bluuebunny/crossref_metadata_2025 huggingface dataset:
Polarity orientation of microtubules in hippocampal neurons: uniformity in the axon and nonuniformity in the dendrite.
Authors: Baas P W , Deitch J S , Black M M , Banker G A
Published: May 2006

We have analyzed the polarity orientation of microtubules in the axons and dendrites of cultured rat hippocampal neurons. As previously reported of axons from other neurons, microtubules in these axons are uniform with respect to polarity; (+)-ends are directed away from the cell body toward the growth cone. In sharp contrast, microtubules in the mid-region of the dendrite, approximately 75 microns from the cell body, are not of uniform polarity orientation. Roughly equal proportions of these microtubules are oriented with (+)-ends directed toward the growth cone and (+)-ends directed toward the cell body. At distances within 15 micron of the growth cone, however, microtubule polarity orientation in dendrites is similar to that in axons; (+)-ends are uniformly directed toward the growth cone. These findings indicate a clear difference between axons and dendrites with respect to microtubule organization, a difference that may underlie the differential distribution of organelles within the neuron.
BRL 34915 (cromakalim) activates ATP-sensitive K+ current in cardiac muscle.
Authors: Sanguinetti M C , Scott A L , Zingaro G J , Siegl P K
Published: May 2006

The mechanism by which the antihypertensive agent BRL 34915 (cromakalim) affects action potential duration (APD) and effective refractory period (ERP) in isolated cardiac muscle was investigated. BRL 34915 (greater than or equal to 3 microM) shortened ERP of ferret (Mustela putorius furo) and guinea pig (Cavia porcellus) papillary muscles in a concentration-dependent fashion. The reduction in ERP resulted from a decrease in APD. ERP and APD of papillary muscles were also reduced during hypoxia produced by bubbling the physiological bathing solution with N2 instead of O2. Reduction of APD during hypoxia has previously been attributed to activation of ATP-sensitive K+ channels in heart. Glyburide, an inhibitor of ATP-sensitive K+ channels, prevented or reversed the shortening of ERP and APD produced by hypoxia and BRL 34915, respectively. These results suggest that BRL 34915 acts by opening ATP-sensitive K+ channels in heart. The actions of BRL 34915 were temperature-dependent, decreasing ERP 64% at 37 degrees C, but having no effect at 22 degrees C. The effect of BRL 34915 on K+ currents was tested directly in voltage-clamped guinea pig ventricular myocytes. As observed with the papillary muscles, BRL 34915 was without effect at 22 degrees C. At 36 degrees C, BRL 34915 (after a delay) increased outward currents positive to, and less so at potentials negative to, the K+ current reversal potential. The normal inwardly rectifying current-voltage relationship for peak K+ currents during 200-msec pulses was changed to one that was nearly ohmic. The current activated by BRL 34915 was blocked by glyburide. The data support the hypothesis that BRL 34915, like hypoxia, activates ATP-sensitive K+ channels in the heart. Based upon the profound temperature sensitivity of BRL 34915 action, this activation may be indirect, perhaps by means of modulation of an enzymatic activity that regulates gating of these channels. BRL 34915 and glyburide will be valuable tools for studying the role of ATP-sensitive K+ channels in normal and abnormal cardiac function.
Regulation of beta-adrenergic receptors by "permissive" hormones: glucocorticoids increase steady-state levels of receptor mRNA.
Authors: Hadcock J R , Malbon C C
Published: May 2006

Incubation of DDT1 MF-2 hamster vas deferens cells with glucocorticoids results in a marked increase in beta-adrenergic receptor (beta AR) number. The increase in receptor number was visualized by indirect immunofluorescence with antiserum specific for the beta AR and was verified by radioligand binding. The steady-state levels of beta AR mRNA were quantified in untreated (control) and glucocorticoid-treated cells by DNA-excess solution hybridization using a single-stranded probe corresponding to nucleotides +12 to 182 of the hamster beta 2AR cDNA coding region. The steady-state level increased from 0.37 pg of beta AR mRNA per microgram of total cellular RNA in untreated cells to 1.05 pg of beta AR mRNA per microgram of RNA in cells treated with dexamethasone (500 nM) for 2-4 hr. After this sharp transient peak, the steady-state level of receptor mRNA declined by 6 hr to a level approximately twice that of the untreated cells. Half-maximal effects were achieved at 20-40 nM dexamethasone. Testosterone (500 nM) and 17 beta-estradiol (500 nM), in contrast, did not alter the steady-state levels of beta AR mRNA. Actinomycin D, a potent inhibitor of transcription, abolished the dexamethasone-induced increase in beta AR mRNA, suggesting that the permissive hormone effect was exerted on gene transcription. The half-life of the receptor mRNA measured in the presence of actinomycin D was found to be 12 hr in both the untreated and the dexamethasone-treated cells. These studies provide a molecular explanation for the well-known regulation of GTP-binding protein (G-protein)-linked cell-surface receptors by permissive hormones.
Acidic intracellular pH shift during Caenorhabditis elegans larval development.
Authors: Wadsworth W G , Riddle D L
Published: May 2006

During recovery from the developmentally arrested, nonfeeding dauer stage of the nematode Caenorhabditis elegans, metabolic activation is accompanied by a decrease in intracellular pH (pHi). Phosphorus-31 nuclear magnetic resonance (31P NMR) analyses of perchloric acid extracts show that inorganic phosphate predominates in dauer larvae, whereas ATP and other high-energy metabolites are abundant within 6 hr after dauer larvae have been placed in food to initiate development. Although metabolic activation has been associated with an alkaline pHi shift in other organisms, in vivo 31P NMR analysis of recovering dauer larvae shows a pHi decrease from approximately 7.3 to approximately 6.3 within 3 hr after the animals encounter food. This shift occurs before feeding begins, and it coincides with, or soon follows, the developmental commitment to recover from the dauer stage, suggesting that control of pHi may be important in the regulation of larval development in nematodes.
Lymphoid V(D)J recombination: nucleotide insertion at signal joints as well as coding joints.
Authors: Lieber M R , Hesse J E , Mizuuchi K , Gellert M
Published: May 2006

The coding regions of antigen receptor genes assembled by variable-diversity-joining region [V(D)J] recombination are known in many cases to have undergone deletions of several nucleotides and also to contain insertions of noncoded nucleotides at the recombined junction (the coding joint). By using extrachromosomal recombination substrates to transfect lymphoid cell lines, we show that the signal joint (the fusion of the corresponding recognition signal sequences) can also contain insertions; however, nucleotide loss from the signals is very rare. The frequency of nucleotide addition varies among pre-B-cell lines in a manner proportional to their content of terminal deoxynucleotidyltransferase. We also find frequent nucleotide additions (and deletions) at coding joints, but in this case there is no strong correlation with the level of terminal deoxynucleotidyltransferase activity. Inserts at both signal and coding joints are rich in G + C, consistent with the base utilization preference of this enzyme.
Characterization and expression of the murine CD3-epsilon gene.
Authors: Clevers H , Dunlap S , Saito H , Georgopoulos K , Wileman T , Terhorst C
Published: May 2006

The receptor for antigen on the surface of T lymphocytes consists of a variable disulfide-bridged hetero-dimer (TCR-alpha/beta or -gamma/delta) associated with invariant CD3 proteins (CD3-gamma, -delta, -epsilon, and -zeta). The genes coding for the CD3 proteins are expressed in the earliest recognizable thymocytes, preceding the rearrangement and expression of the TCR genes. The isolation, characterization, and in vitro expression of the murine CD3-epsilon gene, as reported here, represent obligatory steps toward our understanding of the complex rules that govern T-cell-specific gene expression. The CD3-epsilon gene was transcribed from a non-TATA promoter and consisted of eight exons, two of which were unusually small (18 and 15 base pairs). The transmembrane exon was found to be homologous to the transmembrane exons of the CD3-gamma and CD3-delta genes. In transient-transfection experiments, a genomic fragment comprising 4 kilobases of upstream sequence and extending into the second exon sufficient to drive the expression of a reporter gene in murine T cells.
Identification of arginine as a precursor of endothelium-derived relaxing factor.
Authors: Sakuma I , Stuehr D J , Gross S S , Nathan C , Levi R
Published: May 2006

Nitric oxide (NO) is a major endothelium-derived relaxing factor (EDRF) released in response to vasodilating amines, peptides, proteins, ionophores, and nucleotides. EDRF is an important regulator of smooth muscle tone and platelet aggregation and adhesion. Histamine and acetylcholine relax the intact norepinephrine-constricted guinea pig pulmonary artery by an EDRF-dependent mechanism in a medium free of amino acids. N omega-Monomethylarginine (N-MeArg; 0.25 mM) inhibited this relaxation by 64-73%. Inhibition by N-MeArg developed rapidly and was immediately and completely reversed by excess L-arginine but not by D-arginine or by citrulline. N-MeArg did not diminish relaxation induced by nitroprusside, an NO-generating agent, indicating that N-MeArg acts on endothelium rather than on smooth muscle. These observations strongly suggest that, in the intact guinea pig pulmonary artery, EDRF originates from enzymatic action on the guanido nitrogen(s) of an endogenous pool of arginine. This is strikingly similar to the origin of reactive nitrogen intermediates in activated macrophages.
Immunohistochemical evidence for the existence of a dopamine- and cyclic AMP-regulated phosphoprotein (DARPP-32) in brown adipose tissue of pigs.
Authors: Meister B , Fried G , Hökfelt T , Hemmings H C , Greengard P
Published: May 2006

The indirect immunofluorescence technique was used to study the cellular localization of DARPP-32, a dopamine- and cyclic AMP-regulated phosphoprotein, in brown adipose tissue of newborn piglets. Clusters of strongly DARPP-32-immunoreactive cells were found in brown adipose tissue from the interscapular area and around lymph nodes close to the kidneys, adrenal glands, descending aorta, and great veins in the neck. The DARPP-32-immunoreactive cells contained multilocular lipid droplets, had round, centrally located nuclei, and were polygonal in shape, thus possessing characteristics and location sites typical for brown fat cells. The results indicate that brown adipose tissue from the newborn pig contains DARPP-32, an intracellular third messenger for dopamine. Together with recent functional data, these results strongly suggest that dopaminergic D1 mechanisms--i.e., activation of adenylate cyclase and formation of cyclic AMP--may be involved in cold-induced, nonshivering, and/or diet-induced thermogenesis.
Magnetic localization of neuronal activity in the human brain.
Authors: Yamamoto T , Williamson S J , Kaufman L , Nicholson C , Llinás R
Published: May 2006

The performance of a cryogenic system that monitors the extracranial magnetic field simultaneously at 14 positions over the scalp has been evaluated to determine the accuracy with which neuronal activity can be located within the human brain. Initially, measurements were implemented on two model systems, a lucite sphere filled with saline and a model skull. With a magnetic field strength similar to that of a human brain, the measurement and analysis procedures demonstrated a position accuracy better than 3 mm, for a current dipole 3 cm beneath the surface. Subsequently, measurements of the magnetic field pattern appearing 100 ms after the onset of an auditory tone-burst stimulus were obtained in three human subjects. The location of the current dipole representing intracellular ionic current in active neurons of the brain was determined, with 3-mm accuracy, to be within the cortex forming the floor of the Sylvian fissure of the individual subjects, corresponding closely to the Heschl gyrus as determined from magnetic resonance images. With the sensors placed at appropriate positions, the locations of neuronal sources for different tone frequencies could be obtained without moving the recording instrument. Adaptation of activity in human auditory cortex was shown to reveal long-term features with a paradigm that compared response amplitudes for three tones randomly presented.
A glutamate receptor regulates Ca2+ mobilization in hippocampal neurons.
Authors: Murphy S N , Miller R J
Published: May 2006

We investigated the effect of various excitatory amino acids on intracellular free Ca2+ concentration ( [Ca2+]i) in single mouse hippocampal neurons in vitro by using the Ca2+-sensitive dye fura-2. In normal physiological solution, glutamate, kainate, N-methyl-D-aspartate, and quisqualate all produced increases in [Ca2+]i. When all extracellular Ca2+ was removed, kainate and N-methyl-D-aspartate were completely ineffective, but quisqualate and glutamate were able to produce a spike-like Ca2+ transient, presumably reflecting the release of Ca2+ from intracellular stores. Ca2+ transients of similar shape could also be produced by the alpha 1-adrenergic agonist phenylephrine. After the production of a Ca2+ transient a second addition of quisqualate was ineffective unless intracellular stores were refilled by loading the cell with Ca2+ following depolarization in Ca2+-containing medium. None of the conventional excitatory amino acid receptor antagonists inhibited the Ca2+-mobilizing effects of quisqualate. Furthermore alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) was unable to produce Ca2+ mobilization in Ca2+-free medium, although it could produce Ca2+ influx in Ca2+-containing medium. Thus, glutamate can produce mobilization of Ca2+ from intracellular stores in hippocampal neurons by acting on a quisqualate-sensitive but AMPA-insensitive receptor. This receptor is therefore distinct from the quisqualate receptor that produces cell depolarization. The possibility that this Ca2+-mobilizing effect is mediated by inositol triphosphate production is discussed.
Detection of nucleic acid hybridization by nonradiative fluorescence resonance energy transfer.
Authors: Cardullo R A , Agrawal S , Flores C , Zamecnik P C , Wolf D E
Published: May 2006

Three approaches were used to study hybridization of complementary oligodeoxynucleotides by nonradiative fluorescence resonance energy transfer. (i) Fluorescein (donor) and rhodamine (acceptor) were covalently attached to the 5' ends of complementary oligodeoxynucleotides of various lengths. Upon hybridization of the complementary oligodeoxynucleotides, energy transfer was detected by both a decrease in fluorescein emission intensity and an enhancement in rhodamine emission intensity. In all cases, fluorescein emission intensity was quenched by about 26% in the presence of unlabeled complement. Transfer efficiency at 5 degrees C decreased from 0.50 to 0.22 to 0.04 as the distance between donor and acceptor fluorophores in the hybrid increased from 8 to 12 to 16 nucleotides. Modeling of these hybrids as double helices showed that transfer efficiency decreased as the reciprocal of the sixth power of the donor-acceptor separation R, as predicted by theory with a corresponding R0 of 49 A. (ii) Fluorescence resonance energy transfer was used to study hybridization of two fluorophore-labeled oligonucleotides to a longer, unlabeled oligodeoxynucleotide. Two 12-mers were prepared that were complementary to two adjacent sequences separated by four bases on a 29-mer. The adjacent 5' and 3' ends of the two 12-mers labeled with fluorescein and rhodamine exhibited a transfer efficiency of approximately 0.60 at 5 degrees C when they both hybridized to the unlabeled 29-mer. (iii) An intercalating dye, acridine orange, was used as the donor fluorophore to a single rhodamine covalently attached to the 5' end of one oligodeoxynucleotide in a 12-base-pair hybrid. Under these conditions, the transfer efficiency was approximately 0.47 at 5 degrees C. These results establish that fluorescence modulation and nonradiative fluorescence resonance energy transfer can detect nucleic acid hybridization in solution. These techniques, with further development, may also prove useful for detecting and quantifying nucleic acid hybridization in living cells.
DNA damage-inducible transcripts in mammalian cells.
Authors: Fornace A J , Alamo I , Hollander M C
Published: May 2006

Hybridization subtraction at low ratios of RNA to cDNA was used to enrich for the cDNA of transcripts increased in Chinese hamster cells after UV irradiation. Forty-nine different cDNA clones were isolated. Most coded for nonabundant transcripts rapidly induced 2- to 10-fold after UV irradiation. Only 2 of the 20 cDNA clones sequenced matched known sequences (metallothionein I and II). The predicted amino acid sequence of one cDNA had two localized areas of homology with the rat helix-destabilizing protein. These areas of homology were at the two DNA-binding sites of this nucleic acid single-strand-binding protein. The induced transcripts were separated into two general classes. Class I transcripts were induced by UV radiation and not by the alkylating agent methyl methanesulfonate. Class II transcripts were induced by UV radiation and by methyl methanesulfonate. Many class II transcripts were induced also by H2O2 and various alkylating agents but not by heat shock, phorbol 12-tetradecanoate 13-acetate, or DNA-damaging agents which do not produce high levels of base damage. Since many of the cDNA clones coded for transcripts which were induced rapidly and only by certain types of DNA-damaging agents, their induction is likely a specific response to such damage rather than a general response to cell injury.
Human papillomavirus type 16 cooperates with activated ras and fos oncogenes in the hormone-dependent transformation of primary mouse cells.
Authors: Crook T , Storey A , Almond N , Osborn K , Crawford L
Published: May 2006

The effect of human papillomavirus (HPV) and activated oncogenes on the growth and morphology of primary baby mouse kidney (BMK) cells has been studied. Early region DNA from HPV types 16, 18, 31, and 33, but not type 6, under the transcriptional control of a heterologous, retroviral promoter cooperated with EJ-ras to produce cell lines that gave rise to carcinomas in syngeneic immunocompetent animals. The same HPV constructs, when cotransfected with a plasmid containing sequences from the Finkel-Biskis-Jinkins murine sarcoma virus provirus (v-fos), produced cell lines that were tumorigenic in nude mice. None of the other activated oncogenes tested, including activated c-myc, displayed any activity with HPV in this cotransfection assay. When the heterologous promoter was replaced by the homologous HPV16 promoter, the transforming effect of HPV16 with either EJ-ras or v-fos required the presence of either glucocorticoid or progestogen. Cell lines derived from transfection of HPV16 with either EJ-ras or v-fos required the continued presence of hormones for proliferation.
Additional low-abundance human small nuclear ribonucleoproteins: U11, U12, etc.
Authors: Montzka K A , Steitz J A
Published: May 2006

Two-dimensional gel fractionation has revealed the existence of a number (greater than or equal to 8) of additional species of HeLa cell small RNAs that have 5' trimethylguanosine cap structures and are bound by proteins containing Sm epitopes. Therefore, these low-abundance (10(3)-10(4) per cell) RNAs belong to the Sm class of small nuclear ribonucleoproteins (snRNPs), whose best-known members are the four highly abundant (approximately 10(6) per cell) particles required for pre-mRNA splicing. The complexity of Sm snRNPs in mammalian cells is thus not greatly different from that previously established for lower eukaryotes. Two of the new RNAs, designated U11 (131 nucleotides) and U12 (150 nucleotides), have been sequenced. The U11 and U12 snRNPs have been characterized further by examining their nuclease sensitivity and their possible interactions with other snRNPs. Potential roles for the low-abundance snRNPs in aspects of pre-mRNA processing are discussed.
Down-regulation of c-myb gene expression is a prerequisite for erythropoietin-induced erythroid differentiation.
Authors: Todokoro K , Watson R J , Higo H , Amanuma H , Kuramochi S , Yanagisawa H , Ikawa Y
Published: May 2006

The role of nuclear protooncogenes during erythroid cell differentiation was examined by transfecting exogenous c-fos and c-myb genes into mouse erythroleukemia cells, which can be induced to differentiate either with erythropoietin (Epo) or dimethyl sulfoxide. Expression of exogenous c-myb or c-fos oncogene completely inhibited Epo-induced erythroid differentiation but only partially inhibited dimethyl sulfoxide-induced differentiation. Normally Epo-induced differentiation leads to a drastic decline of c-myb mRNA levels and an increase of c-myc transcripts in the early stage of differentiation. Cells expressing exogenous c-fos gene, however, maintained high levels of c-myb mRNA after Epo treatment. This high level of c-myb transcripts was found to be due to block of transcription shutoff (or transcriptional activation) rather than to mRNA stabilization. It is concluded that the down-regulation of endogenous c-myb gene expression is a prerequisite for commitment of Epo-induced erythroid differentiation and that expression of c-myb gene may be indirectly regulated by c-fos gene product. We also concluded that early down-regulation of c-myc gene expression is not essential for erythroid differentiation and that gene regulation of chemically induced erythroid differentiation may differ from that of Epo-induced differentiation.
Human DNA topoisomerase I is encoded by a single-copy gene that maps to chromosome region 20q12-13.2.
Authors: Juan C C , Hwang J L , Liu A A , Whang-Peng J , Knutsen T , Huebner K , Croce C M , Zhang H , Wang J C , Liu L F
Published: May 2006

cDNA clones of the human TOP1 gene encoding DNA topoisomerase I (EC 5.99.1.2) have been obtained by immunochemical screening of phage lambda libraries expressing human cDNA segments, using rabbit antibodies raised against purified HeLa DNA topoisomerase I. Hybridization patterns between the cloned cDNA sequences and human cellular DNA and cytoplasmic mRNAs indicate that human TOP1 is a single-copy gene. The chromosomal location of the gene has been mapped to the long arm of chromosome 20, in the region q12-13.2, by hybridization of a radioactively labeled TOP1 cDNA probe to human metaphase chromosomes and to a panel of rodent-human somatic hybrids retaining overlapping subsets of human chromosomes.
Overproduction of the epsilon subunit of DNA polymerase III counteracts the SOS mutagenic response of Escherichia coli.
Authors: Jonczyk P , Fijalkowska I , Ciesla Z
Published: May 2006

It has been found that the mutator phenotype of the recA441 and recA730 strains that express the SOS response constitutively is suppressed by pIP1, a high-copy plasmid carrying the dnaQ gene encoding the 3'----5' exonuclease subunit (epsilon) of DNA polymerase III. We have constructed plasmid pIP11, in which the dnaQ gene is fused to the strong tac (trp-lac) promoter. Enhanced synthesis of the epsilon subunit stimulated by isopropyl beta-D-thiogalactopyranoside, the inducer of tac, prevents expression of the mutator phenotype of recA441 and markedly decreases the frequency of UV-induced mutations. These results strongly suggest that a loss of editing capacity by the epsilon subunit of DNA polymerase III holoenzyme plays a crucial role in generation of mutations during the SOS response.
Detection of benzo[a]pyrene diol epoxide-DNA adducts in human placenta.
Authors: Manchester D K , Weston A , Choi J S , Trivers G E , Fennessey P V , Quintana E , Farmer P B , Mann D L , Harris C C
Published: May 2006

Human placenta is a readily available organ that responds to maternal environmental insult and has been previously used to investigate metabolism and bioactivation of procarcinogens, for example, benzo[a]pyrene. HPLC in combination with synchronous fluorescence spectroscopy was used to examine 28 placentas for the presence of benzo[a]pyrene diol epoxide-DNA adducts, and 10 of these were found to be positive. DNA samples from these placentas were subsequently pooled and subjected to partial enzymatic digestion to oligonucleotide fragments. Concentration of those DNA fragments containing benzo[a]pyrene diol epoxide-DNA adducts was achieved by immunoaffinity chromatography with polyclonal antibodies raised against these adducts. Column eluates were hydrolyzed under mild acid conditions and extracted with an organic solvent. The presence of benzo[a]pyrene-7,10/8,9-tetrahydrotetrol residues in the extracts was determined by HPLC and synchronous fluorescence spectroscopy and was confirmed by GC/MS. The results unequivocally confirm bioactivation and formation of DNA adducts from benzo[a]pyrene in human placenta in vivo and establish a methodological approach to direct measurement of carcinogen-DNA adducts that are formed as a result of human environmental exposure.
Reversion toward an earlier stage of differentiation and loss of polarity during progression of N-methyl-N-nitrosourea-induced rat mammary tumours.
Authors: Dulbecco R , Armstrong B , Allen R
Published: May 2006

To work toward an understanding of the mechanisms of cancer progression, we have followed the changes in expression of several genes in rat mammary carcinomas during the development of invasion. Tumors were induced by N-methyl-N-nitrosourea and serially transplanted to isogeneic rats. Gene expression in individual cells was followed by immunofluorescence microscopy. We show that invading cells do not express the same genes as the primary tumor or normal adult mammary gland but are similar to the primitive mammary duct cells present in late embryos. In the most advanced tumors, the most fundamental epithelial characteristic, polarity, is lost. Relative to the mechanism of invasion, we observe that the basal lamina, often thinner than normal, always coats the strands of invading cells and may be entirely absent from single invasive cells only. These observations suggest that the basal lamina ultimately disappears because of reduced expression of the genes or the failure to assemble the components. In these tumors it is the progressive alteration in gene expression rather than the enzymatic breakdown of the basal lamina that leads to invasion. These results show that the most important aspect of cancer progression is the progressive alteration of gene expression. They also raise questions about the mechanism that causes tumor cells to retrace the steps in their differentiation.
Aberrant expression of receptors for platelet-derived growth factor in an anaplastic thyroid carcinoma cell line.
Authors: Heldin N E , Gustavsson B , Claesson-Welsh L , Hammacher A , Mark J , Heldin C H , Westermark B
Published: May 2006

Receptors for platelet-derived growth factor (PDGF) have previously only been found on cells of mesenchymal and glial origin. This study shows PDGF receptors on an anaplastic thyroid carcinoma cell line, C 643, that was found to express thyroglobulin mRNA, confirming its origin from thyroid epithelium. Northern blot analysis of poly(A)+ RNA hybridized with a human PDGF B-type receptor cDNA probe revealed a 5.4-kilobase transcript in the C 643 cells. The existence of receptor protein on the cell surface was shown by immunofluorescence microscopy with a PDGF receptor monoclonal antibody. Binding experiments with 125I-labeled dimeric forms of PDGF indicated that the cells contain B-, but not A-, type PDGF receptors. The addition of PDGF to C 643 membranes in the presence of [32P]ATP induced phosphorylation of the receptor. A polyclonal PDGF B-type receptor peptide antiserum was used to immunoprecipitate a receptor protein from metabolically labeled C 643 cells; the receptor was found to be 5-10 kDa larger than that in normal human fibroblasts. Removal of N-linked carbohydrates using endoglycosidase H resulted in deglycosylated receptor proteins of similar size in C 643 cells and fibroblasts, indicating differences in glycosylation patterns of the two receptor proteins. The aberrant expression of receptors might be crucial in tumor development by conferring a selective growth advantage to the cancer cells.
Mammalian cerebral cortical tissue responds to low-intensity visible light.
Authors: Wade P D , Taylor J , Siekevitz P
Published: May 2006

Low levels of visible light directed onto slices of rat cerebral cortical tissue enhanced net potassium-induced release of the neurotransmitter gamma-aminobutyric acid (GABA) from these brain slices. At higher light intensity, net potassium-induced release was suppressed. These effects were apparently not from increased temperature. The amount of light enhancing this neurotransmitter release is approximately equal to the amount of light that can penetrate the head and reach the brain at the intensities of sunlight; this was determined by measuring the light entering the rat head through fur, scalp, skull, and dura mater and considering several natural lighting conditions. These results suggest that ambient light may be sufficient to alter the release of transmitters from mammalian cerebral cortex in vivo.
Patch-clamp study of gamma-aminobutyric acid receptor Cl- channels in cultured astrocytes.
Authors: Bormann J , Kettenmann H
Published: May 2006

The membrane channels operated by gamma-aminobutyric acid (GABA) were studied in cultured astrocytes from rat cerebral hemispheres by using patch-clamp techniques. The channel properties appeared to be very similar, in many respects, to those present in neuronal cell membranes. The Cl- -selective channels were activated after the sequential binding of two GABA molecules to the receptor, as deduced from the slope of the dose-response curve. Single-channel currents displayed multiple conductance states of 12 pS, 21 pS, 29 pS, and 43 pS, with the main-state conductance being 29 pS. The gating properties could be described by a sequential reaction scheme for agonist-activated channels. GABA-induced whole-cell currents were potentiated by the benzodiazepine receptor agonist diazepam and also, to a lesser extent, by methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate an inverse agonist. In neurons and chromaffin cells, methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate reduces the sensitivity of the GABA receptor, indicating that neuronal and glial GABA/benzodiazepine receptor--Cl- channel complexes are different. Glial GABA receptor channels could be of functional importance in buffering extracellular Cl- in the cleft of the GABAergic synapse.
Measurement of local cerebral protein synthesis in vivo: influence of recycling of amino acids derived from protein degradation.
Authors: Smith C B , Deibler G E , Eng N , Schmidt K , Sokoloff L
Published: May 2006

A quantitative autoradiographic method for the determination of local rates of protein synthesis in brain in vivo is being developed. The method employs L-[1-14C]leucine as the radiolabeled tracer. A comprehensive model has been designed that takes into account intracellular and extracellular spaces, intracellular compartmentation of leucine, and the possibility of recycling of unlabeled leucine derived from steady-state degradation of protein into the precursor pool for protein synthesis. We have evaluated the degree of recycling by measuring the ratio of the steady-state precursor pool distribution space for labeled leucine to that of unlabeled leucine. The values obtained were 0.58 in whole brain and 0.47 in liver. These results indicate that there is significant recycling of unlabeled amino acids derived from steady-state protein degradation in both tissues. Any method for the determination of rates of cerebral protein synthesis in vivo with labeled tracers that depends on estimation of precursor pool specific activity in tissue from measurements in plasma must take this recycling into account.
Two putative active centers in human angiotensin I-converting enzyme revealed by molecular cloning.
Authors: Soubrier F , Alhenc-Gelas F , Hubert C , Allegrini J , John M , Tregear G , Corvol P
Published: May 2006

The amino-terminal amino acid sequence and several internal peptide sequences of angiotensin I-converting enzyme (ACE; peptidyl-dipeptidase A, kininase II; EC 3.4.15.1) purified from human kidney were used to design oligonucleotide probes. The nucleotide sequence of ACE mRNA was determined by molecular cloning of the DNA complementary to the human vascular endothelial cell ACE mRNA. The complete amino acid sequence deduced from the cDNA contains 1306 residues, beginning with a signal peptide of 29 amino acids. A highly hydrophobic sequence located near the carboxyl-terminal extremity of the molecule most likely constitutes the anchor to the plasma membrane. The sequence of ACE reveals a high degree of internal homology between two large domains, suggesting that the molecule resulted from a gene duplication. Each of these two domains contains short amino acid sequences identical to those located around critical residues of the active site of other metallopeptidases (thermolysin, neutral endopeptidase, and collagenase) and therefore bears a putative active site. Since earlier experiments suggested that a single Zn atom was bound per molecule of ACE, only one of the two domains should be catalytically active. The results of genomic DNA analysis with the cDNA probe are consistent with the presence of a single gene for ACE in the haploid human genome. Whereas the ACE gene is transcribed as a 4.3-kilobase mRNA in vascular endothelial cells, a 3.0-kilobase transcript was detected in the testis, where a shorter form of ACE is synthesized.
Transcription induces gyration of the DNA template in Escherichia coli.
Authors: Figueroa N , Bossi L
Published: May 2006

We show that transcription modulation of a plasmid sequence in exponentially growing Escherichia coli cells leads to a rapid change in the linking number of plasmid DNA. Activation of transcription is accompanied by an increase in the plasmid's level of negative supercoiling. The added superhelical turns, whose number is proportional to the strength of the promoter and to the length of the transcript, are promptly removed when transcription is turned off. The transcription-induced increase of template supercoiling can still be detected in the presence of an inhibitor of ATP-dependent DNA gyrase [DNA topoisomerase (ATP-hydrolyzing), EC 5.99.1.3]. Altogether, our results indicate that, in addition to being under a general control, DNA superhelicity can be modulated locally in response to the topological perturbations associated with DNA tracking processes. We discuss a model in which supercoiling changes are produced by differential swiveling activities on the opposite sides of a transcriptional flow during transcriptional modulation.
DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA.
Authors: Innis M A , Myambo K B , Gelfand D H , Brow M A
Published: May 2006

The highly thermostable DNA polymerase from Thermus aquaticus (Taq) is ideal for both manual and automated DNA sequencing because it is fast, highly processive, has little or no 3'-exonuclease activity, and is active over a broad range of temperatures. Sequencing protocols are presented that produce readable extension products greater than 1000 bases having uniform band intensities. A combination of high reaction temperatures and the base analog 7-deaza-2'-deoxyguanosine was used to sequence through G + C-rich DNA and to resolve gel compressions. We modified the polymerase chain reaction (PCR) conditions for direct DNA sequencing of asymmetric PCR products without intermediate purification by using Taq DNA polymerase. The coupling of template preparation by asymmetric PCR and direct sequencing should facilitate automation for large-scale sequencing projects.
LR1: a candidate RNA virus of Leishmania.
Authors: Tarr P I , Aline R F , Smiley B L , Scholler J , Keithly J , Stuart K
Published: May 2006

Although viruses are important biological agents and useful molecular tools, little is known about the viruses of parasites. We report here the discovery of a candidate for an RNA virus in a kinetoplastid parasite. This potential virus, which we term LR1, is present in the promastigote form of the human pathogen Leishmania braziliensis guyanensis CUMC1-1A but not in 11 other stocks of Leishmania that were examined nor in Trypanosoma brucei. The candidate viral RNA has a size of approximately 6000 nucleotides, is single-stranded, and is largely, if not exclusively, located in the cytoplasm. No homologous LR1 sequences are detected in genomic DNA. The candidate viral RNA is associated with a spherical particle 32 nm in diameter that has a sedimentation coefficient of approximately 130 S. There is as yet no evident effect of this potential virus on parasite physiology or the disease caused by the parasite.
Replicating myoblasts express a muscle-specific phenotype.
Authors: Kaufman S J , Foster R F
Published: May 2006

During the terminal stage of skeletal myogenesis, myoblasts stop replicating, fuse to form multinucleate fibers, and express the genes that encode the proteins that convey contractile capacity. Because of this dramatic shift in proliferative state, morphology, and gene expression, it has been possible to readily identify and quantitate terminally differentiating myoblasts. In contrast, it is not clear whether the proliferating cells that give rise to postmitotic myoblasts are equally distinct in their phenotype and in fact whether distinct stages in skeletal myogenesis precede the onset of terminal differentiation. To address these questions, monoclonal antibodies and immunofluorescence microscopy were used to determine that replicating myoblasts from newborn rats do express a muscle-specific phenotype. To identify replicating cells, incorporation of 5-bromo-2'-deoxyuridine (BrdUrd) into DNA was assayed by using anti-BrdUrd antibody. The developmentally regulated, muscle-specific, integral membrane protein H36 and the intermediate-filament protein desmin were scored as markers of the myogenic phenotype. The percentage of BrdUrd+ (i.e., proliferative) cells among H36+ and desmin+ myoblasts was equal to the percentage of BrdUrd+ cells in the entire population, indicating that the expression of H36 and desmin is uniformly characteristic of replicating myoblasts. Inhibition of protein synthesis before and during growth in BrdUrd did not alter the frequency of desmin and H36 immunofluorescence in BrdUrd+ cells. Thus, desmin and H36 were present in the replicating myoblasts prior to the onset of growth in BrdUrd. These results were confirmed using H36+ cells selected by flow cytometry: these purified H36+ myoblasts replicate, express desmin, and differentiate. Similar results were obtained with mouse myoblasts. Desmin expression in these mammalian cells differs from that in chicken embryo myoblasts: only a small proportion of replicating chicken embryo myoblasts express desmin. That replicating mammalian myoblasts have a muscle-specific phenotype serves to define a distinct stage in myogenic development and a specific cell in the myogenic lineage. Further, it implies that there is a regulatory event activated during myogenesis that precedes terminal differentiation and that is required for expression of those genes whose products distinguish the replicating myoblast.
Antioxidant defenses and lipid peroxidation in human blood plasma.
Authors: Frei B , Stocker R , Ames B N
Published: May 2006

The temporal disappearance in human blood plasma of endogenous antioxidants in relation to the appearance of various classes of lipid hydroperoxides measured by HPLC postcolumn chemiluminescence detection has been investigated under two types of oxidizing conditions. Exposure of plasma to aqueous peroxyl radicals generated at a constant rate leads immediately to oxidation of endogenous ascorbate and sulfhydryl groups, followed by sequential depletion of bilirubin, urate, and alpha-tocopherol. Stimulating polymorphonuclear leukocytes in plasma initiates very rapid oxidation of ascorbate, followed by partial depletion of urate. Once ascorbate is consumed completely, micromolar concentrations of hydroperoxides of plasma phospholipids, triglycerides, and cholesterol esters appear simultaneously, even though sulfhydryl groups, bilirubin, urate, and alpha-tocopherol are still present at high concentrations. Nonesterified fatty acids, the only lipid class in plasma not transported in lipoproteins but bound to albumin, are preserved from peroxidative damage even after complete oxidation of ascorbate, most likely due to site-specific antioxidant protection by albumin-bound bilirubin and possibly by albumin itself. Thus, in plasma ascorbate and, in a site-specific manner, bilirubin appear to be much more effective in protecting lipids from peroxidative damage by aqueous oxidants than all the other endogenous antioxidants. Hydroperoxides of linoleic acid, phosphatidylcholine, and cholesterol added to plasma in the absence of added reducing substrates are degraded, in contrast to hydroperoxides of trilinolein and cholesterol linoleate. These findings indicate the presence of a selective peroxidase activity operative under physiological conditions. Our data suggest that in states of leukocyte activation and other types of acute or chronic oxidative stress such a simple regimen as controlled ascorbate supplementation could prove helpful in preventing formation of lipid hydroperoxides, some of which cannot be detoxified by endogenous plasma activities and thus might cause damage to critical targets.
Hemoglobin adducts of aromatic amines: associations with smoking status and type of tobacco.
Authors: Bryant M S , Vineis P , Skipper P L , Tannenbaum S R
Published: May 2006

Hemoglobin adducts of 15 aromatic amines were determined in nonsmokers and smokers of blond- or black-tobacco cigarettes living in Turin, Italy. The subjects were all males age 55 or less and were representative of the population previously examined in a case/control study of bladder cancer. 4-Aminobiphenyl adduct levels were found to be significantly different in the three groups, and the differences were approximately proportional to the relative risk of each group. Adjustment for age and cigarette consumption did not materially influence the differences. A significant correlation of adduct levels with cigarette consumption was also observed for all smokers as well as for smokers of blond tobacco. Other amines for which significant differences between smokers and nonsmokers were observed were 3-aminobiphenyl, 2-naphthylamine, o- and p-toluidine, 2,4-dimethylaniline, and 2-ethylaniline. Some of these amines are human bladder carcinogens, and their occurrence in blood as hemoglobin adducts is evidence for their metabolic activation. Thus, by a combination of traditional epidemiological methods and modern chemical analyses, we have provided evidence for a biochemical basis for the often observed association between cigarette smoking and bladder cancer.
Two classes of N-methyl-D-aspartate recognition sites: differential distribution and differential regulation by glycine.
Authors: Monaghan D T , Olverman H J , Nguyen L , Watkins J C , Cotman C W
Published: May 2006

The N-methyl-D-aspartate (NMDA) receptor, a subtype of excitatory amino acid receptor, mediates synaptic responses in many regions of the central nervous system. This receptor plays a critical role in the mechanisms of both synaptic plasticity and excitotoxicity. Although these receptors were generally thought to be a single homogeneous receptor population, we report observations indicating that two anatomically distinct forms of the NMDA-receptor complex exist. (i) The distribution of NMDA receptors, as labeled by the NMDA agonist L-[3H]glutamate, differs from that obtained with the radiolabeled antagonist 3H-labeled 3-[(+/-)2-carboxypiperazine-4-yl]propyl-1-phosphonic acid [( 3H]CPP). Relative to L-[3H]glutamate, [3H]CPP binding is low in the striatum and septum and high in the thalamus and inner cerebral cortex. (ii) NMDA antagonists are relatively more potent than agonists at displacing L-[3H]glutamate binding in the thalamus and cerebral cortex; agonists are relatively more potent in the striatum and cerebellum. (iii) Glycine, which potentiates NMDA-receptor responses to glutamate, causes a greater percentage increase in L-[3H]glutamate binding to NMDA receptors in the thalamus and cerebral cortex than in the striatum, septum, and cerebellum. Radiolabeled NMDA-antagonist binding, in contrast, is inhibited by glycine. Thus, as observed for gamma-aminobutyric acid type A receptors, NMDA receptors have an agonist-preferring binding-site population and an antagonist-preferring binding site population. These may represent two distinct receptors and/or two interconverting forms. It could be of significant clinical importance if these two sites differ in their response to NMDA.
Selective visualization of gene structure with ultraviolet light.
Authors: Wang Z , Becker M M
Published: May 2006

The ability of the ultraviolet (UV) "footprinting" technique to detect chromatin has been investigated in vitro. Two basic types of chromatin, a phased nucleosome and a phased nucleosome containing a phased H1 protein, have been reconstituted onto a cloned 5S ribosomal RNA gene from sea urchin. The histone-DNA interactions in each complex have been probed with exonuclease III, DNase I, dimethyl sulfate, and UV light. Whereas DNase I and exonuclease III readily detect interactions between histones and DNA, UV light and dimethyl sulfate do not. In contrast to histone-DNA interactions, we demonstrate that intimate sequence-specific contacts between the same sea urchin 5S DNA and the Xenopus laevis transcription factor IIIA (TFIIIA) are readily detected with UV light. Since the sensitivity of UV light for TFIIIA contacts is similar to its sensitivity for other regulatory protein-DNA contacts, these studies demonstrate the feasibility of using UV light to selectively visualize regulatory protein-DNA interactions in vivo with little or no interference from histone-DNA interactions.
Extracellular creatine regulates creatine transport in rat and human muscle cells.
Authors: Loike J D , Zalutsky D L , Kaback E , Miranda A F , Silverstein S C
Published: May 2006

Muscle cells do not synthesize creatine; they take up exogenous creatine by specific Na+-dependent plasma membrane transporters. We found that extracellular creatine regulates the level of expression of these creatine transporters in L6 rat muscle cells. L6 myoblasts maintained for 24 hr in medium containing 1 mM creatine exhibited 1/3rd of the creatine transport activity of cells maintained for 24 hr in medium without creatine. Down-regulation of creatine transport was partially reversed when creatine-fed L6 cells were incubated for 24 hr in medium lacking creatine. Down-regulation of creatine transport occurred independently of amino acid and glucose transport. Furthermore, the down-regulation of creatine transporters by extracellular creatine was slowed by inhibitors of protein synthesis. These results suggest that creatine induces the expression of a protein that functionally inactivates the creatine transporters. Regulation of creatine transport by extracellular creatine also was observed in L6 myotubes and in cultures of human myoblasts and myotubes. Hence, the activity of creatine transport represents another site for the regulation of creatine homeostasis.