bluuebunny/crossref_metadata_2025 huggingface dataset:
Transcription of oxygen-regulated photosynthetic genes requires DNA gyrase in Rhodobacter capsulatus.
Authors: Zhu Y S , Hearst J E
Published: May 2006

The regulation of the photosynthetic genes by DNA supercoiling in Rhodobacter capsulatus has been studied by using gyrase inhibitors in vivo and by measurement of mRNA levels of more than a dozen genes. The results demonstrate that the levels of mRNA for light-harvesting (I, II) and reaction center (L, M, H) proteins, bacteriochlorophyll biosynthetic enzymes, ribulose-bisphosphate carboxylase (EC 4.1.1.39), and the mRNAs from the open reading frames Q and R decreased immediately and dramatically upon addition of novobiocin and coumermycin. In contrast, the mRNAs for carotenoid biosynthetic enzymes, the cytochrome bc1 complex, and constitutively expressed mRNA under aerobic conditions for light-harvesting I and for reaction center (L, M) proteins are less sensitive to the inhibitors. In accordance with these results, the biosynthesis of bacteriochlorophyll is markedly repressed by gyrase inhibitors novobiocin, coumermycin, nalidixic acid, and oxolinic acid either under anaerobic conditions or during a shift from aerobic to anaerobic conditions. The synthesis of light-harvesting (I, II) bacteriochlorophyll complexes is also inhibited by novobiocin and coumermycin. The kinetics of specific mRNA changes and the differential sensitivity of anaerobic and aerobic genes to the gyrase inhibitors strongly suggest that DNA supercoiling is involved in the differential expression of photosynthetic genes in response to the level of oxygen in R. capsulatus.
No strict alignment is required between a transcriptional activator binding site and the "TATA box" of a yeast gene.
Authors: Ruden D M , Ma J , Ptashne M
Published: May 2006

GAL4 is a transcriptional activator of the galactose metabolism genes in the yeast Saccharomyces cerevisiae. We show that GAL4 expressed in yeast activated transcription equally well when a single GAL4 binding site was placed at any of nine positions upstream of the GAL1 (galactokinase gene) "TATA box." We chose a sufficient number of positions for the binding site to ensure that, in several of these positions, GAL4 was on the opposite side of the DNA helix with respect to the TATA box. Smaller GAL4 derivatives were similar to wild-type GAL4 in that they also activated transcription in a manner independent of the side of the DNA helix they bound with respect to the TATA box. Unlike wild-type GAL4, however, these smaller GAL4 derivatives activated transcription better when we placed a binding site progressively closer to the TATA box over a distance of 34 base pairs.
Probing the salt bridge in the dihydrofolate reductase-methotrexate complex by using the coordinate-coupled free-energy perturbation method.
Authors: Singh U C
Published: May 2006

The importance of the ionic interaction due to the formation of the salt bridge between the Asp-27 and the pteridine ring in Escherichia coli dihydrofolate reductase-methotrexate complex has been studied by using the free-energy perturbation method. The calculation suggests that the ion-pair contribution to the binding energy is insignificant, as the enzyme surroundings do not stabilize the salt bridge to the extent of the desolvation of the charged groups. The activation barrier for the proton exchange between the pteridine ring and the Asp-27 is calculated to be 20.1 kcal/mol (1 cal = 4.184 J) by using the coordinate-coupled perturbation method, implying that this may be a channel to the proton exchange from the pteridine ring to the solvent. The Gibbs-energy difference of binding between the Asn-27 and Ser-27 is calculated to be 3.2 kcal/mol and is mainly due to the electrostatic interactions.
Recognition of galactosylgloboside by monoclonal antibodies derived from patients with primary lung cancer.
Authors: Schrump D S , Furukawa K , Yamaguchi H , Lloyd K O , Old L J
Published: May 2006

Lymph node lymphocytes from patients with primary lung cancer were immortalized with Epstein-Barr virus, and culture supernatants were screened for cell-surface reactivity against allogeneic cancer cell lines. The percentage of wells containing detectable antibodies in initial screening ranged from 1 to 17%, but the vast majority of the cultures lost antibody activity on subsequent expansion. Two antibody-secreting clones, J309 and D579, derived from separate individuals and reactive with anaplastic lung cancer cell lines, were successfully expanded and fused with the NS-1 mouse myeloma cell line. The antibodies produced by these clones exhibited identical restricted serologic reactivity against cultured cell lines and detected a carbohydrate antigen present in the neutral glycolipid fraction of MCF-7 breast cancer cells. Serologic, immunochemical, and chemical analyses revealed that the antigen recognized by antibodies J309 and D579 is galactosylgloboside [Gal(beta 1----3)GalNAc(beta 1----3)Gal(alpha 1----4)Gal(beta 1----4)- GlcCer]. Conclusions regarding the significance of these findings with respect to the biology of lung cancer await further information concerning the distribution of galactosylgloboside in normal and malignant tissues and the frequency of antibodies to this structure in normal and tumor-bearing individuals.
Increased prostacyclin and thromboxane A2 biosynthesis in atherosclerosis.
Authors: Mehta J L , Lawson D , Mehta P , Saldeen T
Published: May 2006

It has been proposed that atherosclerotic arteries produce less prostacyclin (PGI2) than nonatherosclerotic arteries do, thereby predisposing arteries to vasospasm and thrombosis in vivo. We reexamined this concept by measuring spontaneous as well as arachidonate-induced PGI2 biosynthesis in aortic segments from nonatherosclerotic and cholesterol-fed atherosclerotic New Zealand White rabbits. Thromboxane A2 (TXA2) generation was also measured. Formation of PGI2, as well as TXA2, as measured by radioimmunoassay (RIA) of their metabolites, was increased in atherosclerotic aortic segments relative to nonatherosclerotic segments (P less than or equal to 0.05) at 0, 5, 10, 15, and 30 min of incubation with arachidonate. Pretreatment of arterial segments with indomethacin inhibited PGI2 as well as TXA2 formation, whereas pretreatment with the selective TXA2 inhibitor OKY-046 inhibited only TXA2 release, thus confirming the identity of icosanoids. To confirm the RIA data, aortic segments were incubated with [14C]arachidonate prior to stimulation with unlabeled arachidonate. The uptake of arachidonate was similar, but the release of incorporated [14C]arachidonate was significantly (P less than or equal to 0.05) greater in atherosclerotic segments than in nonatherosclerotic ones. Conversions of released [14C]arachidonate to 6-keto[14C]prostaglandin F1 alpha and [14C]thromboxane B2 were similar in the two types of aortic segments. Thus, synthesis of PGI2 as well as TXA2 is increased in atherosclerosis, and this alteration in arachidonate metabolism is related to increased release of arachidonate.
Nonenzymatic sequence-specific methyl transfer to single-stranded DNA.
Authors: Iverson B L , Dervan P B
Published: May 2006

2'-Deoxyuridine 5'-triphosphate with a methylthioether moiety at the 5 position can be incorporated into a primer-template DNA complex with Klenow enzyme. Activation with CNBr at 25 degrees C, pH 5.5, followed by treatment with piperidine produces sequence-specific cleavage on the template DNA predominantly at a single guanine position. The mechanism involves methyl-group transfer from sulfur on the modified deoxyuridine of the extended primer to N-7 of guanine on the template DNA. This raises the possibility for the design and synthesis of a nonenzymatic class of sequence-specific methyltransferases for DNA.
Carcinoembryonic antigen is anchored to membranes by covalent attachment to a glycosylphosphatidylinositol moiety: identification of the ethanolamine linkage site.
Authors: Hefta S A , Hefta L J , Lee T D , Paxton R J , Shively J E
Published: May 2006

The COOH-terminal amino acid of carcinoembryonic antigen (CEA) is shown to covalently link with ethanolamine, evidence consistent with the anchorage of CEA to the plasma membrane through a phosphatidylinositol-glycan tail. Purified CEA was digested with trypsin, and the resulting peptides were isolated by reverse-phase HPLC. Tryptic hexapeptide T12, terminating atypically with alanine, corresponded in sequence (Ser-Ile-Thr-Val-Ser-Ala) with the last six residues (637-642) of the third repeating domain in the mature CEA protein. Mass determination of the hexapeptide by fast atom bombardment mass spectrometry suggested the presence of an additional ethanolamine moiety. This finding and the absence of the subsequent 26 hydrophobic residues predicted by cDNA sequence is evidence that hexapeptide T12 is the COOH-terminal peptide of mature CEA. A synthetic peptide identical to hexapeptide T12 was prepared, and ethanolamine was coupled to its COOH-terminal alanine; chromatographic properties of this synthetic ethanolamine-coupled peptide and peptide T12 were the same. B/E-linked-scan mass spectral analysis of the ethanolamine-coupled synthetic peptide and peptide T12 revealed a fragment ion series consistent with the presence of a COOH-terminal ethanolamine. Release of membrane-bound CEA from the CEA-expressing cell line LS 174T was shown by indirect immunofluorescence and flow cytometry after treatment with phosphatidylinositol-specific phospholipase C. We conclude that CEA is processed posttranslationally to remove the hydrophobic COOH-terminal residues (643-668) with subsequent addition of an ethanolamine-glycosylphosphatidylinositol moiety and that treatment of a colonic cell line with phosphatidylinositol-specific phospholipase C releases membrane-bound CEA.
Human HST1 (HSTF1) gene maps to chromosome band 11q13 and coamplifies with the INT2 gene in human cancer.
Authors: Yoshida M C , Wada M , Satoh H , Yoshida T , Sakamoto H , Miyagawa K , Yokota J , Koda T , Kakinuma M , Sugimura T
Published: May 2006

The human HST1 gene, previously designated the hst gene, and now assigned the name HSTF1 for heparin-binding secretory transforming factor in human gene nomenclature, was originally identified as a transforming gene in DNAs from human stomach cancers by transfection assay with mouse NIH 3T3 cells. The amino acid sequence of the product deduced from DNA sequences of the HST1 cDNA and genomic clones had approximately 40% homology to human basic and acidic fibroblast growth factors and mouse Int-2-encoded protein. We have mapped the human HST1 gene to chromosome 11 at band q13.3 by Southern blot hybridization analysis of a panel of human and mouse somatic cell hybrids and in situ hybridization with an HST1 cDNA probe. The HST1 gene was found to be amplified in DNAs obtained from a stomach cancer and a vulvar carcinoma cell line, A431. In all of these samples of DNA, the INT2 gene, previously mapped to human chromosome 11q13, was also amplified to the same degree as the HST1 gene.
Requirement of GTP on somatostatin-induced K+ current in human pituitary tumor cells.
Authors: Yamashita N , Shibuya N , Ogata E
Published: May 2006

The role of GTP on somatostatin-induced K+ current increase was examined in dissociated human pituitary tumor cells obtained from three acromegalic patients. Pituitary cells in culture were voltage-clamped by using the patch clamp technique in the whole-cell configuration. Somatostatin (100 nM) increased the membrane permeability to K+ ions and inhibited hormone secretion. A current-voltage relation of the somatostatin-induced K+ current showed an inward rectification when the concentration of extracellular K+ ions was increased. The amplitude of the somatostatin-induced K+ current decreased during recording when the patch pipette solution did not contain GTP; addition of 100 microM GTP to the patch pipette solution prevented this reduction. Intracellular application of 100 microM guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S] evoked an inward rectifying K+ conductance in the absence of somatostatin. After the GTP[gamma S]-induced K+ conductance reached a steady level, application of somatostatin did not further increase the K+ conductance. In pertussis toxin-treated cells GTP[gamma S] did not evoke K+ conductance. It was concluded that somatostatin-induced K+ channels were regulated by a GTP-binding protein.
Catalysis of concerted reactions by antibodies: the Claisen rearrangement.
Authors: Hilvert D , Carpenter S H , Nared K D , Auditor M T
Published: May 2006

Monoclonal antibodies were prepared against a transition state analog inhibitor of chorismate mutase (EC 5.4.99.5). One of the antibodies catalyzes the rearrangement of chorismate to prephenate with rate accelerations of more than 2 orders of magnitude compared to the uncatalyzed reaction. Saturation kinetics were observed, and at 25 degrees C the values of kcat and Km were 1.2 X 10(-3) s-1 and 5.1 X 10(-5) M respectively. The transition state analog was shown to be a competitive inhibitor of the reaction with Ki equal to 0.6 microM. These results demonstrate the feasibility of using rationally designed immunogens to generate antibodies that catalyze concerted reactions.
Lipid requirement and kinetic studies of solubilized UDP-galactose:diacylglycerol galactosyltransferase activity from spinach chloroplast envelope membranes
Authors: Covés Jacques , Joyard Jacques , Douce Roland
Published: May 2006

We have demonstrated a lipid requirement for the UDPgalactose:1,2-diacylglycerol 3-β-D-galactosyl-transferase (or monogalactosyldiacylglycerol synthase; EC 2.4.1.46), an enzyme involved in the biosynthesis of monogalactosyldiacylglycerol, solubilized from chloroplast envelope membranes and partially purified by hydroxyapatite chromatography. The enzyme fraction was highly delipidated (<0.1 mg of lipid per mg of protein), and addition of lipids extracted from chloroplast membranes was necessary to reveal the activity. Acidic glycerolipids, and especially phosphatidylglycerol, were the best activators of the enzyme. The preparation of a delipidated enzyme fraction and the development of optimal assay conditions were prerequisites for the determination of the kinetic parameters for the hydrophobic substrate of the enzyme, diacylglycerol. In addition, we have demonstrated the existence of two substrate-binding sites: a hydrophobic one for diacylglycerol and a hydrophilic one for UDP-galactose.
Iron-carbonyl bond geometries of carboxymyoglobin and carboxyhemoglobin in solution determined by picosecond time-resolved infrared spectroscopy.
Authors: Moore J N , Hansen P A , Hochstrasser R M
Published: May 2006

The iron-carbonyl geometries in carboxymyoglobin (MbCO) and carboxyhemoglobin (HbCO) in ambient temperature solution have been investigated using picosecond time-resolved infrared spectroscopy. Polarized infrared and visible beams were used to monitor the change in infrared absorbance of the bound CO stretch bands on photodissociation of the ligand. The ratio of the change in absorbance for perpendicular and parallel relative polarizations of the photolysis and infrared probe beams is directly related to the angle between the ligand bond axis and the normal to the heme plane. Ratios, and hence the angles, have been obtained for the configurations giving rise to the principal CO stretch infrared absorption bands of HbCO and MbCO: 18 degrees for the 1951 cm-1 band of HbCO; 20 degrees and 35 degrees, respectively, for the 1944 cm-1 and 1933 cm-1 bands of MbCO. Structures consistent with x-ray diffraction and the picosecond experiments reported here are proposed for MbCO and HbCO in which the Fe-C bond tilts to the heme normal and the Fe-C-O angle differs significantly from 180 degrees.
Comparative evolution: latent potentials for anagenetic advance.
Authors: Stebbins G L , Hartl D L
Published: May 2006

One of the principles that has emerged from experimental evolutionary studies of microorganisms is that polymorphic alleles or new mutations can sometimes possess a latent potential to respond to selection in different environments, although the alleles may be functionally equivalent or disfavored under typical conditions. We suggest that such responses to selection in microorganisms serve as experimental models of evolutionary advances that occur over much longer periods of time in higher organisms. We propose as a general evolutionary principle that anagenic advances often come from capitalizing on preexisting latent selection potentials in the presence of novel ecological opportunity.
Anti-L3T4 antibody inhibits the lysis of H-2 class II antigen-negative target cells by L3T4+ cytotoxic T lymphocytes.
Authors: Macphail S , Stutman O
Published: May 2006

Anti-L3T4 monoclonal antibodies inhibit the cytotoxic activity of L3T4+ cytotoxic T lymphocytes specific for H-2 class I antigens. The P815 target cells used to detect this population of murine cytolytic cells are shown by immunofluorescence, radioimmunoprecipitation, and RNA blot analysis not to express H-2 class II protein or mRNA. Contrary to previously proposed models regarding its function, we conclude that the L3T4 molecule is involved at some stage of the lytic interaction between the class I-specific L3T4+ effector cell and its target cell by a mechanism for which there is not an obligatory requirement for H-2 class II antigen expression by the target cell. L3T4 may be an early component of the system that transduces the activation signal from the T-cell receptor complex to the cytoplasm, a cell-surface receptor for a yet undefined natural ligand that delivers a negative signal to the killer T cell, or it may modulate the avidity of the antigen-specific T-cell receptor through a direct physical association with it.
Photophysical studies of tin(IV)-protoporphyrin: potential phototoxicity of a chemotherapeutic agent proposed for the prevention of neonatal jaundice.
Authors: Land E J , McDonagh A F , McGarvey D J , Truscott T G
Published: May 2006

The strongly light-absorbing metalloporphyrin tin(IV)-protoporphyrin IX (SnPP) is currently being considered as a chemotherapeutic agent for preventing severe hyperbilirubinemia in newborns, a condition usually treated by phototherapy with visible light. To assess the potential phototoxicity of SnPP we studied the photophysics of the drug in aqueous and nonaqueous solutions using laser flash photolysis and pulse radiolysis. Quantum yields for formation of triplet-state excited SnPP were measured, along with triplet lifetimes and extinction coefficients. In addition, we measured quantum yields for the SnPP-photosensitized formation of singlet oxygen in MeO2H and 2H2O containing cetyltrimethylammonium bromide, using a time-resolved luminescence technique. Quantum yields for formation of triplet SnPP from monomeric ground-state SnPP are high (approximately equal to 0.6-0.8), and triplet lifetimes are long (approximately equal to 0.1-0.2 ms). Efficient quenching of triplet SnPP by molecular oxygen was seen with rate constants greater than 10(9) M-1.s-1. SnPP-photosensitized formation of singlet oxygen in aqueous and nonaqueous solvents was confirmed by the detection of the characteristic luminescence at 1270 nm (phi delta = 0.58 in MeO2H). The photophysical parameters and singlet oxygen-sensitizing efficiency of SnPP are similar to those reported for hematoporphyrin and other metal-free porphyrins known to be phototoxic to humans. These observations suggest that cutaneous photosensitivity arising from singlet-oxygen damage is likely to be an undesirable side-effect of SnPP therapy.
L-glutamate-induced depolarization in solitary photoreceptors: a process that may contribute to the interaction between photoreceptors in situ.
Authors: Tachibana M , Kaneko A
Published: May 2006

L-Glutamate is a leading candidate for the vertebrate photoreceptor transmitter. In addition to the signal transmission to second-order neurons, photoreceptors communicate with each other not only electrically but also chemically. In the present study, by using solitary turtle photoreceptors, we examined the possibility that L-glutamate mediates interreceptor communication. L-Glutamate evoked an inward current in all subtypes of photoreceptors voltage-clamped to the resting potential. The highest glutamate sensitivity was located at the axon terminal. Both stereoisomers of aspartate were effective, whereas kainate, quisqualate, N-methyl-D-aspartate, and D-glutamate were ineffective. The presence of Na+ was essential to response generation; even Li+ could not substitute for Na+. The relation between L-glutamate-induced current and the membrane voltage was strongly inward-rectifying. These results favor the hypothesis that the L-glutamate-induced response is generated by an electrogenic uptake carrier. However, L-glutamate-induced current was always accompanied by an increase in current fluctuations, a phenomenon commonly observed in ion channels but not expected for an uptake carrier. Although the underlying mechanism needs further elucidation, it seems likely that L-glutamate is a transmitter for communication between photoreceptors.
Herpes simplex virus DNA replication: the UL9 gene encodes an origin-binding protein.
Authors: Olivo P D , Nelson N J , Challberg M D
Published: May 2006

Herpes simplex virus 1 contains seven genes that are necessary and sufficient for origin-dependent DNA synthesis in cultured cells. We have expressed the product of one of these genes, UL9, in insect cells by using a baculovirus expression vector. The apparent size of the UL9 protein, both in insect cells and in herpes simplex virus-infected Vero cells, is 82,000 Da. By using an immunoassay for protein-DNA interaction, we have shown that UL9 protein binds specifically to the herpes simplex virus origins of DNA replication, oriS and oriL. DNase I "footprint" analysis has shown that the UL9 protein interacts with two related sites on oriS, located on each arm of a nearly perfect palindrome. Our data strongly suggest that the origin-binding activity described previously by Elias et al. [Elias, P., O'Donnell, M. E., Mocarski, E. S. & Lehman, I. R. (1986) Proc. Natl. Acad. Sci. USA 83, 6322-6326] is the product of the UL9 gene.
Translation initiation controls the relative rates of expression of the bacteriophage lambda late genes.
Authors: Sampson L L , Hendrix R W , Huang W M , Casjens S R
Published: May 2006

The late operon of bacteriophage lambda contains the genes encoding the morphogenetic proteins of the phage. These genes are transcribed equally from the single late promoter. Although the functional half-lives of the mRNA for the various genes of this operon vary less than 2-fold, their relative rates of expression have been shown to vary by nearly 1000-fold. This variation could result from differing rates of translation initiation, from overlapping upstream translation, or from differential elongation rates due to the presence of codons for which the corresponding tRNAs are rare. To distinguish between these possibilities, we have cloned sequences surrounding the initiator codons of several of these genes and measured their ability to drive synthesis of hybrid lambda-beta-galactosidase proteins. The rates of expression of the hybrid genes thus produced correlate very well with the natural rates of expression of the corresponding phage genes, suggesting that the rate of initiation of translation controls the relative expression rates of these genes.
Alternative DNA loops regulate the arabinose operon in Escherichia coli.
Authors: Huo L , Martin K J , Schleif R
Published: May 2006

The araCBAD regulatory region of Escherichia coli contains two divergently oriented promoters and three sites to which AraC, the regulatory protein of the operon, can bind. This paper presents the results of in vivo dimethyl sulfate "footprinting" experiments to monitor occupancy of the three AraC sites and measurements of activity of the two promoters. These measurements were made both in the absence of the inducer arabinose and at various times after arabinose addition to growing cells containing the wild-type ara regulatory region or the regulatory region containing various deletions and point mutations. The data lead to the conclusion that two different DNA loops can form in the ara regulatory region. These loops are generated by AraC protein molecules binding to two different DNA sites and binding to each other. One of these loops predominates in the absence of arabinose and plays a major role in repressing activity of one of the promoters. Upon the addition of arabinose the amount of the first loop type, the repression loop, decreases and the amount of a second loop increases. Formation of this second loop precludes the counterproductive formation of the repression loop.
Relationship between endo- and exopeptidases in a processing enzyme system: activation of an endoprotease by the aminopeptidase B-like activity in somatostatin-28 convertase.
Authors: Gomez S , Gluschankof P , Lepage A , Cohen P
Published: May 2006

The somatostatin-28 convertase activity involved in vitro in the processing of somatostatin-28 into the neuropeptides somatostatin-28-(1-12) and somatostatin-14 is composed of an endoprotease and a basic aminopeptidase. We report herein on the purification to apparent homogeneity of these two constituents and on their functional interrelationship. In particular we observed that after various physicochemical treatments, the 90-kDa endoprotease activity was recovered both at this molecular mass and as a 45-kDa entity. Moreover, the production of [Arg-2,Lys-1]somatostatin-14 from somatostatin-28 by the action of the endoprotease was activated in a cooperative manner by the aminopeptidase B-like enzyme. A 10-fold activation occurred when the exopeptidase was inhibited by 6.5 mM diisopropyl fluorophosphate and allowed the determination of a half-maximal activation constant (K1/2) of approximately equal to 13 nM. These observations strongly suggest that both enzymes act in a concerted manner in vitro and that they may form a complex in vivo.
Antibodies in human serum that precipitate ribonuclease P.
Authors: Gold H A , Craft J , Hardin J A , Bartkiewicz M , Altman S
Published: May 2006

Sera from certain patients with systemic lupus erythematosus (SLE) and related rheumatic diseases contain antibodies that selectively deplete extracts of HeLa cells of RNase P activity. Most of the sera that recognize RNase P, an endoribonuclease with an essential RNA subunit, also contain antibodies against another small ribonucleoprotein known as the Th antigen. A species of RNA about 400 nucleotides in length is the only RNA species found in common in all immunoprecipitates prepared with anti-RNase P antibodies. The discovery of antibodies against RNase P defines a major class of antibodies produced by patients with autoimmune disease.
cDNA sequence encoding the 16-kDa proteolipid of chromaffin granules implies gene duplication in the evolution of H+-ATPases.
Authors: Mandel M , Moriyama Y , Hulmes J D , Pan Y C , Nelson H , Nelson N
Published: May 2006

Vacuolar H+-ATPases function in generating protonmotive force across the membranes of organelles connected with the vacuolar system of eukaryotic cells. This family of H+-ATPases is distinct from the two other families of H+-ATPases, the plasma membrane-type and the eubacterial-type. One of the subunits of the vacuolar H+-ATPase binds N,N'-dicyclohexylcarbodiimide (DCCD) and has been implicated in the proton-conducting activity of these enzymes. We have cloned and sequenced the gene encoding the DCCD-binding protein (proteolipid) of the H+-ATPase of bovine chromaffin granules. The gene encodes a highly hydrophobic protein of 15,849 Da. Hydropathy plots revealed four transmembrane segments, one of which contains a glutamic residue that is the likely candidate for the DCCD binding site. Sequence homology with the vacuolar proteolipid and with the proteolipids of eubacterial-type H+-ATPases was detected. The proteolipids from Escherichia coli, spinach chloroplasts, and yeast mitochondria matched better to the NH2-terminal part of the vacuolar protein. The proteolipids of bovine mitochondria and Neurospora mitochondria matched better to the COOH-terminal end of the vacuolar proteolipid. These findings suggest that the proteolipids of the vacuolar H+-ATPases were evolved in parallel with the eubacterial proteolipid, from a common ancestral gene that underwent gene duplication.
Agrobacterium tumefaciens
-mediated transformation of
Arabidopsis thaliana
root explants by using kanamycin selection
Authors: Valvekens Dirk , Montagu Marc Van , Lijsebettens Mieke Van
Published: May 2006

Culture conditions were developed that induce
Arabidopsis thaliana
(L.) Heynh. root cuttings to regenerate shoots rapidly and at 100% efficiency. The shoots produce viable seeds
in vitro
or after rooting in soil. A transformation procedure for
Arabidopsis
root explants based on kanamycin selection was established. By using this regeneration procedure and an
Agrobacterium
tumor-inducing Ti plasmid carrying a chimeric neomycin phosphotransferase II gene (
neo
), transformed seed-producing plants were obtained with an efficiency between 20% and 80% within 3 months after gene transfer. F
1
seedlings of these transformants showed Mendelian segregation of the kanamycin-resistance trait. The transformation method could be applied to three different
Arabidopsis
ecotypes. In addition to the
neo
gene, a chimeric
bar
gene conferring resistance to the herbicide Basta was introduced into
Arabidopsis
. The expression of the
bar
gene was shown by enzymatic assay.
Properties of a copper-containing cytochrome ba3: a second terminal oxidase from the extreme thermophile Thermus thermophilus.
Authors: Zimmermann B H , Nitsche C I , Fee J A , Rusnak F , Münck E
Published: May 2006

We describe an alternate terminal oxidase found in the plasma membrane of Thermus thermophilus and designate it cytochrome ba3. The enzyme consists of a single approximately equal to 35-kDa polypeptide that binds one heme B molecule, one heme A molecule, and two Cu ions. Optical spectra suggest the presence of cytochrome b, cytochrome a3, and CuA in this protein. Quantitative EPR and Mössbauer studies of the oxidized protein indicate the presence of one low-spin ferric heme, which is assigned to cytochrome b. Mössbauer studies of the reduced protein show the presence of one low-spin ferrous heme, assigned to cytochrome b, and a predominant high-spin ferrous heme that reacts quantitatively with CO to yield an additional low-spin ferrous heme. The latter Fe atom is associated with the heme A and is designated cytochrome a3. The EPR spectrum of the oxidized protein also reveals the presence of a CuA-type center that accounts for half the total Cu. The remainder of the Cu would appear to be present as CuB that is magnetically coupled to the heme A. Amino acid analyses of cytochrome ba3 show the presence of eight to nine histidine residues and one cysteine residue.
X gene-related sequences in the core gene of duck and heron hepatitis B viruses.
Authors: Feitelson M A , Miller R H
Published: May 2006

The genomes of all known mammalian hepadnaviruses contain an open reading frame (ORF), designated X, located just upstream from the gene encoding the major viral nucleocapsid polypeptide. This gene is believed to have one or more roles central to the life cycle of these viruses. Consequently, it is surprising that avian hepadnaviruses appear to lack this ORF. However, the observation that the size and position of the core gene in the duck hepatitis B virus (DHBV) genome and the heron hepatitis B virus (HHBV) genome is comparable to the combined X and core genes of the mammalian hepadnaviruses suggests that X function(s) may be performed by the major nucleocapsid polypeptide of DHBV and HHBV. Computer-assisted analyses were carried out to test the hypothesis that the primary and secondary structural characteristics of the X gene product are also present in the major core gene product of the duck (DHBcAg) and heron (HHBcAg) viruses. Primary sequence comparison of the major core-associated polypeptides encoded by the avian and mammalian hepadnaviruses demonstrates considerable homology at both the amino- and carboxyl-terminal regions of these components. However, the middle portion of the DHBcAg and HHBcAg polypeptide, spanning about half the molecule, is unique. Comparison of this region with the carboxyl-terminal half of the X gene sequences from mammalian hepadnaviruses demonstrates similarities in both primary sequence and secondary structural characteristics. These results suggest that X-like gene product sequences are present in the core gene products of DHBV and HHBV. In addition, a sequence of about two dozen residues at the amino terminus of the mammalian X gene product, overlapping the polymerase gene product, is found in the corresponding position in DHBV. This is consistent with the conclusion that the relationship between the DHBV and HHBV core genes compared to the X and core genes of the mammalian hepadnaviruses may be explained by one or more translocations in the this region of the viral genome. The previous finding of X antigen determinants associated with one or more core-related polypeptides in the mammalian hepadnaviruses, combined with the results of this study, suggests that X gene product function is conserved among these viruses.
Differentiation of bipotential glial precursors into oligodendrocytes is promoted by interaction with type-1 astrocytes in cerebellar cultures.
Authors: Aloisi F , Agresti C , D'Urso D , Levi G
Published: May 2006

The differentiation of bipotential precursors of oligodendrocytes (OL) and type-2 astrocytes (AS) was followed in primary cultures from 8-day postnatal rat cerebellum by labeling the cells with the antibodies LB1 (which binds to the surface disialoganglioside GD3 present in glial precursors, type-2 AS, and immature OL), O4 (a marker of immature and mature OL binding to surface sulfatide), anti-galactocerebroside (GalCer, a marker of OL), and anti-glial fibrillary acidic protein (GFAP, a marker of AS). Two hours after plating, hardly any LB1+, GFAP+ cells were detectable, 40% of the O4+ cells were GalCer+, and none of the O4+ cells were GFAP+. Upon culturing cells plated at a density of 1 x 10(5) cells per cm2 in the presence of fetal calf serum, most of the LB1+ precursors differentiated into type-2 AS, even if most of them had already expressed the O4 antigen. Thus, in culture, most type-2 AS seem to derive from progenitor cells that were differentiating in vivo into OL. In higher density cultures (2.5 x 10(5) cells per cm2), however, many precursors differentiated into GalCer+ OL, rather than into AS. As a possible source of the signals responsible for the behavior of the glial precursors in high-density cultures, we focused our attention on type-1 AS, the most abundant cell type in the cultures. We found that, in low-density cultures maintained for 5-7 days in a medium conditioned by type-1 AS, the proliferation of the precursors was enhanced and their differentiation into OL or AS was prevented. In contrast, when cerebellar cells were coplated with type-1 AS dissociated from purified cultures, not only did the precursors proliferate more than in control cultures, but also a larger proportion of them differentiated into GalCer+ OL. In conclusion, type-1 AS appear to facilitate the differentiation of bipotential glial precursors into OL through direct cell-cell interactions. The influence of type-1 AS on the differentiation of the LB1+ and O4+ precursors is supported also by experiments with glial cortical cultures.
Early blindness results in a degraded auditory map of space in the optic tectum of the barn owl.
Authors: Knudsen E I
Published: May 2006

The optic tectum of the barn owl (Tyto alba) contains a neural map of auditory space consisting of neurons that are sharply tuned for sound source location and organized precisely according to their spatial tuning. The importance of vision for the development of this auditory map was investigated by comparing space maps measured in normal owls with those measured in owls raised with both eyelids sutured closed. The results demonstrate that owls raised without sight, but with normal hearing, develop auditory space maps with degraded precision and with aspects of topography that are abnormal.
Mouse lysozyme M gene: isolation, characterization, and expression studies.
Authors: Cross M , Mangelsdorf I , Wedel A , Renkawitz R
Published: May 2006

We have isolated and characterized both cDNA and genomic DNA of the mouse lysozyme M gene. Derivation of the amino acid sequence from the nucleotide sequences revealed six positions in the carboxyl terminus that differ from partial sequences previously published. The differential detection of specific mRNAs from the closely related lysozyme M and P genes has revealed different but overlapping tissue specificities of expression. The M gene is expressed weakly in myeloblasts, moderately in immature macrophages, and strongly in both mature macrophages and macrophage-rich tissues, while high levels of P transcripts are present only in small intestine. Sites of protein accumulation, rather than gene expression, have been identified by comparative quantitation of mRNA and enzyme levels.
Purification and partial characterization of a receptor protein for mouse interferon gamma.
Authors: Basu M , Pace J L , Pinson D M , Hayes M P , Trotta P P , Russell S W
Published: May 2006

A receptor protein for mouse interferon gamma has been purified from solubilized plasma membranes of the mouse monomyelocytic cell line WEHI-3. Sequential wheat germ agglutinin and ligand affinity chromatography of membranes extracted with octyl beta-D-glucopyranoside resulted in at least a 680-fold purification of the receptor, as measured by precipitating it in association with liposomes composed of phosphatidylcholine. The purified receptor bound 125I-labeled recombinant mouse interferon gamma (rMuIFN-gamma) with a Kd of 10 nM, a value comparable to that obtained with isolated membranes (3.5 nM). PAGE analysis of radiolabeled (with either 35S or 125I) receptor preparations consistently revealed a major band of 95 kDa. This species was degraded with time to smaller fragments, principally one of 60 +/- 5 kDa. Treatment with peptide:N-glycosidase F reduced the apparent molecular masses of the proteins in the 95- and 60-kDa regions by 15-20 kDa each. GR-20, a monoclonal antibody against the receptor, completely inhibited specific binding of 125I-labeled rMuIFN-gamma to WEHI-3 cells, blocked the induction of priming by rMuIFN-gamma of macrophage-mediated tumor cell killing, removed binding activity for 125I-labeled rMuIFN-gamma from solubilized membranes, and immunoprecipitated a single 95-kDa protein from the extract of surface labeled (125I) WEHI-3 cells. Cross-linking of 125I-labeled rMuIFN-gamma to its receptor yielded a complex of 125 +/- 5 kDa, consistent with the binding of the dimeric form of mouse interferon gamma (32 kDa) to a membrane protein of 95 kDa. These data suggest that the receptor for mouse interferon gamma (or a ligand-binding subunit thereof) is a glycoprotein of 95 kDa.
Expression of a human placental alkaline phosphatase gene in transfected cells: use as a reporter for studies of gene expression.
Authors: Henthorn P , Zervos P , Raducha M , Harris H , Kadesch T
Published: May 2006

The human placental alkaline phosphatase gene has been cloned and reintroduced into mammalian cells. When a plasmid carrying the gene under control of the simian virus 40 early promoter (pSV2Apap) is transfected into a variety of different cell types, placental alkaline phosphatase activity can readily be detected by using whole cell suspensions or cell lysates. Alkaline phosphatase activity can also be visualized directly in individual transfected cells by histochemical staining. The gene is appropriate for use as a reporter in studies of gene regulation since its expression is dependent on the presence of exogenous transcription control elements. The overall assay to detect the expression of the gene is quantitative, very rapid, and inexpensive. Cotransfections of cells with pSV2Apap and a related plasmid carrying the bacterial chloramphenicol acetyltransferase gene (pSV2Acat) indicate that transcription of these two genes is detected with roughly the same sensitivity.
Interaction of a gibberellin-induced factor with the upstream region of an alpha-amylase gene in rice aleurone tissue.
Authors: Ou-Lee T M , Turgeon R , Wu R
Published: May 2006

The interaction between the DNA sequences of an alpha-amylase (EC 3.2.1.1) gene and a tissue-specific factor induced in rice (Oryza sativa L.) aleurone tissue by gibberellin was studied. DNA mobility-shift during electrophoresis indicated that a 500-base-pair sequence (HS500) of a rice alpha-amylase genomic clone (OSamy-a) specifically interacted with a factor from gibberellin-induced rice aleurone tissue. The amount of complex formed between the HS500 DNA fragment and the gibberellin-induced factor increased in proportion to increasing amounts of aleurone extract and decreased with the addition of unlabeled HS500 DNA fragment. A specific segment of the HS500 fragment was protected from exonuclease III digestion by protein(s) in the aleurone extract, revealing the approximate position of the protein-DNA interaction. In the protected region, there is a direct repeat that overlaps with a potential stem-loop structure. Protein in extracts from leaves, roots, or deembryonated seeds incubated without gibberellin A3 did not bind to the HS500 DNA fragment. However, the binding factor was induced in deembryonated seeds when exogenous gibberellin A3 was added during seed imbibition. This suggests that activation of alpha-amylase synthesis by gibberellin may be mediated by the formation of a factor that interacts with a specific sequence of the alpha-amylase gene.
Tn5tac1, a derivative of transposon Tn5 that generates conditional mutations.
Authors: Chow W Y , Berg D E
Published: May 2006

Conditional lethal mutations are valuable for analyzing essential genes. We describe here a derivative of the bacterial transposon Tn5 called Tn5tac1 and its use in an innovative strategy for making mutations with conditional phenotypes. The 4.6-kilobase Tn5tac1 element contains a strong, regulatable, outward-facing promoter (Ptac) near one end and is polar on the expression of distal genes when the inducer of Ptac [isopropyl beta-D-thiogalactoside (IPTG)] is absent. Our results show that two unusual conditional mutant phenotypes can result from Tn5tac1 insertion in Escherichia coli: one is corrected by IPTG while the other is induced by IPTG. The broad host range of Tn5 and the conditional nature of these mutant phenotypes makes Tn5tac1 well suited for identifying essential genes in diverse bacterial species.
Ligand-induced desensitization of B-cell membrane immunoglobulin-mediated Ca2+ mobilization and protein kinase C translocation.
Authors: Cambier J , Chen Z Z , Pasternak J , Ransom J , Sandoval V , Pickles H
Published: May 2006

Binding of ligand to B-cell membrane immunoglobulin (mIg) can lead to activation of a number of distinct biologic responses, including altered expression of genes encoding c-fos, c-myc, and Ia, as well as proliferation and immunologic tolerance. Tolerance could reflect a functional uncoupling of receptors from systems that generate intracellular second messengers (i.e., receptor desensitization). To better understand the molecular basis of immune regulation, we examined the ability of mIg to function as a signal transducer after the cell's initial contact with mIg-binding ligand. The results show that ligand binding to as little as 2-10% of mIgM or mIgD renders the cell unresponsive to ligand binding to the reciprocal isotype as judged by Ca2+ mobilization and protein kinase C translocation responses. This heterologous receptor desensitization lasts longer than 24 hr and does not reflect loss of receptor from the cell surface. Studies with the calcium ionophore ionomycin, 1,2-dioctanoyl-sn-glycerol, and the protein kinase inhibitor staurosporine indicate that both protein kinase C-dependent and protein kinase C-independent (staurosporine-insensitive) mechanisms mediate heterologous desensitization after mIg crosslinking.
In vitro assay for protein-protein interaction: carboxyl-terminal 40 residues of simian virus 40 structural protein VP3 contain a determinant for interaction with VP1.
Authors: Gharakhanian E , Takahashi J , Clever J , Kasamatsu H
Published: May 2006

Intermolecular interactions between polypeptide chains play essential roles in the functioning of proteins. We describe here an in vitro assay system for identifying and characterizing such interactions. Such interactions are difficult to study in vivo. We have translated synthetic, nonmethyl-capped RNAs in a cell-free protein-synthesizing system. The translation products were allowed to interact posttranslationally to form protein-protein complexes. The chemical nature of the protein interaction(s) was determined by coimmunoprecipitation of associating proteins, sedimentation through sucrose gradients, followed by NaDodSO4/polyacrylamide gel electrophoresis or by nonreducing NaDodSO4/polyacrylamide gel electrophoresis. The system has been utilized to show the self-assembly of monomeric VP1, the major structural protein of simian virus 40, into disulfide-linked pentamers and to show the noncovalent interaction of another structural protein, VP3, with VP1 at low monomer concentrations. Additionally, we show that the carboxyl-terminal 40 amino acids of VP3 are essential and sufficient for its interaction with VP1 in vitro. The in vitro assay system described here provides a method for identifying the domains involved in, and the molecular nature of, protein-protein interactions, which play an important role in such biological phenomena as replication, transcription, translation, transport, ligand binding, and assembly.
Nerve growth factor induces protein-tyrosine phosphorylation.
Authors: Maher P A
Published: May 2006

When the sympathetic nerve-like cell line PC12 is exposed to nerve growth factor (NGF), there is a rapid and transient phosphorylation of tyrosine residues in cellular proteins, as demonstrated by immunoblotting of cell extracts with high-affinity polyclonal antibodies specific for phosphotyrosine residues. Epidermal growth factor (EGF), which does not cause the morphological differentiation of PC12 cells that is produced by NGF, also induces protein-tyrosine phosphorylation. The methyltransferase inhibitor, 5'-methylthioadenosine, which is known to block the NGF-mediated morphological differentiation of PC12 cells, also inhibits the induction of protein-tyrosine phosphorylation by NGF. 5'-Methylthioadenosine has no effect, however, on EGF-stimulated phosphorylation of tyrosine residues in cellular proteins. In addition, low temperature markedly slows the rate of protein-tyrosine phosphorylation stimulated by NGF, but it has no effect on the time course of protein-tyrosine phosphorylation induced by EGF. These data suggest that NGF and EGF induce protein-tyrosine phosphorylation in PC12 cells by different mechanisms.