bluuebunny/crossref_metadata_2025 huggingface dataset:
Adenylate cyclase mediates olfactory transduction for a wide variety of odorants.
Authors: Lowe G , Nakamura T , Gold G H
Published: May 2006

An odor-stimulated adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] is thought to mediate olfactory transduction in vertebrates. However, it is not known whether the adenylate cyclase serves this function for all odorants or for only certain classes of odorants. To investigate this question, we have compared the abilities of 35 odorants to stimulate the adenylate cyclase and to elicit an electrophysiological response. We report a strong positive correlation between the magnitude of adenylate cyclase stimulation and the summated electrical response of the olfactory epithelium (electro-olfactogram) evoked by individual odorants. We also show that the adenylate cyclase stimulator forskolin equally attenuates the electro-olfactogram response for all odorants tested. These data provide evidence that the adenylate cyclase mediates transduction for a wide variety of odorants.
Molecular cloning of a neuron-specific transcript and its regulation during normal and aberrant cerebellar development.
Authors: Sangameswaran L , Hempstead J , Morgan J I
Published: May 2006

PEP-19 is a brain-specific polypeptide whose levels increase dramatically during the late maturation of the rodent nervous system. By using immunocytochemistry, PEP-19 is shown to be localized to several regions of the central nervous system, notably cerebellum, thalamus caudate putamen, and olfactory bulb. We have isolated a 0.5-kilobase cDNA clone that encodes the entire PEP-19 protein sequence, making this one of the smallest primary transcripts and translation products ever identified in eukaryotes. The cDNA was used to investigate the developmental expression of PEP-19 in rodent brain. PEP-19 mRNA rises from low levels at embryonic day 17 of gestation in the rat to a plateau value by day 18 postpartum. This mirrored the levels of the protein determined by radioimmunoassay. Since the rise coincided with the formation of synaptic contacts onto Purkinje cells (a major site of PEP-19 expression), the hypothesis was tested that the activity and/or presence of afferent input modulated PEP-19 expression. Parallel fiber innervation was disrupted either by killing granule cells with the cytostatic agent methylazoxymethanol or by examining PEP-19 levels in cerebellar granuloprival mutant mice (reeler and weaver). The influence of climbing fiber input was assessed by either eliminating them with 3-acetylpyridine or stimulating them with harmaline in both neonatal and mature rats. None of the above altered PEP-19 gene expression in cerebellum, leading us to propose that the signals triggering the PEP-19 gene do not emanate from granule cells or neurons in the olivary nucleus. However, preliminary evidence suggests that PEP-19 is under posttranscriptional regulation.
Evidence that brain prostaglandin E2 is involved in physiological sleep-wake regulation in rats.
Authors: Matsumura H , Honda K , Choi W S , Inoué S , Sakai T , Hayaishi O
Published: May 2006

We reported in previous studies that prostaglandin E2 (PGE2) has central effects of augmenting wakefulness and suppressing slow-wave sleep (SWS) and paradoxical sleep (PS) in rats. In the present study, we tested the effect of AH 6809, an antagonist of PGE2 receptors, on sleep-wake activities. AH 6809 in saline was infused continuously into the third ventricle of freely moving rats at a rate of 2.1, 6.3, and 21 pmol/min from 2300 to 0500 hr. During the infusion at 21 pmol/min, wakefulness decreased to 82%, and SWS and PS increased to 122% and 161%, of the respective baseline values. These changes can be explained by AH 6809 antagonizing the endogenous PGE2 that acts to augment wakefulness in the brain. This explanation is supported by the fact that the infusion of AH 6809 at 21 pmol/min inhibited the wakefulness-promoting effect of PGE2 infused at 10 pmol/min. Moreover, the PGE2-related mechanisms for regulating sleep-wake activities may be different from those producing hyperthermia, because AH 6809 at 21 pmol/min had no primary effect on brain temperature and did not antagonize the hyperthermia produced by the PGE2 infusion. A diurnal infusion (1200 to 1800 hr) of AH 6809 at 21 pmol/min produced similar effects on sleep-wake activities compared with the nocturnal infusion (2300 to 0500 hr), although the PS increase was not significant, suggesting that the PGE2-related mechanisms are acting all day long with or without a circadian rhythm. These findings strongly suggest that endogenous PGE2 in the brain is involved in the physiological mechanisms for regulating sleep-wake activities.
Expression of bovine beta-1,4-galactosyltransferase cDNA in COS-7 cells.
Authors: Masibay A S , Qasba P K
Published: May 2006

A bovine beta-1,4-galactosyltransferase (GT; EC 2.4.1.90) cDNA in an Okayama-Berg vector, pLsGT, was constructed from a partial cDNA clone and a genomic fragment. We report that the cDNA sequence of pLsGT, in a transient expression assay in COS-7 cells, codes for an enzymatically active GT protein. There is an approximately 12-fold increase in the GT activity in pLsGT-transfected cells compared to cells transfected with the antisense bovine GT construct, pLasGT, or pSV2Neo or mock-transfected cells. The increased activity is correlated with the increase in bovine GT mRNA, which is distinguishable from COS GT mRNA with a 3'-end-specific probe of pLsGT. The expressed GT activity is modulated by alpha-lactalbumin, which changes the acceptor specificity to glucose to synthesize lactose. Polyclonal antibody raised against SDS/PAGE-purified bovine milk GT and a monoclonal antibody (mAb 4-10) directed against a synthetic peptide corresponding to the amino-terminal region of the protein encoded by pLsGT bind the expressed protein, and the resulting immunoprecipitates exhibit GT enzymatic activity.
Tryptophan fluorescence quenching as a monitor for the protein conformation changes occurring during the photocycle of bacteriorhodopsin under different perturbations.
Authors: Jang D J , el-Sayed M A
Published: May 2006

The rates of the quenching and recovery of tryptophan fluorescence are determined in the microsecond-millisecond time scale during the photocycle of bacteriorhodopsin under different perturbations. The kinetics suggest the presence of two quenching processes, a rapid one (on the time scale of photocycle intermediate L550 formation or faster) and a slow one (slightly slower than the slow component of intermediate M412 formation). The slow quenching process is found to respond to different perturbations in the same manner as the slow component of M412 formation. It has the same activation energy, it is inhibited if metal cations are removed, it is negligible at pH values greater than the pKa of tyrosine, and its rate is slowed down when 75% of the lipids are removed. These results, together with the observed value of the quenching activation energy, suggest that the rates of the tryptophan fluorescence quenching, like those of tyrosinate and M412 formations during the cycle, are all determined by the rates of the protein conformation changes. The pH studies of the slow quenching process show that the maximum quenching probability occurs at neutral pH. A rapid decrease in quenching occurs at lower pH (approximately 3 and approximately 5.5) and higher pH (approximately 9). Two quenching mechanisms involving energy transfer to either retinal or to tyrosinate are considered. Protein conformation changes resulting from a change in the ionization state of amino acids of different pKa values could change the tryptophan-retinal (or tryptophan-tyrosinate) coupling and thus the quenching efficiency.
Effects of lipid packing on polymorphic phase behavior and membrane properties.
Authors: Hui S W , Sen A
Published: May 2006

The self assembly of phospholipid molecules in the bilayer form was considered in terms of equivalent molecular shapes representing intermolecular forces. The equivalent size of each phospholipid headgroup was approximated by the net atomic volume plus the volume of the associated water molecules, which was derived from water/hydrocarbon partitioning experiments. The equivalent lengths of unsaturated acyl chains were derived from the retention time data from chromatographic measurements. The spontaneous curvature of various phospholipid monolayers was calculated from their equivalent molecular shapes, and the energy required to flatten them to the bilayer plane was calculated, using the known bending modulus. With increasing bending energy, the mixtures showed increasing susceptibility to phospholipase A2, facilitated lipid transfer rate by phospholipid exchange proteins, permeability to carboxyfluorescein, incorporation of human erythrocyte proteins, and calcium transport by Ca-ATPase from sarcoplasmic reticulum in reconstituted vesicles. When the calculation was applied to known lipid compositions of nine cellular membranes, the protein/lipid ratio and phospholipid/cholesterol ratio were found to have a positive and a negative correlation, respectively, with the latent bending energy of the phospholipids. The energy expense in conforming to a bilayer phase may be an important physical parameter regarding the activity and the biogenesis of membranes.
Purification of scatter factor, a fibroblast-derived basic protein that modulates epithelial interactions and movement.
Authors: Gherardi E , Gray J , Stoker M , Perryman M , Furlong R
Published: May 2006

Scatter factor is a fibroblast-derived protein that causes separation of contiguous epithelial cells and increased local mobility of unanchored cells. Highly purified scatter factor has been obtained by a combination of ion-exchange and reverse-phase chromatography from serum-free medium conditioned by a ras-transformed clone (D4) of mouse NIH 3T3 fibroblasts. Under nonreducing conditions scatter factor has a pI of approximately 9.5 and migrates in SDS/polyacrylamide gels as a single band at approximately 62 kDa from which epithelial scatter activity can be recovered. Treatment with reducing agents destroys biological activity and is associated with the appearance of two major bands at approximately 57 and approximately 30 kDa. Whether both the 57-kDa and 30-kDa polypeptides are required for biological activity remains to be established. All the activities observed in crude medium conditioned by cells producing scatter factor are retained by highly purified preparations of scatter factor. These include (i) increased local movement, modulation of morphology, and inhibition of junction formation by single epithelial cells and (ii) disruption of epithelial interactions and cell scattering from preformed epithelial sheets. These changes occur with picomolar concentrations of purified scatter factor and without an effect on cell growth.
Tetrahydrobiopterin, the cofactor for aromatic amino acid hydroxylases, is synthesized by and regulates proliferation of erythroid cells.
Authors: Tanaka K , Kaufman S , Milstien S
Published: May 2006

The only known role for 6(R)-5,6,7,8-tetrahydrobiopterin (BH4) is as the cofactor for the aromatic amino acid hydroxylases. However, BH4 has been shown to be synthesized by cells that do not contain any hydroxylase activity, suggesting that it may have still undiscovered functions. Our finding of much higher levels of BH4 and GTP cyclohydrolase, the first enzyme of de novo BH4 biosynthesis, in rat reticulocytes compared to mature erythrocytes raised the possibility that BH4 might play a role in erythrocyte maturation. We have now demonstrated, by using murine erythroleukemia (MEL) cells as a model for erythrogenesis, that BH4 synthesis is required for proliferation of these cells. Inhibition of BH4 biosynthesis in rapidly dividing MEL cells with N-acetylserotonin, a potent inhibitor of sepiapterin reductase, the terminal enzyme in the BH4 biosynthetic pathway, results in inhibition of DNA synthesis and mitogenesis without induction of hemoglobin synthesis. The inhibition of DNA synthesis is reversed by repletion of cellular BH4 levels with sepiapterin, a pterin that is readily taken up by the cells and converted to BH4 by the sequential reductions of sepiapterin reductase and dihydrofolate reductase. Treatment of MEL cells with hexamethylene bisacetamide, an inducer of differentiation, results in a decrease in BH4 synthesis accompanied by a cessation of growth and concomitant hemoglobin synthesis. The inhibition of proliferation induced by hexamethylene bisacetamide can be reversed by maintaining high intracellular levels of BH4, which also decreases the amount of hemoglobin. The mechanism of the BH4 effect has not yet been elucidated, but it appears as though BH4 synthesis is more intimately linked with cell proliferation than with the differentiation process.
In situ autoradiography and ligand-dependent tyrosine kinase activity reveal insulin receptors and insulin-like growth factor I receptors in prepancreatic chicken embryos.
Authors: Girbau M , Bassas L , Alemany J , de Pablo F
Published: May 2006

We previously reported specific cross-linking of 125I-labeled insulin and 125I-labeled insulin-like growth factor I (IGF-I) to the alpha subunit of their respective receptors in chicken embryos of 20 somites and older. To achieve adequate sensitivity and localize spatially the receptors in younger embryos, we adapted an autoradiographic technique using whole-mounted chicken blastoderms. Insulin receptors and IGF-I receptors were expressed and could be localized as early as gastrulation, before the first somite is formed. Relative density was analyzed by a computer-assisted image system, revealing overall slightly higher binding of IGF-I than of insulin. Structures rich in both types of receptors were predominantly of ectodermal origin: Hensen's node in gastrulating embryos and neural folds, neural tube and optic vesicles during neurulation. The signal transduction capability of the receptors in early organogenesis was assessed by their ability to phosphorylate the exogenous substrate poly(Glu80Tyr20). Ligand-dependent tyrosine phosphorylation was demonstrable with both insulin and IGF-I in glycoprotein-enriched preparations from embryos at days 2 through 6 of embryogenesis. There was a developmentally regulated change in ligand-dependent tyrosine kinase activity, with a sharp increase from day 2 to day 4, in contrast with a small increase in the ligand binding. Binding of 125I-labeled IGF-I was, with the solubilized receptors, severalfold higher than binding of 125I-labeled insulin. However, the insulin-dependent phosphorylation was as high as the IGF-I-dependent phosphorylation at each developmental stage.
Cardiac and neurological abnormalities in v-fps transgenic mice.
Authors: Yee S P , Mock D , Maltby V , Silver M , Rossant J , Bernstein A , Pawson T
Published: May 2006

Transgenic mice that widely express the v-fps protein-tyrosine kinase develop several independent pathological conditions, in addition to a high tumor incidence. v-fps expression and protein-tyrosine kinase activity in the heart were directly correlated with cardiac enlargement. This cardiomegaly was accompanied by severe myocardial and endocardial damage, which was concentrated in the left ventricular wall, and characterized by a progressive atrophy and necrosis of cardiac muscle fibers with concomitant fibrosis. This pathology was associated with congestive heart failure. Mice from five lines developed a marked trembling, correlated with expression of the v-fps transgene in the brain, and two lines showed a striking bilateral enlargement of the trigeminal nerves. Unlike tumor formation, these cardiac and neurological phenotypes were evident shortly after birth and showed 100% penetrance. The pleiotropic effects of the v-fps transgene suggest the involvement of protein-tyrosine kinases in mammalian neural development and cardiac function.
Tumor necrosis factor alpha activates human immunodeficiency virus type 1 through induction of nuclear factor binding to the NF-kappa B sites in the long terminal repeat.
Authors: Duh E J , Maury W J , Folks T M , Fauci A S , Rabson A B
Published: May 2006

Expression of human immunodeficiency virus type 1 (HIV-1) can be activated in a chronically infected T-cell line (ACH2 cells) by a cytokine, human tumor necrosis factor alpha (TNF-alpha). TNF-alpha treatment of ACH2 cells resulted in an increase in steady-state levels of HIV RNA and HIV transcription. Gel mobility shift assays demonstrated that the transcriptional activation of the HIV long terminal repeat (LTR) by TNF-alpha was associated with the induction of a nuclear factor(s) binding to the NF-kappa B sites in the LTR. Deletion of the NF-kappa B sites from the LTR eliminated activation by TNF-alpha in T cells transfected with plasmids in which the HIV LTR directed the expression of the bacterial chloramphenicol acetyltransferase gene. Thus, TNF-alpha appears to activate HIV RNA and virus production by ACH2 cells through the induction of transcription-activating factors that bind to the NF-kappa B sequences in the HIV LTR.
Dynamic properties of the Ca2+/calmodulin-dependent protein kinase in Drosophila: identification of a synapsin I-like protein.
Authors: Mitschulat H
Published: May 2006

Visual adaptation with blue light induces a change in a special light/dark choice behavior in Drosophila. On the molecular level adaptation induces long-term modulation of the in vitro autophosphorylation capacity of a Ca2+/calmodulin-dependent protein kinase. Here I describe a Drosophila phosphoprotein that is a substrate of this protein kinase. The molecular mass and phosphopeptide composition of this protein are similar to those of rat synapsin I. Furthermore, the Drosophila protein shows immunological cross-reactivity with monoclonal antibodies against rat synapsin I. I conclude that this 86-kDa protein in Drosophila is homologous to the vertebrate synapsin I.
Translation in Saccharomyces cerevisiae: initiation factor 4A-dependent cell-free system.
Authors: Blum S , Mueller M , Schmid S R , Linder P , Trachsel H
Published: May 2006

Yeast Saccharomyces cerevisiae genes TIF1 and TIF2 (translation initiation factor) encode a protein tentatively called translation initiation factor (Tif) due to the similarity of its amino acid sequence and its molecular weight to mammalian eukaryotic initiation factor 4A. To clarify whether Tif is involved in translation, we produced an affinity-purified anti-Tif antibody by using Tif isolated from a Tif-overproducing yeast strain as immunogen and an Escherichia coli strain expressing Tif from an expression vector to provide the extract for affinity purification of the antibody. By using chromatographic procedures and the affinity-purified anti-Tif antibody as probe to identify Tif-containing fractions, we purified Tif from wild-type yeast cells. When yeast cells containing the only TIF1 gene on a plasmid under the control of the galactose-inducible CYC1-GAL10 promoter were grown in medium containing glucose as the carbon source, the production of Tif was shut off and growth was arrested. Lysates made from these cells were inactive in in vitro translation. Addition of Tif to these lysates restored in vitro protein synthesis. These results show that Tif is a translation factor, the yeast homologue of mammalian translation initiation factor 4A.
Purification and characterization of the nifN and nifE gene products from Azotobacter vinelandii mutant UW45.
Authors: Paustian T D , Shah V K , Roberts G P
Published: May 2006

The nifN and -E gene products are involved in the synthesis of the iron-molybdenum cofactor of dinitrogenase, the enzyme responsible for the reduction of dinitrogen to ammonia. By using the in vitro iron-molybdenum cofactor biosynthesis assay, we have followed the purification of these gene products 450-fold to greater than 95% purity. An overall recovery of 20% was obtained with the purified protein having a specific activity of 6900 units/mg of protein. The protein (hereafter referred to as NIFNE) was found to contain equimolar amounts of the nifN and -E gene products and have a native molecular mass of 200 +/- 10 kDa, which indicates an alpha 2 beta 2 structure. NIFNE was oxygen labile with a half-life of 1 min in air. A UV-visible spectrum of the dye-oxidized protein showed an absorption maximum at 425 nm that could be bleached by reduction of NIFNE with sodium dithionite, suggesting the presence of an Fe center in NIFNE.
Dissection of functional domains of adenovirus DNA polymerase by linker-insertion mutagenesis.
Authors: Chen M , Horwitz M S
Published: May 2006

Linker-insertion mutations were introduced into the cloned adenovirus DNA polymerase gene and the functional effects on the initiation and elongation of DNA in vitro were measured. Essential regions of the polymerase appear to be scattered in patches across the entire molecule and are not limited to the five regions of homology shared with a variety of other replicating polymerases. Thus, the adenovirus DNA polymerase presumably contains active sites that must be formed by distant interactions across the polymerase molecule.
Cap-independent translation of mRNA conferred by encephalomyocarditis virus 5' sequence improves the performance of the vaccinia virus/bacteriophage T7 hybrid expression system.
Authors: Elroy-Stein O , Fuerst T R , Moss B
Published: May 2006

A recombinant vaccinia virus that directs the synthesis of bacteriophage T7 RNA polymerase provides the basis for the expression of genes that are regulated by T7 promoters in mammalian cells. The T7 transcripts, which account for as much as 30% of the total cytoplasmic RNA at 24 hr after infection, are largely uncapped. To improve the translatability of the uncapped RNA, the encephalomyocarditis virus (EMCV) untranslated region (UTR) was inserted between the T7 promoter and the chloramphenicol acetyltransferase (CAT) gene. Experiments with a reticulocyte extract demonstrated that the EMCV UTR conferred efficient and cap-independent translatability to CAT RNA synthesized in vitro by T7 RNA polymerase. In cells infected with recombinant vaccinia viruses containing the T7 promoter-regulated CAT gene, the EMCV UTR increased the amount of CAT RNA on polyribosomes. The polyribosome-derived CAT RNA, which contained the EMCV UTR, was translated in vitro in a cap-independent fashion as well. Use of the EMCV UTR significantly enhanced the vaccinia/T7 hybrid expression system as it resulted in a 4- to 7-fold increase in total CAT activity. A further approximately 2-fold improvement was achieved by incubating the cells in hypertonic medium, which favors the translation of uncapped picornavirus RNA over cellular mRNAs. With this newly modified expression system, CAT was the predominant protein synthesized by infected cells and within 24 hr accounted for greater than 10% of the total cell protein.
Stimulatory effects of opioid neuropeptides on locomotory activity and conformational changes in invertebrate and human immunocytes: evidence for a subtype of delta receptor.
Authors: Stefano G B , Cadet P , Scharrer B
Published: May 2006

The presence of opioid neuropeptides was shown to stimulate conformational changes and locomotory activity in immunocytes of two representatives of invertebrates as well as in human leukocytes. Cells were examined by use of phase-contrast and Nomarski optics coupled with a Zeiss Axiophot microscope, and of the Zeiss Videoplan/Vidas Image Analysis system. Immunocompetent blood cells, activated by exogenous opioids or stressful stimuli presumed to engage endogenous opioids, showed flattening, elongation, and formation of pseudopodia. In the mollusc Mytilus edulis, ameboid movements resulted in the formation of cell clusters, an activity not observed in untreated controls, or in immunocytes simultaneously exposed to opioid and naloxone. Tests with nine immunoreactive substances revealed immunocyte stimulation by delta, mu-, kappa-, and epsilon(?)-selective ligands. One of these, [D-Ala2,D-Met5]enkephalinamide (DAMA), active at a concentration of 10 pM, proved to be considerably more effective than the rest. The high pharmacological potency of DAMA, observed in both human and invertebrate immunocytes, sets this opioid apart from the closely related [D-Ala2,D-Leu5]enkephalin, a discrepancy not occurring in the mammalian nervous system. This suggests a specific function for [Met]enkephalin in immunoregulation, mediated perhaps by a special subtype of delta receptor.
Selenium deficiency induced by zinc deprivation in a crustacean
Authors: Keating K. Irwin , Caffrey P. Barone
Published: May 2006

For intact daphnids reared in circumstances of controlled trace element exposure, one consequence of insufficient zinc (Zn) is an increased demand on the animal's pool of available selenium (Se). This demand is manifested by the type of cuticle deterioration associated with Se deficiency and by a depression of reproduction. In the presence of 25 parts per billion (ppb) Zn, 1 ppb Se eliminates these symptoms. In the absence of detectable Zn, 5 ppb Se eliminates overt cuticle damage and substantially increases reproduction. A shortening of life span resulting from Zn deprivation is not ameliorated by Se addition. We suggest that the interplay between Zn and Se concentrations reflects an underlying interplay between interdependent, but distinct, metabolic pathways; i.e., (for Se) glutathione peroxidase and (for Zn) Cu,Zn-superoxide dismutase—each offering protection against free radical damage. Because they are not necessarily localized in a given tissue, the key to recognition of such subtle, complex trace nutrient interactions has been use of intact animals in circumstances of control previously attainable only in tissue cultures.
Critical shape transitions of monolayer lipid domains
Authors: Rice Peter A. , McConnell Harden M.
Published: May 2006

Fluorescence microscopy can be used to visualize coexisting fluid phases in lipid monolayers composed of cholesterol and dipalmitoyl phosphatidylcholine under specified conditions of temperature, composition, and lateral pressure. At a critical composition of ≈30 mol% cholesterol, decreasing the average molecular area below

a
c

[unk]50 Å
2
per molecule forces the binary mixture through a critical point, where the monolayer becomes homogeneous. At molecular areas ≈10% above this critical area, we observe shape transitions from liquid domains with circular shapes to domains with less symmetrical shapes. Shape transitions and critical shape fluctuations can also be triggered with light, due to photochemical effects on the monolayer. Shape fluctuations of lipid domains can thus be used to sense chemical events at the air-water interface.
The noncovalent complex between DNA and the bifunctional intercalator ditercalinium is a substrate for the UvrABC endonuclease of Escherichia coli.
Authors: Lambert B , Jones B K , Roques B P , Le Pecq J B , Yeung A T
Published: May 2006

We have demonstrated that the noncovalent complex formed between DNA and an antitumor bifunctional intercalator, ditercalinium, is recognized in vitro as bulky covalent DNA lesions by the purified Escherichia coli UvrABC endonuclease. It was established that no covalent drug-DNA adduct was formed during the incubation of the drug with DNA or during subsequent incubation with the UvrAB proteins. The nucleoprotein-ditercalinium complexes appear different from those generated by repair of pyrimidine dimers. The UvrA protein is able to form a stable complex with ditercalinium-intercalated DNA in the presence of ATP, whereas both UvrA and UvrB proteins are required to form a stable complex with pyrimidine dimer-containing DNA. The apparent half-life of the UvrA- and UvrAB-ditercalinium-DNA complexes following removal of free ditercalinium is 5 min. However, if the free ditercalinium concentration is maintained to allow the intercalation of one molecule of ditercalinium per 3000 base pairs, the half-life of the UvrA- or UvrAB-ditercalinium-DNA complex is 50 min, comparable to that of the complex of UvrAB proteins formed with pyrimidine dimer-containing DNA. UvrABC endonuclease incises ditercalinium-intercalated DNA as efficiently as pyrimidine dimer-containing DNA. However, unlike repair of pyrimidine dimers, the incision reaction is strongly favored by the supercoiling of the DNA substrate. Because UvrA- or UvrAB-ditercalinium-DNA complexes can be formed with relaxed DNA without leading to a subsequent incision reaction, these apparently dead-end nucleoprotein complexes may become lesions in themselves resulting in the cytotoxicity of ditercalinium. Our results show that binding of excision repair proteins to a noncovalent DNA-ligand complex may lead to cell toxicity.
Identification of the A2 adenosine receptor binding subunit by photoaffinity crosslinking.
Authors: Barrington W W , Jacobson K A , Hutchison A J , Williams M , Stiles G L
Published: May 2006

A high-affinity iodinated agonist radioligand for the A2 adenosine receptor has been synthesized to facilitate studies of the A2 adenosine receptor binding subunit. The radioligand 125I-labeled PAPA-APEC (125I-labeled 2-[4-(2-[2-[(4- aminophenyl)methylcarbonylamino]ethylaminocarbonyl]- ethyl)phenyl]ethylamino-5'-N-ethylcarboxamidoadenosine) was synthesized and found to bind to the A2 adenosine receptor in bovine striatal membranes with high affinity (Kd = 1.5 nM) and A2 receptor selectivity. Competitive binding studies reveal the appropriate A2 receptor pharmacologic potency order with 5'-N-ethylcarboxamidoadenosine (NECA) greater than (-)-N6-[(R)-1-methyl- 2-phenylethyl]adenosine (R-PIA) greater than (+)-N6-[(S)-1-methyl-2- phenylethyl]adenosine (S-PIA). Adenylate cyclase assays, in human platelet membranes, demonstrate a dose-dependent stimulation of cAMP production. PAPA-APEC (1 microM) produces a 43% increase in cAMP production, which is essentially the same degree of increase produced by 5'-N- ethylcarboxamidoadenosine (the prototypic A2 receptor agonist). These findings combined with the observed guanine nucleotide-mediated decrease in binding suggest that PAPA-APEC is a full A2 agonist. The A2 receptor binding subunit was identified by photoaffinity-crosslinking studies using 125I-labeled PAPA-APEC and the heterobifunctional crosslinking agent N-succinimidyl 6-(4'-azido-2'-nitrophenylamino)hexanoate (SANPAH). After covalent incorporation, a single specifically radiolabeled protein with an apparent molecular mass of 45 kDa was observed on NaDodSO4/PAGE/autoradiography. Incorporation of 125I-labeled PAPA-APEC into this polypeptide is blocked by agonists and antagonists with the expected potency for A2 receptors (see above) and is decreased in the presence of 10(-4) M guanosine 5'-[beta, gamma-imido]triphosphate. Photoaffinity crosslinking of the A1 adenosine receptor binding subunit with 125I-labeled 8-[4-[2-(4- aminophenylacetylamino)ethyl]carbonylmethyloxyphenyl]-1,3-di propylxanthine (PAPAXAC) (an A1 selective photoaffinity probe) in the same tissue reveals a 38-kDa peptide that exhibits the appropriate A1 receptor pharmacology. 125I-labeled PAPA-APEC, therefore, has identified the A2 receptor binding subunit as a 45-kDa protein that is unique and distinct from the A1 binding subunit.
Genetically engineered mutant of the cyanobacterium
Synechococcus
PCC 7942 defective in nitrate transport
Authors: Omata Tatsuo , Ohmori Masayuki , Arai Nobuyuki , Ogawa Teruo
Published: May 2006

Nitrate-grown cells of
Synechococcus
PCC 7942 (
Anacystis nidulans
R2) contain a 45-kDa protein as a major protein in the cytoplasmic membrane but ammonium-grown cells lack it. A mutant (M45) was constructed by inactivating the gene encoding the 45-kDa protein. M45 did not grow under low concentrations of nitrate but high concentrations of nitrate could support its growth, with the optimal concentration being 40-70 mM. The growth rate of M45 was as high as that of the wild-type cells when ammonium was the nitrogen source. The 45-kDa protein was absent in M45 irrespective of the growth conditions. The activities of nitrate and nitrite reductases were higher in M45 than in wild type. The rate of nitrate-dependent O
2
evolution in wild type measured in the presence of L-methionine D,L-sulfoximine and D,L-glyceraldehyde showed saturation kinetics with respect to nitrate concentration in the external medium. The nitrate concentration required to produce half the maximal rate was 1 μM. In M45, the rate of nitrate-dependent O
2
evolution was nearly zero at nitrate concentrations <1 mM and was linearly increased as the concentration increased. The presumed absence of nitrate transport in M45 demonstrated by these results suggested that the 45-kDa protein is a nitrate transporter.
Separate photosensitizers mediate degradation of the 32-kDa photosystem II reaction center protein in the visible and UV spectral regions.
Authors: Greenberg B M , Gaba V , Canaani O , Malkin S , Mattoo A K , Edelman M
Published: May 2006

A component of the photosystem II reaction center, the 32-kDa protein, is rapidly turned over in the light. The mechanism of its light-dependent metabolism is largely unknown. We quantified the rate of 32-kDa protein degradation over a broad spectral range (UV, visible, and far red). The quantum yield for degradation was highest in the UVB (280-320 nm) region. Spectral evidence demonstrates two distinctly different photosensitizers for 32-kDa protein degradation. The data implicate the bulk photosynthetic pigments (primarily chlorophyll) in the visible and far red regions, and plastoquinone (in one or more of its redox states) in the UV region. A significant portion of 32-kDa protein degradation in sunlight is attributed to UVB irradiance.
In vitro attachment of glycosyl-inositolphospholipid anchor structures to mouse Thy-1 antigen and human decay-accelerating factor.
Authors: Fasel N , Rousseaux M , Schaerer E , Medof M E , Tykocinski M L , Bron C
Published: May 2006

Glycosyl-inositolphospholipid (GPL) anchoring structures are incorporated into GPL-anchored proteins immediately posttranslationally in the rough endoplasmic reticulum, but the biochemical and cellular constituents involved in this "glypiation" process are unknown. To establish whether glypiation could be achieved in vitro, mRNAs generated by transcription of cDNAs encoding two GPL-anchored proteins, murine Thy-1 antigen and human decay-accelerating factor (DAF), and a conventionally anchored control protein, polymeric-immunoglobulin receptor (IgR), were translated in a rabbit reticulocyte lysate. Upon addition of dog pancreatic rough microsomes, nascent polypeptides generated from the three mRNAs translocated into vesicles. Dispersal of the vesicles with Triton X-114 detergent and incubation of the hydrophobic phase with phosphatidylinositol-specific phospholipases C and D, enzymes specific for GPL-anchor structures, released Thy-1 and DAF but not IgR protein into the aqueous phase. The selective incorporation of phospholipase-sensitive anchoring moieties into Thy-1 and DAF but not IgR translation products during in vitro translocation indicates that rough microsomes are able to support and regulate glypiation.
Three cytoplasmic loops of rhodopsin interact with transducin.
Authors: König B , Arendt A , McDowell J H , Kahlert M , Hargrave P A , Hofmann K P
Published: May 2006

Rhodopsin is a member of an ancient class of receptors that transduce signals through their interaction with guanine nucleotide-binding proteins (G proteins). We have mapped the sites of interaction of rhodopsin with its G protein, which by analogy suggests how other members of this class of receptors may interact with their G proteins. Three regions of rhodopsin's cytoplasmic surface interact with the rod cell G protein transducin (Gt). These are (i) the second cytoplasmic loop, which connects rhodopsin helices III and IV, (ii) the third cytoplasmic loop, which connects rhodopsin helices V and VI, and (iii) a putative fourth cytoplasmic loop formed by amino acids 310-321, as the carboxyl-terminal sequence emerges from helix VII and anchors to the lipid bilayer via palmitoylcysteines 322 and 323. Evidence for these regions of interaction of rhodopsin and Gt comes from the ability of synthetic peptides comprising these regions to compete with metarhodopsin II for binding to Gt. A spectroscopic assay that measures the "extra MII" caused by Gt binding was used to measure the extent of binding of Gt in the presence of competing peptides. The three peptides corresponding to the second, third, and fourth cytoplasmic loops competed effectively with metarhodopsin II, exhibiting Kd values in the 2 microM range; 11 additional peptides comprising all remaining surface regions of rhodopsin failed to compete even at 200 microM. Any two peptides that were effective competitors showed a synergistic effect, having 15 times higher effectiveness when mixed than when assayed separately. A mathematical model was developed to describe this behavior.
Host-independent evolution and a genetic classification of the hepadnavirus family based on nucleotide sequences.
Authors: Orito E , Mizokami M , Ina Y , Moriyama E N , Kameshima N , Yamamoto M , Gojobori T
Published: May 2006

An analysis of molecular phylogeny was undertaken to examine whether the evolution of the hepadnavirus family is host-dependent. Using the nucleotide sequences of 18 strains, we constructed phylogenetic trees. The trees obtained show that all 12 strains of hepatitis B virus can be classified into four subgroups that are not compatible with conventional subtypes. We estimated the rate of synonymous (silent) substitution for hepatitis B virus to be 4.57 x 10(-5) per site per year. Applying this rate to the phylogenetic tree, we estimated that duck hepatitis B virus diverged from a common ancestor about 30,000 years ago at the earliest, that woodchuck hepatitis virus and ground squirrel hepatitis virus diverged about 10,000 years ago, and that hepatitis B virus diverged within the last 3000 years. Because these divergence times of the viruses are much more recent than those of the host species, it suggests that the hepadnavirus family evolved independently of host-species divergence.
Organization of the gene encoding common acute lymphoblastic leukemia antigen (neutral endopeptidase 24.11): multiple miniexons and separate 5' untranslated regions.
Authors: D'Adamio L , Shipp M A , Masteller E L , Reinherz E L
Published: May 2006

The common acute lymphoblastic leukemia antigen (CALLA) is a 749-amino acid type II integral membrane protein that has been identified recently as the neutral endopeptidase 24.11 [NEP (EC 3.4.24.11)]. Herein, we characterize the organization of the human CALLA/NEP gene and show that it spans more than 80 kilobases (kb) and is composed of 24 exons. Exons 1 and 2 encode 5' untranslated sequences; exon 3 [170 base pairs (bp)] encodes the initiation codon and transmembrane and cytoplasmic domain; 20 short exons (exons 4-23), ranging in size from 36 to 162 bp, encode most of the extracellular portion of the enzyme; and exon 24 (approximately 3400 bp) encodes the COOH-terminal 32 amino acids of the protein and contains the entire 3' untranslated region (UTR). Of note, the pentapeptide sequence (His-Glu-Ile-Thr-His) associated with metalloprotease zinc binding and substrate catalysis is encoded within a single exon (exon 19). Three types of CALLA/NEP cDNAs have been identified: these clones contain 5' UTR sequences differing from one another upstream of exon 3. These human 5' sequences are homologous to those found in rat brain and rabbit kidney NEP cDNAs. The three human CALLA cDNA types result from alternative splicing of exons 1, 2a, or 2b to the common exon 3. Moreover, exons 2a and 2b share the same 5' sequence but differ from each other by the use of two distinct donor splice sites 171 bp apart in the gene. The substantial conservation of 5' untranslated sequences among species and the existence of 5' alternative splicing suggest that CALLA gene expression may be differentially controlled in a tissue-specific and/or developmentally regulated fashion.
Binding site for IgE of the human lymphocyte low-affinity Fc epsilon receptor (Fc epsilon RII/CD23) is confined to the domain homologous with animal lectins.
Authors: Bettler B , Maier R , Rüegg D , Hofstetter H
Published: May 2006

The lymphocyte low-affinity receptor for IgE (Fc epsilon RII) is involved in two seemingly unrelated processes: (i) promotion of general B-cell growth and (ii) isotype-specific IgE synthesis. To characterize domains of Fc epsilon RII important for effector function, we have expressed Fc epsilon RII mutants in mammalian cells. The results show that the IgE-binding region of Fc epsilon RII corresponds almost exactly to a domain of 123 amino acid residues homologous with the carbohydrate-binding domain of C-type animal lectins. With the recent demonstration that Fc epsilon RII binds to IgE independently of any lectin-like activity [Vercelli, D., Helm, B., Marsh, P., Padlan, E., Geha, R.S. & Gould, H. (1989) Nature (London) 338, 649-651], it is now clear that, in this case, the lectin module has evolved to interact with a protein rather than a carbohydrate moiety. The epitopes of several independent monoclonal antibodies that inhibit the binding of IgE to Fc epsilon RII are clustered within the lectin-like domain. Some of these antibodies are also known to suppress, isotype-specifically, the interleukin 4-promoted IgE synthesis from peripheral blood mononuclear cells or the spontaneous synthesis of IgE by B cells isolated from atopic donors. The epitope of MHM6, an anti-F epsilon RII monoclonal antibody delivering an epitope-restricted growth-promoting effect on B cells, is also located within the lectin-like domain. Thus, the lectin module of Fc epsilon RII not only acts as a carbohydrate-independent, isotype-specific Fc receptor but may also participate in the general regulation of B-cell growth.
T-type calcium channels mediate the transition between tonic and phasic firing in thalamic neurons.
Authors: Suzuki S , Rogawski M A
Published: May 2006

Thalamic neurons undergo a shift from tonic to phasic (burst) firing upon hyperpolarization. This state transition results from deinactivation of a regenerative depolarizing event referred to as the low-threshold spike. Isolated adult guinea pig thalamic (dorsal lateral geniculate) neurons exhibited low-threshold spikes that could be blocked by low concentrations of nickel but were unaffected by the dihydropyridine nimodipine. Whole-cell voltage-clamp recordings from these cells demonstrated a low-threshold, rapidly inactivating (T) Ca2+ current that manifested similar voltage dependency and time course as the low-threshold spike. Like low-threshold spikes, the T-type Ca2+ current was eliminated by nickel but was unaffected by nimodipine. In thalamic neurons, T-type Ca2+ channels underlie the low-threshold spike and, therefore, play a critical role in regulating the firing pattern of these cells.
Multiplier ideal sheaves and existence of Kähler-Einstein metrics of positive scalar curvature
Authors: Nadel Alan Michael
Published: May 2006

To study
C
0
a priori
estimates for solutions to certain complex Monge—Ampère equations, I introduce a coherent sheaf of ideals and show that it satisfies various global algebrogeometric conditions, including a cohomology vanishing theorem. This technique is used to establish the existence of Kähler-Einstein metrics of positive scalar curvature on a very large class of compact complex manifolds.
In vitro tyrosine phosphorylation studies on RAS proteins and calmodulin suggest that polylysine-like basic peptides or domains may be involved in interactions between insulin receptor kinase and its substrate.
Authors: Fujita-Yamaguchi Y , Kathuria S , Xu Q Y , McDonald J M , Nakano H , Kamata T
Published: May 2006

We have investigated the in vitro tyrosine phosphorylation of the HRAS and KRAS proteins by human placental insulin receptor kinase. Purified HRAS proteins are not phosphorylated by purified insulin receptor kinase. Since the tyrosine phosphorylation of calmodulin by the insulin receptor kinase in vitro requires cofactors such as protamine and poly(L-lysine), we examined the possibility that poly(L-lysine) may also potentiate the interaction between RAS proteins and the insulin receptor. We found that purified HRAS proteins are indeed phosphorylated by purified insulin receptor kinase in the presence of poly(L-lysine). In contrast, the KRAS protein, which carries an extremely basic domain (residues 172-182, Lys-Asp-Glu-Lys6-Ser-Arg), is phosphorylated by the receptor kinase without the addition of basic proteins. We then determined whether the KRAS basic domain peptide plays a role similar to that of poly(L-lysine) and found that both the HRAS protein and calmodulin are phosphorylated by the receptor kinase in the presence of the KRAS basic domain peptide. Further examination of the role of poly(L-lysine) in potentiating tyrosine phosphorylation of the HRAS protein and calmodulin by purified insulin receptor kinase indicates that poly(L-lysine) affects the conformation of these protein substrates as well as that of the receptor kinase domain. These studies suggest that polylysine-like basic proteins or domains are required to establish the interaction between insulin receptor kinase and its substrate.
Differential binding of nuclear factors to the intron 1 sequences containing the transcriptional pause site correlates with c-myb expression.
Authors: Reddy C D , Reddy E P
Published: May 2006

The molecular mechanisms that modulate c-myb mRNA levels in hematopoietic cells appear to involve premature termination of transcription in the first intron of the gene. We have examined the DNA-protein interactions within the first intron of the c-myb gene and identified a 1.0-kilobase region that could be responsible for its transcriptional regulation. Using the mobility-shift assay, we show a direct correlation between the extent of sequence-specific protein binding to intron 1 DNA fragments, and c-myb mRNA levels in different cell types. During dimethyl sulfoxide-induced differentiation of mouse erythroleukemic cells, there was a dramatic decrease in these nuclear factors that correlated with the decrease in the levels of c-myb mRNA. Nucleotide sequence analysis and DNase I footprinting revealed the presence of putative regulatory elements that are implicated in the binding of these nuclear factors. We propose that binding of nuclear factors to the site of transcriptional pause could play an important role in the regulation of c-myb transcription.
Prolyl 4-hydroxylase: molecular cloning and the primary structure of the alpha subunit from chicken embryo.
Authors: Bassuk J A , Kao W W , Herzer P , Kedersha N L , Seyer J , DeMartino J A , Daugherty B L , Mark G E , Berg R A
Published: May 2006

Prolyl 4-hydroxylase (EC 1.14.11.2) is a key enzyme required for the posttranslational hydroxylation of proline residues in collagen. The enzyme is a tetramer composed of two pairs of nonidentical subunits (alpha 2 beta 2). The beta subunit is protein disulfide-isomerase, a ubiquitous enzyme found in the endoplasmic reticulum of many cell types. We report here the amino acid sequence of the alpha subunit. One cDNA clone (alpha 1) was isolated from a chicken embryo cDNA expression library in lambda gt11 by screening with anti-alpha-subunit polyclonal immunoglobulins. This alpha 1 cDNA contains an open reading frame of 1401 base pairs. A comparison of the translation of the nucleotide sequence with protein sequences obtained from the purified chicken alpha-subunit polypeptide verified that alpha 1 cDNA encoded the alpha subunit. Polymerase chain reactions were used to extend the sequence of alpha 1 cDNA toward the 5' end of alpha-subunit mRNA. The mature alpha subunit is composed of 516 amino acids with a calculated molecular mass of 59,373 Da. The compiled amino acid sequence contains two potential glycosylation sites, an observation that agrees with a previous demonstration that the alpha subunit contains two N-linked oligosaccharide chains. Blot hybridization analysis of total chicken embryo RNA detected an mRNA of 3.5 kilobases, a size that closely resembles the size of the cloned cDNA. Since the expression of the alpha subunit is confined to cell types that synthesize and secrete collagens, the regulation of the synthesis of the alpha subunit may play a central role in determining the expression of prolyl 4-hydroxylase activity.