bluuebunny/crossref_metadata_2025 huggingface dataset:
Antibodies to synthetic peptides as probes for the binding site on the alpha subunit of the acetylcholine receptor.
Authors: Neumann D , Gershoni J M , Fridkin M , Fuchs S
Published: May 2006

Synthetic peptides and their respective antibodies were used in an attempt to localize and identify the ligand-binding site of the nicotinic acetylcholine receptor. Two peptides of the receptor alpha subunit were synthesized, the first corresponding to the NH2-terminal domain (positions 1-20) and the other, to a segment (residues 126-143) that contains the first two cysteine residues. Specific antipeptide antibodies were elicited in rabbits after immunization with the peptides conjugated to bovine serum albumin. The antipeptide antibodies thus obtained cross-reacted with the receptor and bound specifically to its alpha subunit. The antipeptide antibodies were used to test whether the peptide sequences corresponded to the alpha-bungarotoxin (alpha-BTX)-binding site. Staphylococcus aureus V8-protease digestion of the isolated receptor alpha subunit generated several fragments. Antipeptide (1-20) and antipeptide (126-143) both bound a 26-kDa fragment, whereas only antipeptide (126-143) bound a 17-kDa fragment. None of these fragments were found to bind alpha-BTX. On the other hand, alpha-BTX bound to an 18-kDa fragment that did not react with either of the antipeptide antibodies. Moreover, the 26-kDa and 17-kDa fragments were also found to contain the endoglycosidase H-susceptible oligosaccharide chain. Our results indicate that the toxin-binding site lies beyond the first possible V8 protease cleavage site after residues 126-143: i.e., Asp-152. This location is in agreement with the possibility that cysteine residues 192 and/or 193 are in close proximity to or contiguous with the ligand-binding site.
Characterization of cultured rat oligodendrocytes proliferating in a serum-free, chemically defined medium.
Authors: Saneto R P , de Vellis J
Published: May 2006

A serumless, chemically defined medium has been developed for the culture of oligodendrocytes isolated from primary neonatal rat cerebral cultures. Combined together, insulin, transferrin, and fibroblast growth factor synergistically induced an essentially homogenous population (95-98%) of cells expressing glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) activity to undergo cell division. Proliferating cels were characterized by several criteria: (i) ultrastructural analysis by transmission electron microscopy identified the cell type as an oligodendrocyte; (ii) biochemical assays showed expression of three oligodendrocyte biochemical markers, induction of both glycerol phosphate dehydrogenase and lactate dehydrogenase (EC 1.1.1.27), and presence of 2',3'-cyclic nucleotide 3'-phosphodiesterase (EC 3.1.4.37); and (iii) immunocytochemical staining showed cultures to be 95-98% positive for glycerol phosphate dehydrogenase, 90% for myelin basic protein, 60-70% for galactocerebroside, and 70% for A2B5. Few cells (less than 5%) stained positive for glial fibrillary acidic protein, and none were detected positive for fibronectin.
Nucleotide sequence of bean golden mosaic virus and a model for gene regulation in geminiviruses
Authors: Howarth Alan J. , Caton John , Bossert Monet , Goodman Robert M.
Published: May 2006

We have sequenced the genome of bean golden mosaic virus, which comprises two circular single-stranded DNA molecules (2646 and 2587 nucleotides long) of mostly unique sequence. Comparison of the sequences of bean golden mosaic virus and of cassava latent virus, which share serological relationship but are very different in host range and geographical origin, shows that each virus has a unique 200-nucleotide sequence (common region) on each 2.6-kilobase molecule of its genome. The common regions of the two viruses have no sequence homology except for a short inverted repeat near the 3′ end. Six open reading frames were identified that possess considerable sequence homology between the two viruses and, in bean golden mosaic virus, may encode proteins of 15.6, 19.6, 27.7, 29.7, 33.1, and 40.2 kDa. Conserved open reading frames are found in both the viral strand and the complementary strand, are approximately the same size, and are in the same orientation with respect to the common region in both viruses. We propose that temporal regulation in geminiviruses depends on the polarity of transcription and that the common region represents a replication origin and contains elements that serve to modulate gene expression.
Characterization of an almost full-length cDNA coding for human blood coagulation factor X.
Authors: Fung M R , Hay C W , MacGillivray R T
Published: May 2006

A human liver cDNA library was screened by colony hybridization with a bovine factor X cDNA probe. Three of the positive plasmids contained overlapping DNA that coded for most of human factor X mRNA. DNA sequence analysis of these three clones allowed the prediction of the complete amino acid sequence of plasma factor X. From these studies, we predict that human factor X is synthesized as a single polypeptide chain precursor in which the light and heavy chains of plasma factor X are linked by the tripeptide Arg-Lys-Arg. The cDNA sequence also predicts that human factor X is synthesized as a preproprotein having an amino-terminal leader peptide of at least 28 amino acid residues. A comparison of the amino acid sequences of human and bovine factor X shows high sequence identity around the calcium-binding regions and catalytic regions but low sequence identity around the nonfunctional regions.
Anomalous properties of a simple small-volume membrane transport system at steady state
Authors: Hill Terrell L. , Chen Yi-der
Published: May 2006

We investigate the transport of a single ligand L, between two macroscopic baths with different concentrations of L, by a simple membrane system. The center of this system is a very small volume
V
that might contain
N
= 0, 1, 2,... molecules of L. Access to
V
from either bath is by means of a single binding site for L. The steady-state properties of this simple system are studied exactly by a discrete-
N
master-equation approach. It is found that the mean concentration of L in
V
, ¯c = ¯N/
V
, is not a significant quantity either kinetically or thermodynamically. Further, the chemical potential of L in
V
is not defined and hence the overall thermodynamic force on L between the two baths cannot be subdivided at
V
. In fact, because
V
is small, the transport system must be treated as an indivisible system with interdependent parts. In the limit when
V
and ¯N are very large,
V
becomes in effect a third macroscopic bath and two well-defined transport subsystems emerge (between
V
and either bath).
Intrinsic curvature hypothesis for biomembrane lipid composition: a role for nonbilayer lipids.
Authors: Gruner S M
Published: May 2006

A rationale is presented for the mix of "bilayer" and "nonbilayer" lipids, which occurs in biomembranes. A theory for the L alpha-HII phase transition and experimental tests of the theory are reviewed. It is suggested that the phase behavior is largely the result of a competition between the tendency for certain lipid monolayers to curl and the hydrocarbon packing strains that result. The tendency to curl is quantitatively given by the intrinsic radius of curvature, Ro, which minimizes the bending energy of a lipid monolayer. When bilayer (large Ro) and nonbilayer (small Ro) lipids are properly mixed, the resulting layer has a value of Ro that is at the critical edge of bilayer stability. In this case, bilayers may be destabilized by the protein-mediated introduction of hydrophobic molecules, such as dolichol. An x-ray diffraction investigation of the effect of dolichol on such a lipid mixture is described. This leads to the hypothesis that biomembranes homeostatically adjust their intrinsic curvatures to fall into an optimum range. Experimental strategies for testing the hypothesis are outlined.
Processing of donor DNA during Haemophilus influenzae transformation: analysis using a model plasmid system.
Authors: Pifer M L , Smith H O
Published: May 2006

A plasmid system was used to investigate the processing of donor DNA during transformation of competent Haemophilus influenzae. Using biochemical and genetic methods, we have determined that portion of a donor plasmid molecule that, on average, can become integrated into a homologous recipient plasmid during transformation. Our results show that (i) transformation efficiency decreases linearly with donor DNA length over the range of 11 to 3.5 kilobase pairs, (ii) transformation efficiency decreases exponentially with size for donor molecules less than 3.5 kilobase pairs in length, and (iii) 5'-end label, but not 3'-end label, can be specifically incorporated into the resident homologous region. We present a model for donor processing during entry that encompasses and explains these observations.
Isolation and characterization of mouse Thy-1 genomic clones.
Authors: Chang H C , Seki T , Moriuchi T , Silver J
Published: May 2006

The mouse Thy-1.2 gene was isolated from a C57Bl/6 cosmid library and its nucleotide sequence was determined from an 8-kilobase-long EcoRI fragment. The predicted amino acid sequence indicates that the mouse Thy-1 molecule contains a 19 amino acid leader peptide and the 112 amino acids reported previously from protein sequence analysis, plus 31 extra amino acids at the carboxyl terminus. These 31 amino acids contain a stretch of 20 amino acids, at positions 124-143, which is highly hydrophobic. RNA transfer blot analysis of RNA from mouse tissues indicates that the sequence coding for these 31 amino acids is present on poly(A)-containing RNA of brain and thymus tissues. This hydrophobic segment very likely provides the basis for integration of Thy-1 within the plasma membrane. The entire coding sequence of Thy-1 is distributed among three exons, encoding amino acid residues -19 to 8, -7 to 106, and 107 to 143, respectively. Comparison of the mouse and rat Thy-1 genes shows that both have a similar gene organization and encode a highly conserved transmembrane segment.
Asbestos-associated chromosomal changes in human mesothelial cells.
Authors: Lechner J F , Tokiwa T , LaVeck M , Benedict W F , Banks-Schlegel S , Yeager H , Banerjee A , Harris C C
Published: May 2006

Replicative cultures of human pleural mesothelial cells were established from noncancerous adult donors. The cells exhibited normal mesothelial cell characteristics including keratin, hyaluronic acid mucin, and long branched microvilli, and they retained the normal human karyotype until senescence. The mesothelial cells were 10 and 100 times more sensitive to the cytotoxic effects of asbestos fibers than normal human bronchial epithelial or fibroblastic cells, respectively. In addition, cultures of mesothelial cells that survived two cytotoxic exposures of amosite fibers were aneuploid with consistent specific chromosomal losses indicative of clonal origin. These aneuploid cells exhibit both altered growth control properties and a population doubling potential of greater than 50 divisions beyond the culture life span (30 doublings) of the control cells.
Neuropeptide Y injected in the paraventricular hypothalamus: a powerful stimulant of feeding behavior.
Authors: Stanley B G , Leibowitz S F
Published: May 2006

Neuropeptide Y (NPY) was injected directly into the paraventricular nucleus of the hypothalamus (PVN) of satiated, brain-cannulated rats, and food and water intake were measured 0.5, 1, 2, 4, and 22 hr postinjection. NPY (24, 78, 235, 783, and 2351 pmol/0.3 mul) produced a large, dose-dependent increase in food intake as well as small increase in water intake. The latency to eat was about 10 min, with substantial feeding occurring in the first 30 min. At dose below 78 pmol, the eating generally occurred only within the first hour. At doses above 235 pmol, however, the subjects' food intake continued to increase such that by 4 hr postinjection they had consumed the equivalent of normal 22-hr intake, and 22 hr postinjection they had also eaten significantly more than control subjects. Previous studies have shown that norepinephrine injected into the PVN stimulates feeding through alpha-adrenergic receptors. To investigate a possible interaction, subjects were given PVN injections of phentolamine (60 nmol) prior to injections of either NPY (78 pmol) or norepinephrine (20 nmol). Phentolamine pretreatment significantly decreased feeding elicited by norepinephrine without affecting feeding elicited by NPY. This suggests that NPY does not stimulate feeding through the release of endogenous norepinephrine. The powerful stimulation of feeding elicited by this neuropeptide suggests an important role for hypothalamic NPY, or a structurally related peptide, in the regulation of feeding behavior.
Purification, induction, and distribution of placental glutathione transferase: a new marker enzyme for preneoplastic cells in the rat chemical hepatocarcinogenesis.
Authors: Satoh K , Kitahara A , Soma Y , Inaba Y , Hatayama I , Sato K
Published: May 2006

A polypeptide of Mr 26,000 and pI 6.7 that was markedly increased in rat livers bearing hyperplastic nodules (HNs) induced by chemical carcinogens was identified immunochemically as the subunit of neutral glutathione (GSH) transferase (GSHTase; RX:glutathione R-transferase, EC 2.5.1.18; also called GSH S-transferase) purified from placenta (GSHTase-P) and was demonstrated immunohistochemically to be localized in preneoplastic foci and HNs. In the present study, GSHTase-P has been purified from the HN-bearing liver, and the distribution and inducibility have been examined quantitatively using anti-GSHTase-P antibody. Elevation of GSHTase-P in the HN-bearing livers was also confirmed by in vitro translation of mRNAs isolated from the HN-bearing livers. The purified GSHTase-P was homogeneous in size but had two charge isomers on two-dimensional gel electrophoresis. In normal tissues, including liver, placenta, and fetal liver, the protein content of GSHTase-P was generally low but was significantly high in kidney and pancreas. In contrast, the amount of GSHTase-P in HN-bearing livers (primary hepatomas) and transplantable Morris hepatoma 5123D were several 10-fold higher than that in normal liver but were undetectably low in transplantable Yoshida ascites hepatoma AH 130. Different from ordinary drug-metabolizing enzymes, GSHTase-P was uninducible by administration of drugs and carcinogens prior to appearance of the preneoplastic foci and HNs. In addition, species specificity of GSHTase-P was low as it was crossreactive among rat, hamster, and human.
Cloning of cDNA coding for peroxisomal acyl-CoA oxidase from the yeast Candida tropicalis pK233.
Authors: Rachubinski R A , Fujiki Y , Lazarow P B
Published: May 2006

Candida tropicalis pK233 cells were grown with n-alkanes as carbon source to induce the synthesis of peroxisomal proteins and the proliferation of peroxisomes. Poly-(A)+ RNA was isolated and used to construct a cDNA library by insertion of double-stranded reverse transcripts into the Pst I site of pBR322 followed by cloning in Escherichia coli. Clones complementary to mRNAs induced by growth on alkanes were selected by differential DNA dot-blot analysis using [32P]cDNA reverse-transcribed from poly(A)+ RNA of glucose-grown cells (which contain few peroxisomes) or of alkane-grown cells. Among these clones, one containing a 1.7-kilobase insert coding for acyl-CoA oxidase (the first enzyme in the peroxisomal Beta-oxidation pathway) was identified by hybridization-selection translation and immunoprecipitation. By RNA blot analysis, the acyl-CoA oxidase mRNA was estimated to be approximately equal to 2.2 kilobases long, of which 2.1 kilobases are required to code for the approximately equal to 76-kDa protein. Since the mRNA is polyadenylylated, there appears to be little additional nontranslated region. Cell-free mRNA translation and RNA dot-blot hybridization analyses demonstrated that, whereas glucose-grown C. tropicalis contained little or no acyl-CoA oxidase mRNA, alkane-grown cells contained so much of this mRNA as to make acyl-CoA oxidase one of the major in vitro translation products.
Response of patients in classes III and IV of cardiomyopathy to therapy in a blind and crossover trial with coenzyme Q10.
Authors: Langsjoen P H , Vadhanavikit S , Folkers K
Published: May 2006

Coenzyme Q10 (CoQ10), a biochemically established redox component of respiration including the coupled mechanisms of electron transfer and oxidative phosphorylation, is naturally present in the human myocardium. A double-blind and double-crossover trial has been conducted by administering CoQ10 and a matching placebo orally to two groups of patients having class III or IV cardiomyopathy (classification according to criteria of the New York Heart Association). Group A received CoQ10 and then placebo; group B received placebo and then CoQ10. Blood levels of CoQ10 and cardiac function were determined at 0 and 4 weeks (control stabilization period) and at 16 and 28 weeks (after the 12-week CoQ/placebo-treatment periods). For group A, significant increases in CoQ10 blood levels and cardiac function occurred during CoQ10 treatment and then decreased during crossover to placebo. For group B, there was no change in CoQ10 blood levels and cardiac function during placebo treatment, but increases in both parameters occurred in crossover to CoQ10. These patients, steadily worsening and expected to die within 2 years under conventional therapy, generally showed an extraordinary clinical improvement, indicating that CoQ10 therapy might extend the lives of such patients. This improvement could be due to correction of a myocardial deficiency of CoQ10 and to enhanced synthesis of CoQ10-requiring enzymes.
Regulation by vitamin D metabolites of messenger ribonucleic acid for preproparathyroid hormone in isolated bovine parathyroid cells.
Authors: Silver J , Russell J , Sherwood L M
Published: May 2006

We have recently determined that high calcium concentrations, in parallel with their suppressive effects on parathyroid hormone (PTH) secretion, reversibly and specifically decrease preproPTH mRNA in cultured bovine parathyroid cells. In order to determine whether vitamin D metabolites also regulate the content of preproPTH mRNA, we tested their effects on bovine parathyroid cells in the same culture system. Levels of preproPTH mRNA were determined by dot-blot hybridization or blot hybridization with a labeled cloned cDNA probe. Incubation with 1,25-dihydroxycholecalciferol at doses varying from 10 pM to 0.1 microM caused a direct decrease in mRNA down to 50% of control values at 48 hr. There was no evidence that 1,25-dihydroxycholecalciferol, even at the highest concentrations, had any toxic effects on cell number or viability or on total RNA or RNA synthesis. Levels of alpha-actin mRNA did not change in the same experiments, and the suppression of preproPTH mRNA was reversible. When the relative potency of various vitamin D metabolites in suppressing preproPTH mRNA was evaluated, 1,25-dihydroxycholecalciferol greater than 24,25-dihydroxycholecalciferol greater than 25-hydroxycholecalciferol greater than vitamin D3 (cholecalciferol). These effects were highly specific and suggest that vitamin D metabolites play an important role in regulating the production of PTH.
Dual modulation of K channels by thyrotropin-releasing hormone in clonal pituitary cells.
Authors: Dubinsky J M , Oxford G S
Published: May 2006

Transmembrane electrical activity in pituitary tumor cells can be altered by substances that either stimulate or inhibit their secretory activity. Using patch recording techniques, we have measured the resting membrane potentials, action potentials, transmembrane macroscopic ionic currents, and single Ca2+-activated K channel currents of GH3 and GH4/C1 rat pituitary tumor cells in response to thyrotropin-releasing hormone (TRH). TRH, which stimulates prolactin secretion, causes a transient hyperpolarization of the membrane potential followed by a period of elevated action potential frequency. In single cells voltage clamped and internally dialyzed with solutions containing K+, TRH application results in a transient increase in Ca2+-activated K currents and a more protracted decrease in voltage-dependent K currents. However, in cells internally dialyzed with K+-free solutions, TRH produces no changes in inward Ca2+ or Ba2+ currents through voltage-dependent Ca channels. The time courses of the effects on Ca2+-activated and voltage-dependent K currents correlate with the phases of hyperpolarization and hyperexcitability, respectively. During application of TRH to whole cells, single Ca2+-activated K channel activity increases in cell-attached patches not directly exposed to TRH. In contrast, TRH applied directly to excised membrane patches produces no change in single Ca2+-activated K channel behavior. We conclude that TRH (i) triggers intracellular Ca2+ release, which opens Ca2+-activated K channels, (ii) depresses voltage-dependent K channels during the hyperexcitable phase, which further elevated intracellular Ca2+, and (iii) does not directly modulate Ca channel activity.
Isolation and characterization of two fractions from HeLa cells required for mRNA splicing in vitro.
Authors: Furneaux H M , Perkins K K , Freyer G A , Arenas J , Hurwitz J
Published: May 2006

A nuclear extract from HeLa cells has been separated by DEAE-cellulose chromatography into two fractions, both of which are required for mRNA splicing in vitro. Both fractions are heat labile and sensitive to N-ethylmaleimide. The activity of one of the fractions was abolished by preincubation with micrococcal nuclease, while the other fraction was unaffected by this treatment. This abolition indicates an essential nucleic acid component. Fractions I and II are required for the in vitro splicing of human beta-globin and adenovirus transcripts.
Inositol trisphosphate modification of ion transport in rough endoplasmic reticulum.
Authors: Muallem S , Schoeffield M , Pandol S , Sachs G
Published: May 2006

The ion transport properties of the rough endoplasmic reticulum (RER) from liver have been defined by using measurements of active and potential gradient-driven transport. The Ca2+ pump is shown to be electrogenic, and both ATP and potential difference is able to drive vanadate-inhibitable Ca2+ uptake into the RER. ATP-dependent Ca2+ transport into the RER depends on the presence of tetraethylammonium-sensitive cation conductance and a furosemide-inhibited cation/chloride cotransport pathway. Inositol trisphosphate does not affect either of the monovalent ion translocation systems but activates a Ca2+ conductance in the RER, allowing efflux of RER Ca2+ stores into the cytosol in exchange for K+ uptake.
Dietary saturated triacylglycerols suppress hepatic low density lipoprotein receptor activity in the hamster.
Authors: Spady D K , Dietschy J M
Published: May 2006

The liver plays a key role in the regulation of circulating levels of low density lipoproteins (LDL) because it is both the site for the production of and the major organ for the degradation of this class of lipoproteins. In this study, the effects of feeding polyunsaturated or saturated triacylglycerols on receptor-dependent and receptor-independent hepatic LDL uptake were measured in vivo in the hamster. In control animals, receptor-dependent LDL transport manifested an apparent Km value of 85 mg/dl (plasma LDL-cholesterol concentration) and reached a maximum transport velocity of 131 micrograms of LDL-cholesterol/hr per g, whereas receptor-independent uptake increased as a linear function of plasma LDL levels. Thus, at normal plasma LDL-cholesterol concentrations, the hepatic clearance rate of LDL equaled 120 and 9 microliter/hr per g by receptor-dependent and receptor-independent mechanisms, respectively. As the plasma LDL-cholesterol was increased, the receptor-dependent (but not the receptor-independent) component declined. When cholesterol (0.12%) alone or in combination with polyunsaturated triacylglycerols was fed for 30 days, receptor-dependent clearance was reduced to 36-42 microliter/hr per g, whereas feeding of cholesterol plus saturated triacylglycerols essentially abolished receptor-dependent LDL uptake (5 microliter/hr per g). When compared to the appropriate kinetic curves, these findings indicated that receptor-mediated LDL transport was suppressed approximately equal to 30% by cholesterol feeding alone and this was unaffected by the addition of polyunsaturated triacylglycerols to the diet. In contrast, receptor-dependent uptake was suppressed approximately equal to 90% by the intake of saturated triacylglycerols. As compared to polyunsaturated triacylglycerols, the intake of saturated lipids was also associated with significantly higher plasma LDL-cholesterol concentrations and lower levels of cholesteryl esters in the liver.
Sequence-dependent termination of in vitro DNA synthesis by cis- and trans-diamminedichloroplatinum (II).
Authors: Pinto A L , Lippard S J
Published: May 2006

Inhibition of DNA replication by the antitumor drug cis-diamminedichloroplatinum (II) (cis-DDP) has been proposed to be responsible for its cytotoxicity. Treatment of primed phage M13 mp8 viral DNA templates with the drug followed by second-strand synthesis using large fragment DNA polymerase I reveals that cis-DDP forms an adduct with DNA that inhibits DNA synthesis in vitro. This inhibition occurs at all (dG)n (n greater than or equal to 2) sequences in the template strand, confirming that these regions are the major cis-DDP binding sites on DNA. trans-Diamminedichloroplatinum (II), which is inactive as a drug, also forms adducts that inhibit DNA synthesis. Although considerably lower specificity is observed with the trans isomer, there appears to be a preference for d(GpNpG) sequences, where N is any intervening nucleotide. The monofunctional adduct formed between chlorodiethylenetriamineplatinum(II) chloride and DNA does not inhibit DNA synthesis in this system.
Electron microscopic visualization of trp operon expression in Salmonella typhimurium.
Authors: French S , Martin K , Patterson T , Bauerle R , Miller O L
Published: May 2006

Transcriptional activity of plasmids carrying wild-type and mutant trp operons was visualized in cell lysates of Salmonella typhimurium. Plasmid and transcription-unit sizes varied with the size of the cloned operon. Following 3-(3-indolyl)acrylic acid derepression, all operons of a particular type exhibited the same high level of transcriptional activity. An estimated 11-14 transcripts must be initiated each minute to maintain the 190-base-pair spacing of RNA polymerases observed on the promoter-proximal half of the wild-type trp operon. A decline in RNA polymerase density was observed on promoter-distal portions of cloned trp operons, which may be attributable to premature transcription termination accompanying translation inhibition due to indolylacrylic acid's interference with normal tryptophanyl-tRNA synthetase activity.
Translation activates the paused transcription complex and restores transcription of the trp operon leader region.
Authors: Landick R , Carey J , Yanofsky C
Published: May 2006

It has been proposed that RNA polymerase pausing in the leader region of the tryptophan (trp) operon of Escherichia coli is responsible for the synchronization of transcription and translation essential to attenuation control. In this report we use an in vitro coupled transcription/translation system to study the effect of trp leader peptide synthesis on RNA polymerase pausing in the trp leader region. Wild-type and translation-defective trp leader templates of E. coli and Serratia marcescens were employed, and pause RNA synthesis and paused complex release (activation) were quantified relative to synthesis of the terminated leader transcript. It was observed that pausing in the trp leader region was prolonged when translation of the leader transcript was reduced by mutations in the leader region or by addition of the translation inhibitor kasugamycin or chloramphenicol. Experiments with S-30 extracts from a mutant strain that is inefficient in translating the tryptophan codons in the leader transcript indicated that ribosome movement to these codons also releases the paused transcription complex. These findings indicate that the paused trp leader transcription complex resumes transcription when released by ribosome movement over the leader peptide coding region. This release would facilitate the coupling of transcription and translation essential to attenuation control.
Origin and turnover of mitochondrial glutathione.
Authors: Griffith O W , Meister A
Published: May 2006

Mitochondrial glutathione in liver does not arise by intramitochondrial synthesis, but rather from the cytoplasm, by a process characterized by slow net transport and more rapid exchange transport.
Localization of galactosyl- and sialyltransferase by immunofluorescence: evidence for different sites.
Authors: Berger E G , Hesford F J
Published: May 2006

Polyclonal rabbit antisera against soluble human milk galactosyltransferase and bovine colostrum sialyltransferase were used to localize by indirect immunofluorescence the respective intracellular enzymes in primary cultures from bovine fetal kidneys and established cell lines of human and bovine fibroblasts. Staining for galactosyltransferase was juxtanuclear and crescent shaped in epitheloid cells; a similar staining, occasionally perinuclear and sparsely distributed in the cytoplasm, was found in fibroblasts. In contrast, staining for sialyltransferase in epitheloid kidney cells derived from the same primary culture was observed predominantly in cytoplasmic vesicles that were spread over the whole cytoplasm. Sialyltransferase-positive vesicles had a similar distribution in fibroblasts and often appeared concentrated around an unstained Golgi area. Thus, in both cell types galactosyl- and sialyltransferase were localized in different subcellular compartments. Since both galactosyl- and sialyltransferase participate in formation of the terminal glycan NeuAc(alpha 2----6)Gal(beta 1----4)GlcNAc(Neu, neuraminic acid) present in many N-glycosidic complex types of glycans, different subcellular compartments for these enzymes support a model of functional compartmentalization of the Golgi apparatus that is compatible with an assembly-line model for glycan chain elongation and termination.
Molecular cloning of the genome of a cardiotropic Coxsackie B3 virus: full-length reverse-transcribed recombinant cDNA generates infectious virus in mammalian cells.
Authors: Kandolf R , Hofschneider P H
Published: May 2006

The molecular cloning of double-stranded cDNA synthesized from the single-stranded RNA genome of the cardiotropic Coxsackie B3 virus (Nancy strain) is reported. Full-length reverse-transcribed cloned viral cDNA of approximately equal to 7500 nucleotides generated infectious antigenically identical Coxsackie B3 virus upon transfection of recombinant plasmid DNA into mammalian cells, demonstrating the molecular cloning of a biologically active viral cDNA copy. Furthermore, the cloned cDNA is characterized by restriction enzyme analysis and partial nucleotide sequencing of the 5' end. The Coxsackie B3 virus cDNA described can now be used to study the molecular basis of human enteroviral heart disease, and it provides a valuable diagnostic means for patients with suspected viral heart disease.
Monoclonal antibodies reveal structural homogeneity of gamma-aminobutyric acid/benzodiazepine receptors in different brain areas.
Authors: Häring P , Stähli C , Schoch P , Takács B , Staehelin T , Möhler H
Published: May 2006

Monoclonal antibodies (mAb) against a gamma-aminobutyric acid/benzodiazepine receptor complex (GABAA/BZR) were produced by using spleen cells from a mouse immunized with GABAA/BZR purified from bovine cerebral cortex. The mAb, most of which were of the IgG1 isotype could be divided into four groups (I-IV) specifying different antigenic structures. On immunoblots, group I mAb recognized exclusively the Mr 55,000 beta-subunit, while groups II and IV mAb recognized the Mr 50,000 alpha-subunit of bovine GABAA/BZR. Three of the four groups of mAb (I, III, and IV) crossreacted with both human and rat GABAA/BZR with the same subunit specificity as in bovine brain; the fourth group (II) crossreacted with human but not with the rat receptor. The binding sites for benzodiazepines as well as the high and low affinity GABA sites reside on the same structural complex as shown by immunoprecipitation. Ligand binding to these sites was not inhibited by mAb. Since quantitative immunoprecipitation of GABAA/BZR was achieved with mAb selective for either the alpha- or beta-subunit, both subunits occur in each individual receptor complex. The pattern of immunoblot staining suggests that the smaller alpha-subunit is not a processing product of the larger beta-subunit. Both alpha- and beta-subunits were present in all brain areas and species tested (rat cerebral cortex, cerebellum, and hippocampus; bovine cerebral cortex and cerebellum; human cerebral cortex). This suggests a uniform subunit composition of the receptor throughout the brain in contrast to earlier evidence for a heterogeneous subunit composition based on photoaffinity labeling.
Sequence and evolution of the human T-cell antigen receptor beta-chain genes.
Authors: Tunnacliffe A , Kefford R , Milstein C , Forster A , Rabbitts T H
Published: May 2006

We present the nucleotide sequences of the two genomic constant (C)-region gene segments, C beta 1 and C beta 2, encoding the beta chain of the human T-cell antigen receptor. The two C beta genes are organized identically to each other and to the corresponding mouse genes, both having four exons, whose boundaries were confirmed from the sequence of a C beta 2 cDNA clone from the T-cell line MOLT-4. The predicted amino acid sequences of human C beta 1 and C beta 2 differ at only five positions, which suggests that the proteins have very similar functions. This similarity is the result of strong nucleotide-sequence conservation in protein-coding regions, which extends to silent positions. A quantitative analysis of an alignment of the nucleotide sequences of the two human genes shows that whereas the 5' ends (including the first exon) are extremely homologous, the 3' ends are widely divergent, with other regions having intermediate levels of homology. Analysis of published data [Gascoigne, N.R.J., Chien, Y., Becker, D.M., Kavaler, J. & Davis, M.M. (1984) Nature (London) 310, 387-391] shows that the mouse C beta 1 and C beta 2 genes are also virtually identical in their first exons but more divergent in the remaining coding regions. Therefore, partial gene conversion events may have occurred during the evolution of both human and mouse C beta genes.
Carbonic anhydrase is aberrantly and constitutively expressed in both human and murine erythroleukemia cells.
Authors: Frankel S R , Walloch J , Hirata R K , Bondurant M C , Villanueva R , Weil S C
Published: May 2006

The levels of the erythrocyte proteins carbonic anhydrase (CA) and hemoglobin (Hb) change coordinately during human ontogeny. To further probe the coordinate gene expression of these two proteins in vitro, we used an immunoblotting technique to measure their levels during erythroid differentiation in normal human and murine erythroid progenitors, in human and murine erythroleukemia cells, and in normal murine erythroid progenitors infected with Friend virus. Levels of CA and Hb seem to gradually increase in normal differentiating stem cells. In contrast, both human and murine erythroleukemia cells show high levels of CA, but not of Hb, prior to induction of differentiation. Friend virus infection of normal murine progenitors appears to stimulate CA synthesis as an initial and integral step in transformation. In addition, both the erythroleukemia cells and the erythroid progenitors transformed with Friend virus seem to contain much higher levels of CA than Hb during the early stages of differentiation. This relationship is in marked contrast to normal erythroid differentiation, in which Hb levels are always higher than CA levels. Thus, neoplastic transformation seems to be associated with aberrant production of CA that does not correspond to a maturation arrest of the normal differentiation sequence.
Effect of dietary ascorbic acid on the incidence of spontaneous mammary tumors in RIII mice.
Authors: Pauling L , Nixon J C , Stitt F , Marcuson R , Dunham W B , Barth R , Bensch K , Herman Z S , Blaisdell B E , Tsao C
Published: May 2006

A study of the effect of different amounts of L-ascorbic acid (vitamin C), between 0.076% and 8.3%, contained in the food has been carried out with ten groups of RIII mice (seven ascorbic acid and three control groups), with 50 mice in each group. With an increase in the amount of ascorbic acid there is a highly significant decrease in the first-order rate constant for appearance of the first spontaneous mammary tumor after the lag time to detection by palpation. There is also an increase in the lag time. The mean body weight and mean food intake were not significantly different for the seven ascorbic acid groups. Striking differences were observed between the 0.076% ascorbic acid and the control groups (which synthesize the vitamin): smaller food intake, decreased lag time, and increased rate constant of appearance of the first mammary tumor. This comparison cannot be made experimentally for guinea pigs and primates because the control groups would develop scurvy.
Identification of immediate early genes from herpes simplex virus that transactivate the virus thymidine kinase gene.
Authors: Gelman I H , Silverstein S
Published: May 2006

A HeLa cell transient-expression assay system was used to determine if isolated immediate early (alpha) genes from herpes simplex virus (HSV) could transcriptionally activate (transactivate) the type 1 (HSV-1) thymidine kinase (TK) gene [an early (beta) gene]. Cells transfected with the TK gene alone transcribed very low levels of TK RNA. Cells cotransfected with plasmids bearing the sequences that encode the alpha-gene product infected cell protein 0 or 4 (ICP0 or ICP4) and the TK gene faithfully transcribed high levels of TK RNA. The plasmid containing the sequences encoding ICP0 was a more potent transactivator than the plasmid containing the sequences for ICP4.
Protonic conductivity of hydrated lysozyme powders at megahertz frequencies.
Authors: Careri G , Geraci M , Giansanti A , Rupley J A
Published: May 2006

Dielectric losses were measured for lysozyme powders of varied hydration level by a dielectric-gravimetric technique in the frequency range of 10 kHz to 10 MHz. The relaxation showed an isotope effect and pH dependence, indicating that the inferred conductivity is protonic. The transport process is likely restricted to the surface of individual macromolecules and involves shifting of protons between ionizable side chain groups of the protein. The time constant of the relaxation shows cooperativity in its seventh-order dependence on bound protons. The process develops in the hydration region critical for the onset of the catalytic properties of the enzyme. The binding of a substrate increases the relaxation time by a factor of 2. These observations suggest that the megahertz dispersion reflects behavior at the protein surface, specifically the cooperative channeling of proton flow through the active site, that may be of particular significance for the enzymatic and other functional properties of proteins.
Chlorophyll biosynthesis and assembly into chlorophyll-protein complexes in isolated developing chloroplasts
Authors: Bhaya Devaki , Castelfranco Paul A.
Published: May 2006

Isolated developing plastids from greening cucumber cotyledons or from photoperiodically grown pea seedlings incorporated
14
C-labeled 5-aminolevulinic acid (ALA) into chlorophyll (Chl). Incorporation was light dependent, enhanced by
S
-adenosylmethionine, and linear for 1 hr. The
in vitro
rate of Chl synthesis from ALA was comparable to the
in vivo
rate of Chl accumulation. Levulinic acid and dioxoheptanoic acid strongly inhibited Chl synthesis but not plastid protein synthesis. Neither chloramphenicol nor spectinomycin affected Chl synthesis, although protein synthesis was strongly inhibited. Components of thylakoid membranes from plastids incubated with [
14
C]ALA were resolved by electrophoresis and then subjected to autoradiography. This work showed that (
i
) newly synthesized Chl was assembled into Chl-protein complexes and (
ii
) the inhibition of protein synthesis during the incubation did not alter the labeling pattern. Thus, there was no observable short-term coregulation between Chl synthesis (from ALA) and the synthesis of membrane proteins in isolated plastids.
Cultured psoriatic fibroblasts from involved and uninvolved sites have a partial but not absolute resistance to the proliferation-inhibition activity of 1,25-dihydroxyvitamin D3.
Authors: MacLaughlin J A , Gange W , Taylor D , Smith E , Holick M F
Published: May 2006

We examined the responsiveness of cultured dermal fibroblasts from biopsies of uninvolved and involved areas of skin from six patients with psoriasis to the cell-proliferation-inhibition activity of 1,25-dihydroxyvitamin D3 (1,25-(OH)2-D3). Cultured fibroblasts from age-matched controls responded to 1,25-(OH)2-D3 (at 0.01, 1, 10, and 100 microM) in a dose-dependent fashion, whereas cultured psoriatic fibroblasts from involved or uninvolved skin showed no inhibition of proliferation when exposed to 0.01 or 1 microM of 1,25-(OH)2-D3. However, 1,25-(OH)2-D3 did inhibit proliferation of cultured psoriatic fibroblasts when the concentrations were increased to 10 and 100 microM. An analysis of the 1,25-(OH)2-D3 receptors in cultured psoriatic fibroblasts from uninvolved skin revealed that the Kd, nmax, and sedimentation coefficient were identical to the receptors found in the fibroblasts from age-matched controls. Therefore, cultured psoriatic fibroblasts from involved and uninvolved skin have a partial resistance to 1,25-(OH)2-D3, suggesting that there may be a biochemical defect that is inherent in the dermal fibroblasts of psoriatic patients. Recognition of this defect may provide a new approach for the evaluation of the cause and treatment of this disfiguring skin disorder.
Quantal mechanism of long-term synaptic potentiation.
Authors: Baxter D A , Bittner G D , Brown T H
Published: May 2006

Intracellular recordings were used to demonstrate the occurrence and to analyze the microphysiology of long-term synaptic potentiation (LTP) in the crayfish opener neuromuscular synapse. Brief stimulation of the single excitor motor axon enhanced the amplitudes of subsequent postsynaptic potentials for several hours. Three methods of quantal analysis were used to evaluate the mechanism responsible for LTP. The results of all three methods supported predictions of the hypothesis that LTP results from a presynaptic mechanism that increases the average of neurotransmitter quanta evoked by nerve impulses in the excitor axon.
Differentiation-specific regulation of Schwann cell expression of the major myelin glycoprotein.
Authors: Poduslo J F , Windebank A J
Published: May 2006

Previous experiments demonstrated that Schwann cells from permanently transected sciatic nerves of adult rats synthesize basal levels of the major myelin glycoprotein (P0). This denervated preparation at 35 days after transection was characterized by the absence of both axons and myelin assembly. The present investigation demonstrates that production of P0 continues after culture of the Schwann cells as endoneurial slices for 14 days. Thus, the level of differentiation is unchanged in culture even though only basal levels of P0 are produced and post-translational processing is incomplete. In contrast, Schwann cells from 4-day-old rat sciatic nerves actively synthesized P0 and assembled myelin membrane; however, after only 4 days in culture biosynthesis of P0 ceased. Because the same culture conditions and precursor incorporation procedures were used for both neonatal and transected nerves, it is proposed that neonatal Schwann cells in culture return to a progenitor state that is not capable of P0 gene expression. This comparison, both before and after culture, of neonatal Schwann cells that are programmed to myelinate and Schwann cells from the adult transected nerve that were in a myelin-maintaining mode provides a useful model for investigating the mechanisms by which differential gene expression is controlled. These results confirm that axons are not necessary for specifying P0 gene expression by Schwann cells from the adult transected nerves. The role that axons play in controlling P0 gene expression by neonatal Schwann cells in culture, however, has yet to be determined. It is concluded that the differentiation and maturation of Schwann cells is multistage process that allows the sequential production of specific gene products.
Expression of a cytoplasmic LINE-1 transcript is regulated in a human teratocarcinoma cell line.
Authors: Skowronski J , Singer M F
Published: May 2006

The major primate family of highly repeated, long interspersed DNA sequences (LINE-1, previously Kpn I, family) includes several thousand 6-kilobase-pair long units that terminate in an A-rich stretch. Recent evidence indicates that long open reading frames occur in at least some family members. These results suggested that one or more LINE-1 family members might be structural genes. Accordingly, a variety of human cell lines was analyzed for the presence of a cytoplasmic, polyadenylylated RNA homologous to LINE-1 sequences. Such a transcript was detected in a human pluripotent teratocarcinoma cell line (NTera2 clone D1). The RNA is approximately 6.5 kilobases long and is homologous to the LINE-1 strand with the open reading frames. The abundance of the transcript varies markedly with previously described variations in the phenotype of these cells and is highest when the cells display the embryonal carcinoma morphology. This RNA may represent a mRNA transcribed from one or more functional genes in the LINE-1 family.
A highly polymorphic locus in human DNA revealed by cosmid-derived probes.
Authors: Litt M , White R L
Published: May 2006

Human gene mapping would be greatly facilitated if marker loci with sufficient heterozygosity were generally available. As a source of such markers, we have used cosmids from a human genomic library. We have developed a rapid method for screening random cosmids to identify those that are homologous to genomic regions especially rich in restriction fragment length polymorphisms. This method allows whole cosmids to be used as probes against Southern transfers of genomic DNA; regions of cosmid probes homologous to repeated genomic sequences are rendered unable to hybridize with Southern transfers by prehybridization of the probes with a vast excess of nonradioactive genomic DNA. From one cosmid identified by this procedure, we have isolated three single-copy probes that collectively identify seven polymorphic loci. Of 56 unrelated individuals, 52 were heterozygous at one or more of these marker loci.
Estradiol 16 alpha-hydroxylation in the mouse correlates with mammary tumor incidence and presence of murine mammary tumor virus: a possible model for the hormonal etiology of breast cancer in humans.
Authors: Bradlow H L , Hershcopf R J , Martucci C P , Fishman J
Published: May 2006

In this report, we describe our findings on the relationship between estradiol 16 alpha-hydroxylation and mammary tumor incidence. A close correlation between the two has been demonstrated with 16-hydroxylation being elevated in strains with a high incidence of tumors, such as RIII and C3H, and low in strains with a low incidence of cancer, such as C57BL. The extent of reaction is highly reproducible and unaffected by age or presence of overt mammary tumors. Studies on the inheritance of estradiol 16 alpha-hydroxylase showed that it is inherited as an autosomal dominant and is not correlated with estradiol 2-hydroxylase or androgen and progestin 16 alpha-hydroxylases. In addition, the reaction was shown to be markedly enhanced by the presence of murine mammary tumor virus and diminished in the absence of the virus. These studies establish a relationship between genetics, hormonal factors, and murine mammary tumor virus, the three key factors in mammary tumorigenesis.