. The absence of glutamate-enhanced inositolphospholipid turnover in mice with Purkinje-cell degeneration indicates that the inositolphospholipid-linked quisqualate receptor mediates parallel fiber-Purkinje cell synaptic transmission. The quantitative prominence of this synapse accounts for the massive enrichment of elements of the inositolphospholipid system in cerebellar Purkinje cells.
Clonal contributions of small numbers of retrovirally marked hematopoietic stem cells engrafted in unirradiated neonatal W/Wv mice.
Authors: Capel B , Hawley R , Covarrubias L , Hawley T , Mintz B
Published: May 2006

Mice were repopulated with small numbers of retrovirally marked hematopoietic cells operationally definable as totipotent hematopoietic stem cells, without engraftment of cells at later stages of hematopoiesis, in order to facilitate analysis of stem cell clonal histories. This result depended upon the use of unirradiated W/Wv newborn recipients. Before transplantation, viral integration markers were introduced during cocultivation of fetal liver or bone marrow cells with helper cell lines exporting defective recombinant murine retroviruses of the HHAM series. Omission of selection in culture [although the vector contained the bacterial neomycin-resistance (neo) gene] also limited the proportion of stem cells that were virally labeled. Under these conditions, engraftment was restricted to a small population of marked and unmarked normal donor stem cells, due to their competitive advantage over the corresponding defective cells of the mutant hosts. A relatively simple and coherent pattern emerged, of one or a few virally marked clones, in contrast to previous studies. In order to establish the totipotent hematopoietic stem cell identity of the engrafted cells, tissues were sampled for viral and inbred-strain markers for periods close to one year after transplantation. The virally labeled clones were characterized as stem cell clones by their extensive self-renewal and by formation of the wide range of myeloid and lymphoid lineages tested. Results clearly documented concurrent contributions of cohorts of stem cells to hematopoiesis. A given stem cell can increase or decrease its proliferative activity, become completely inactive or lost, or become active after a long latent period. The contribution of a single clone present in a particular lineage was usually between 5% and 20%.