bluuebunny/crossref_metadata_2025 huggingface dataset:
An auxin-binding protein is localized to the plasma membrane of maize coleoptile cells: identification by photoaffinity labeling and purification of a 23-kda polypeptide.
Authors: Feldwisch J , Zettl R , Hesse F , Schell J , Palme K
Published: May 2006

Plasma membrane vesicles were isolated from maize (Zea mays L.) coleoptile tissue by aqueous two-phase partitioning and assayed for homogeneity by the use of membrane-specific enzymatic assays. Using 5-azido-[7-3H]indole-3-acetic acid ([3H]N3IAA), we identified several IAA-binding proteins with molecular masses of 60 kDa (pm60), 58 kDa (pm58), and 23 kDa (pm23). Using Triton X-114, we were able to selectively extract pm23 from the plasma membrane. We show that auxins and functional analogues compete with [3H]N3IAA for binding to pm23. We found that PAB130, a polyclonal antibody raised against auxin-binding protein 1 (ABP-1), recognized ABP-1 as well as pm23. This suggests that pm23 shares common epitopes with ABP-1. In addition, we identified an auxin-binding protein with a molecular mass of 24 kDa (pm24), which was detected in microsomal but not in plasma membrane vesicle preparations. Like pm23 this protein was extracted from membrane vesicles with Triton X-114. We designed a purification scheme allowing simultaneous purification of pm23 and pm24. Homogeneous pm23 and pm24 were obtained from coleoptile extracts after 7000-fold purification.
Synaptic localization of kappa opioid receptors in guinea pig neostriatum.
Authors: Jomary C , Gairin J E , Beaudet A
Published: May 2006

Distribution of kappa opioid receptors was examined by EM radioautography in sections of guinea pig neostriatum with the selective 125I-labeled dynorphin analog [D-Pro10]dynorphin-(1-11). Most specifically labeled binding sites were found by probability circle analysis to be associated with neuronal membrane appositions. Because of limitations in resolution of the method, the radioactive sources could not be ascribed directly to either one of the apposed plasma membranes. Nevertheless, three lines of evidence favored a predominant association of ligand with dendrites of intrinsic striatal neurons: (i) the high frequency with which labeled interfaces implicated a dendrite, (ii) the enrichment of dendro-dendritic interfaces, and (iii) the occurrence of dendritic profiles labeled at several contact points along their plasma membranes. A small proportion of labeled sites was associated with axo-axonic interfaces, which may subserve the kappa opioid-induced regulation of presynaptic dopamine and acetylcholine release documented in guinea pig neostriatum. Although most membrane-associated kappa sites were found at extrasynaptic locations, approximately 23% were associated with synaptic specializations. This proportion is markedly higher than that previously reported for either mu or delta sites in rat neostriatum. Whether labeled synapses represent preferential sites of action for kappa ligands remains to be established. In any event, these results support the hypothesis that in mammalian brain kappa opioid receptors are conformationally and functionally distinct from mu and delta types.
Methotrexate induces differentiation of human keratinocytes.
Authors: Schwartz P M , Barnett S K , Atillasoy E S , Milstone L M
Published: May 2006

Terminal differentiation is a key element in the maintenance of tissue homeostasis in the epidermis. We show here that methotrexate (MTX) induces differentiation of human epidermal keratinocytes in vitro. MTX inhibits proliferation of keratinocytes and also induces several markers of differentiation: a change in cell morphology, a marked increase in cell size, an increase in the proportion of cells that express involucrin, and an increase in the amount of cornified envelope protein. These effects of MTX are dose- and exposure-time-dependent and become irreversible after 24 hr, approximately one population doubling time. These effects of MTX cannot be attributed to cytotoxicity since keratinocytes not only remain viable but also actively synthesize proteins. MTX causes reproducible changes in the SDS/PAGE profiles of newly synthesized proteins and, in particular, increases the amount of involucrin synthesis. Thymidine completely prevents these effects of MTX, suggesting that they are caused by a depletion of thymine deoxyribonucleotides. The effect of MTX on keratinocytes may provide a model for studying the relationship between deoxyribonucleotide metabolism and differentiation in normal cells. In addition, the ability of MTX to induce differentiation in keratinocytes suggests a mechanism to explain its therapeutic action in psoriasis.
A recombinant retrovirus encoding alkaline phosphatase confirms clonal boundary assignment in lineage analysis of murine retina.
Authors: Fields-Berry S C , Halliday A L , Cepko C L
Published: May 2006

Recombinant retroviruses encoding the histochemically detectable enzyme beta-galactosidase have been used to investigate lineage in the vertebrate nervous system. Identification of the descendants of individual progenitors is straightforward when progeny cells are arranged in a reproducible, clustered pattern, but difficulties in interpretation arise when progeny migrate extensively and/or in an irregular pattern. To better resolve clonal boundaries, additional histochemical marker viruses that engender distinctive reaction products can be used in combination with lacZ-bearing viruses. To this end, we have created a retrovirus vector, DAP, encoding an easily assayable enzyme, human placental alkaline phosphatase. DAP was found to be at least as useful as a lacZ-encoding retrovirus (e.g., BAG) with respect to high viral titer, stability of expression, and in identification of infected cells in vivo. Moreover, it was found to be neutral with respect to postnatal rodent retinal development and offered superior staining characteristics relative to lacZ. Coinfection of rodent retina with DAP and BAG allowed an examination of the clonal nature of radial arrays of labeled retinal cells that previously had been described as products of a single infected progenitor. Of 1100 radial arrays examined for the presence of both DAP- and BAG-infected cells, only 1.2% were the result of infection with more than one virus.
Molecular cloning and structural analysis of genes from Zea mays (L.) coding for members of the ras-related ypt gene family.
Authors: Palme K , Diefenthal T , Vingron M , Sander C , Schell J
Published: May 2006

We have isolated, cloned, and characterized two cDNAs from Zea mays (L.), denoted yptm1 and yptm2, encoding proteins related to the ypt protein family. Amino acid similarity scores with YPT1 from yeast and ypt from mouse are in the range of 70% for yptm1 and 74% for yptm2, respectively, whereas similarities with p21 ras and other ras-related proteins are less than 40%. Most amino acid residues showing identity are clustered in the GTP/GDP binding domain. In addition, two cysteine residues close to the C-terminal ends, known to be palmitoylated and necessary for membrane binding in all eukaryotic ras-related proteins that have been characterized so far, are conserved in the maize genes as well. Northern blot hybridization analysis of poly(A)+ mRNA from etiolated maize coleoptiles revealed single mRNA species of approximately the same size as the isolated cDNAs. The gene for yptm1 is expressed at very low levels in maize coleoptiles and tissue culture cells. The gene for yptm2 is expressed at higher levels and is differentially represented in RNAs isolated from various organs of maize plants, with its highest level in leaves and flowers. The structural similarity of the genes identified suggests that they could be involved in the control of secretory processes.
Small amino acid changes in the V3 hypervariable region of gp120 can affect the T-cell-line and macrophage tropism of human immunodeficiency virus type 1.
Authors: Shioda T , Levy J A , Cheng-Mayer C
Published: May 2006

Human immunodeficiency virus type 1 (HIV-1) strains display a high degree of heterogeneity in their biological properties that correlate with in vivo pathogenesis of the virus. We previously demonstrated that overlapping regions encompassing the third hypervariable domain (V3), within the envelope glycoprotein gp120 determine the tropisms of HIV-1 for T-cell lines and primary macrophages. Studies with mutant viruses carrying one or more amino acid substitutions in the V3 loop have now identified this hypervariable domain as a major determinant for these cellular host range properties. Three to five amino acid changes in this domain, but rarely a single amino acid substitution, can confer macrophage tropism and alter T-cell-line tropism. These findings emphasize the effect on cell tropism of small amino acid differences in the viral envelope and suggest that the overall conformation of the V3 loop plays the major role in determining the ability of HIV-1 to infect T-cell lines and primary macrophages.
Primary electron transfer kinetics in bacterial reaction centers with modified bacteriochlorophylls at the monomeric sites BA,B.
Authors: Finkele U , Lauterwasser C , Struck A , Scheer H , Zinth W
Published: May 2006

The primary electron transfer has been investigated by femtosecond time-resolved absorption spectroscopy in two chemically modified reaction centers (RC) of Rhodobacter sphaeroides, in which the monomeric bacteriochlorophylls BA and BB have both been exchanged by 13(2)-hydroxybacteriochlorophyll a or [3-vinyl]-13(2)-hydroxybacteriochlorophyll a. The kinetics of the primary electron transfer are not influenced by the 13(2)-hydroxy modification. In RCs containing [3-vinyl]-13(2)-hydroxybacteriochlorophyll a the primary rate is reduced by a factor of 10.
Extracellular superoxide dismutase, nitric oxide, and central nervous system O2 toxicity.
Authors: Oury T D , Ho Y S , Piantadosi C A , Crapo J D
Published: May 2006

Although reactive O2 species appear to participate in central nervous system (CNS) O2 toxicity, the exact roles of different reactive O2 species are undetermined. To study the contribution of extracellular superoxide anion (O2-) to CNS O2 toxicity we constructed transgenic mice overexpressing human extracellular superoxide dismutase (ECSOD; superoxide:superoxide oxidoreductase, EC 1.15.1.1) in the brain. Remarkably, when exposed to 6 atm (1 atm = 101.3 kPA) of hyperbaric oxygen for 25 min, transgenic mice demonstrated higher mortality (83%) than nontransgenic litter-mates (33%; P < 0.017). Pretreatment with diethyldithiocarbamate, which inhibits both ECSOD and Cu/Zn superoxide dismutase (Cu/Zn SOD) activity, increased resistance to CNS O2 toxicity, in terms of both survival (100% in transgenics and 93% in nontransgenics) and resistance to seizures (4-fold increase in seizure latency in both transgenic and nontransgenic mice; P < 0.05). Thus, O2- apparently protects against CNS O2 toxicity. We hypothesized that O2- decreased toxicity by inactivating nitric oxide (NO.). To test this, we inhibited NO. synthase (EC 1.14.23) with N omega-nitro-L-arginine to determine whether NO. contributes to enhanced CNS O2 toxicity in transgenic mice. N omega-nitro-L-arginine protected both transgenic and nontransgenic mice against CNS O2 toxicity (100% survival and a 4-fold delay in time to first seizure; P < 0.05), as well as abolishing the difference in sensitivity to CNS O2 toxicity between transgenic and nontransgenic mice. These results implicate NO. as an important mediator in CNS O2 toxicity and suggest that ECSOD increases CNS O2 toxicity by inhibiting O2(-)-mediated inactivation of NO.
Anti-tumor necrosis factor ameliorates joint disease in murine collagen-induced arthritis.
Authors: Williams R O , Feldmann M , Maini R N
Published: May 2006

There is considerable evidence implicating tumor necrosis factor alpha (TNF-alpha) in the pathogenesis of rheumatoid arthritis. This evidence is based not only on the universal presence of TNF-alpha in arthritic joints accompanied by the upregulation of TNF-alpha receptors but also on the effects of neutralizing TNF-alpha in joint cell cultures. Thus, neutralization of TNF-alpha in vitro results in inhibition of the production of interleukin 1, which like TNF-alpha, is believed to contribute to joint inflammation and erosion. To determine the validity of this concept in vivo, the effect of administering TNF-neutralizing antibodies to mice with collagen-induced arthritis has been studied. This disease model was chosen because of its many immunological and pathological similarities to human rheumatoid arthritis. TN3-19.12, a hamster IgG1 monoclonal antibody to murine TNF-alpha/beta, was injected i.p. into mice either before the onset of arthritis or after the establishment of clinical disease. Anti-TNF administered prior to disease onset significantly reduced paw swelling and histological severity of arthritis without reducing the incidence of arthritis or the level of circulating anti-type II collagen IgG. More relevant to human disease was the capacity of the antibody to reduce the clinical score, paw swelling, and the histological severity of disease even when injected after the onset of clinical arthritis. These results have implications for possible modes of therapy of human arthritis.
Purification and characterization of 1-aminocyclopropane-1-carboxylate oxidase from apple fruit.
Authors: Dong J G , Fernández-Maculet J C , Yang S F
Published: May 2006

1-Aminocyclopropane-1-carboxylate (ACC) oxidase catalyzes the oxidation of ACC to ethylene. Following conventional column fractionation, the enzyme was purified 180-fold to near homogeneity with a specific activity of 20 nmol/(mg.min). This purified enzyme preparation migrated as a single protein band with an apparent molecular mass of 35 kDa on SDS/PAGE and 39 kDa on gel filtration. As in vivo, the purified enzyme required CO2 for activity. Removal of CO2 from the reaction mixture completely abolished the enzyme activity, while 0.5% atmospheric CO2 (0.15 mM in the medium) gave half-maximal activity. The purified enzyme displayed an absolute requirement for Fe2+ and ascorbate. The stoichiometry of the enzymatic reaction was determined: ACC + ascorbate + O2-->C2H4 + HCN + CO2 + dehydroascorbate + 2 H2O. A polyclonal antibody was raised against a synthetic tridecapeptide (PDLEEEYRKTMKE) whose sequence was deduced from the apple pAE12 cDNA [Dong, J. G., Olson, D., Silverstone, A. & Yang, S. F. (1992) Plant Physiol. 98, 1530-1531], which is homologous to tomato cDNAs encoding ACC oxidase. On a Western blot, this antibody specifically recognized the purified ACC oxidase protein. The amino acid composition of the purified enzyme agreed well with that deduced from the pAE12 sequence. When the protein was cleaved with CNBr and one of the peptide fragments was isolated and sequenced for 20 cycles, its sequence (KEFAVELEKLAEKLLDLLCE) precisely matched that predicted from pAE12 (residues 115-134). When preclimacteric apple fruit was treated with ethylene, a parallel increase in in vivo and in vitro ACC oxidase activities was observed, and this increase was accompanied by a concomitant increase in the level of pAE12 transcript. These observations support the conclusion that the isolated ACC oxidase protein is encoded by pAE12.
Displacement of BrdUrd-induced YY1 by serum response factor activates skeletal alpha-actin transcription in embryonic myoblasts.
Authors: Lee T C , Shi Y , Schwartz R J
Published: May 2006

Muscle-restricted transcription of the skeletal alpha-actin gene is controlled in part by a positive regulator, serum response factor (SRF), and a negative regulator, F-ACT1, which bind competitively to the most proximal serum response element (SRE1). We show here that F-ACT1 is identical to a transcription factor recently cloned and described as YY1, NF-E1, delta, or UCRBP. We found that although the DNA-binding activity of SRF accumulates during myogenesis, that of YY1 diminishes simultaneously. Myoblasts rendered incapable of differentiation by BrdUrd treatment exhibited the highest level of YY1 and the lowest level of SRF activities. Transfected SRF could directly transactivate the skeletal alpha-actin promoter by overcoming the inhibitory effect of BrdUrd-induced YY1. The transactivation depends on intact SRE DNA elements and requires the DNA-binding/dimerization domain of SRF as well as its C-terminal half rich in serines and threonines. Since the functions of YY1 and SRF appear to be developmentally regulated, the convergence of their binding sites upon the SRE constitutes an integrated mechanism whereby temporal and spatial muscle gene expression may be accomplished.
Oncogenic ras triggers the activation of 42-kDa mitogen-activated protein kinase in extracts of quiescent Xenopus oocytes.
Authors: Shibuya E K , Polverino A J , Chang E , Wigler M , Ruderman J V
Published: May 2006

Quiescent, full-grown Xenopus oocytes, which are arrested at the G2/M border of meiosis, contain an inactive 42-kDa mitogen-activated protein kinase (p42MAPK) that is activated when oocytes are stimulated to resume the meiotic cell cycle. We have made extracts from these oocytes that respond to four cell cycle activators: oncogenic [Val12]Ras protein, clam cyclins A delta 60 and B delta 97, and the phosphatase inhibitor okadaic acid. All four induce the tyrosine phosphorylation and activation of p42MAPK. Both cyclins and okadaic acid, but not [Val12]Ras, also lead to activation of the endogenous cyclin B/cdc2 kinase complexes in extracts of quiescent oocytes. Using extracts prepared from cycloheximide-arrested interphase cells, we show that although p42MAPK activation can occur in response to cyclin-activated cdc2, the Ras-induced activation of p42MAPK occurs without intervening cdc2 activation. Neither the nononcogenic [Gly12]Ras nor [Val12,Arg186]Ras, a mutant that lacks the C-terminal consensus sequence directing prenylation and subsequent membrane association, is an effective activator of p42MAPK in vitro.
A 105-kDa protein is required for yeast mitochondrial RNase P activity.
Authors: Morales M J , Dang Y L , Lou Y C , Sulo P , Martin N C
Published: May 2006

RNase P from the mitochondria of Saccharomyces cerevisiae was purified to near homogeneity > 1800-fold with a yield of 1.6% from mitochondrial extracts. The most abundant protein in the purified fractions is, at 105 kDa, considerably larger than the 14-kDa bacterial RNase P protein subunits. Oligonucleotides designed from the amino-terminal sequence of the 105-kDa protein were used to identify and isolate the 105-kDa protein-encoding gene. Strains carrying a disruption of the gene for the 105-kDa protein are viable but respiratory deficient and accumulate mitochondrial tRNA precursors with 5' extensions. As this is the second gene known to be necessary for yeast mitochondrial RNase P activity, we have named it RPM2 (for RNase P mitochondrial).
Brain-derived neurotrophic factor and neurotrophin 3 mRNAs in the peripheral target fields of developing inner ear ganglia.
Authors: Pirvola U , Ylikoski J , Palgi J , Lehtonen E , Arumäe U , Saarma M
Published: May 2006

In situ hybridization was used to study the site and timing of the expression of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3), and neurotrophin 5 (NT-5) mRNAs in the developing inner ear of the rat. In the sensory epithelia, the levels of NGF and NT-5 mRNAs were below the detection limit. NT-3 and BDNF mRNAs were expressed in the otic vesicle in overlapping but also in distinct regions. Later in development, NT-3 transcripts were localized to the differentiating sensory and supporting cells of the auditory organ and vestibular maculae. In these sensory epithelia, the intensity of NT-3 mRNA expression decreased in parallel with maturation. The expression of BDNF mRNA was restricted to the sensory cells of both the auditory and vestibular organs, including ampullary cristae. In bioassays, BDNF and NT-3, but not NGF, at physiological concentrations induced neurite outgrowth from the statoacoustic ganglion explants. These results demonstrate that NT-3 and BDNF, rather than NGF and NT-5, are the primary neurotrophins present in the target fields of the cochlear and vestibular neurons. Expression of NT-3 and BDNF mRNAs in the otic vesicle before and during the ingrowth of neurites from the statoacoustic ganglion suggests that NT-3 or BDNF or both may serve as chemoattractants for the early nerve fibers. The results also suggest that these neurotrophins have a role in later development of the cochlear and vestibular neurons.
Different missense mutations at the tissue-nonspecific alkaline phosphatase gene locus in autosomal recessively inherited forms of mild and severe hypophosphatasia.
Authors: Henthorn P S , Raducha M , Fedde K N , Lafferty M A , Whyte M P
Published: May 2006

Hypophosphatasia is a heritable form of rickets/osteomalacia with extremely variable clinical expression. Severe forms are inherited in an autosomal recessive fashion; the mode of transmission of mild forms is uncertain. The biochemical hallmark of hypophosphatasia is deficient activity of the tissue-nonspecific isozyme of alkaline phosphatase (TNSALP). Previously, we demonstrated in one inbred infant that an identical missense mutation in both alleles of the gene encoding TNSALP caused lethal disease. We have now examined TNSALP cDNAs from four unrelated patients with the severe perinatal or infantile forms of hypophosphatasia. Each of the eight TNSALP alleles from these four individuals contains a different point mutation that causes an amino acid substitution. These base changes were not detected in at least 63 normal individuals and, thus, appear to be causes of hypophosphatasia in the four patients. (Two additional base substitutions, found in one allele from each of the four patients, are linked polymorphisms.) Twenty-three unrelated patients (of 50 screened), who reflect the entire clinical spectrum of hypophosphatasia, possess one of our of the above eight mutations. In two of these additional patients, mild forms of the disease are also inherited in an autosomal recessive fashion. Our findings indicate that hypophosphatasia can be caused by a number of different missense mutations and that the specific interactions of different TNSALP mutant alleles are probably important for determining clinical expression. Severe forms, perinatal and infantile disease, are largely the result of compound heterozygosity for different hypophosphatasia alleles. At least some cases of childhood and adult hypophosphatasia are inherited as autosomal recessive traits.
Phenotypic and functional immaturity of human umbilical cord blood T lymphocytes.
Authors: Harris D T , Schumacher M J , Locascio J , Besencon F J , Olson G B , DeLuca D , Shenker L , Bard J , Boyse E A
Published: May 2006

Successful implementation of bone marrow transplantation for hematopoietic reconstitution is limited by the lack of suitably HLA-matched donors and by the occurrence of graft-versus-host disease that frequently accompanies this procedure. Recent clinical reports have implied that the use of umbilical cord blood as a source of transplantable stem cells may solve these problems. To date, definitive experiments have not been performed to assess the immunological potential of T cells found in umbilical cord blood, which could mediate graft-versus-host disease. In the present study we have observed that umbilical cord blood contains T lymphocytes that appear to be phenotypically immature. In addition, umbilical cord blood lymphocytes appeared to be functionally immature as shown by minimal responses to stimulation with interleukin 2, phytohemagglutinin, or alloantigens. Thus, umbilical cord blood may be more suitable for allogeneic transplantation than bone marrow in that these cord blood cells may not be as capable of mediating graft-versus-host disease.
Retinoic acid is enriched in Hensen's node and is developmentally regulated in the early chicken embryo.
Authors: Chen Y , Huang L , Russo A F , Solursh M
Published: May 2006

Retinoic acid (RA) has been considered as a potential morphogen in the chicken limb and has also been suggested to be involved in early embryonic development. On the basis of biological activity, previous reports suggest that Hensen's node, the anatomical equivalent in the chicken of the Spemann's organizer, may contain RA. Here, by using a molecular assay system, we demonstrate that Hensen's node contains retinoids in a concentration approximately 20 times more than that in the neighboring tissues. Furthermore, stage 6 Hensen's node contains approximately 3 times more retinoid than that of stage 4 embryos. These endogenous retinoids may establish a concentration gradient from Hensen's node to adjacent tissues and play a role in establishing the primary embryonic axis in the vertebrate. The results also suggest that the retinoid concentration in Hensen's node is developmentally regulated.
Identification of multiple structural domains regulating viroid pathogenicity.
Authors: Sano T , Candresse T , Hammond R W , Diener T O , Owens R A
Published: May 2006

To investigate the role of individual structural domains in viroid pathogenicity and replication, a series of interspecific chimeras was constructed by exchanging the terminal left (TL) and/or pathogenicity (P) domains between tomato apical stunt (TASVd) and citrus exocortis (CEVd) viroids. All six chimeras tested were replicated stably in tomato, and the symptoms exhibited by infected plants were intermediate between those induced by the parental viroids. Quantitative comparisons of symptom development and progeny accumulation revealed that: (i) the TL domain of TASVd contains a determinant required for appearance of severe veinal necrosis in tomato, (ii) the severe epinasty and stunting characteristic of TASVd requires the presence of its TL and P domains, and (iii) the variable (V) and terminal right (TR) domains comprising the right side of the native structure also play an important role in viroid pathogenicity. Chimeras containing the right side of TASVd accumulated to higher levels early in infection, and infected plants developed more severe symptoms than those whose right halves were derived from CEVd. Although the individual contributions of the TL and P domains to symptom induction could not be completely separated from that of viroid titer, the TL domain appears to exert a greater effect upon symptom severity than does the P domain. The TL, P, V, and TR domains of TASVd and CEVd contain three discrete regions of sequence and/or structural variability that may correspond to the pathogenicity determinants uncovered by our genetic analysis.
Ser-752-->Pro mutation in the cytoplasmic domain of integrin beta 3 subunit and defective activation of platelet integrin alpha IIb beta 3 (glycoprotein IIb-IIIa) in a variant of Glanzmann thrombasthenia.
Authors: Chen Y P , Djaffar I , Pidard D , Steiner B , Cieutat A M , Caen J P , Rosa J P
Published: May 2006

Integrins are membrane receptors which mediate cell-cell or cell-matrix adhesion. Integrin alpha IIb beta 3 (glycoprotein IIb-IIIa) acts as a fibrinogen receptor of platelets and mediates platelet aggregation. Platelet activation is required for alpha IIb beta 3 to shift from noncompetent to competent for binding soluble fibrinogen. The steps involved in this transition are poorly understood. We have studied a variant of Glanzmann thrombasthenia, a congenital bleeding disorder characterized by absence of platelet aggregation and fibrinogen binding. The patient's platelets did not bind fibrinogen after platelet activation by ADP or thrombin, though his platelets contained alpha IIb beta 3. However, isolated alpha IIb beta 3 was able to bind to an Arg-Gly-Asp-Ser affinity column, and binding of soluble fibrinogen to the patient's platelets could be triggered by modulators of alpha IIb beta 3 conformation such as the Arg-Gly-Asp-Ser peptide and alpha-chymotrypsin. These data suggested that a functional Arg-Gly-Asp binding site was present within alpha IIb beta 3 and that the patient's defect was not secondary to a blockade of alpha IIb beta 3 in a noncompetent conformational state. This was evocative of a defect in the coupling between platelet activation and alpha IIb beta 3 up-regulation. We therefore sequenced the cytoplasmic domain of beta 3, following polymerase chain reaction (PCR) on platelet RNA, and found a T-->C mutation at nucleotide 2259, corresponding to a Ser-752-->Pro substitution. This mutation is likely to be responsible for the uncoupling of alpha IIb beta 3 from cellular activation because (i) it is not a polymorphism, (ii) it is the only mutation in the entire alpha IIb beta 3 sequence, and (iii) genetic analysis of the family showed that absence of the Pro-752 beta 3 allele was associated with the normal phenotype. Our data thus identify the C-terminal portion of the cytoplasmic domain of beta 3 as an intrinsic element in the coupling between alpha IIb beta 3 and platelet activation.
Isolation of a wheat cDNA clone for an abscisic acid-inducible transcript with homology to protein kinases.
Authors: Anderberg R J , Walker-Simmons M K
Published: May 2006

Increases in the plant hormone abscisic acid (ABA) initiate water-stress responses in plants. We present evidence that a transcript with homology to protein kinases is induced by ABA and dehydration in wheat. A 1.2-kilobase cDNA clone (PKABA1) was isolated from an ABA-treated wheat embryo cDNA library by screening the library with a probe developed by polymerase chain reaction amplification of serine/threonine protein kinase subdomains VIb to VIII. The deduced amino acid sequence of the PKABA1 clone contains the features of serine/threonine protein kinases, including homology with all 12 conserved regions of the catalytic domain. PKABA1 transcript levels are barely detectable in growing seedlings but are induced dramatically when plants are subjected to dehydration stress. The PKABA1 transcript can also be induced by supplying low concentrations of ABA, and coordinate increases in ABA levels and PKABA1 mRNA occur when seedlings are water-stressed. Identification of this ABA-inducible transcript with homology to protein kinases provides a basis for examining the role of protein phosphorylation in plant responses to dehydration.
Tagging of plant potyvirus replication and movement by insertion of beta-glucuronidase into the viral polyprotein.
Authors: Dolja V V , McBride H J , Carrington J C
Published: May 2006

Infectious RNA transcripts were generated from full-length cDNA clones of the tobacco etch potyvirus genome containing an insertion of the bacterial beta-glucuronidase (GUS) gene between the polyprotein-coding sequences for the N-terminal 35-kDa proteinase and the helper component-proteinase. The recombinant virus was able to spread systemically in plants and accumulated to a level comparable with wild-type tobacco etch potyvirus. Proteolytic processing mediated by the 35-kDa proteinase and helper component-proteinase resulted in production of an enzymatically active GUS-helper component-proteinase fusion protein. A virus passage line that retained the GUS insert after numerous plant-to-plant transfers, as well as a line that sustained a deletion of the GUS sequence, was recovered. Use of an in situ histochemical GUS assay in time-course experiments allowed the visualization of virus activity in single, mechanically inoculated leaf epidermal cells, in neighboring epidermal and mesophyll cells, in phloem-associated cells after long-distance transport, and in cells surrounding vascular tissues of organs above and below the site of inoculation. This system represents a powerful tool to study plant virus replication, short- and long-distance virus movement, and virus-host interactions. Additionally, we show that potyviruses may serve as highly efficient, autonomously replicating vectors for the expression of foreign genes in plants.
Copper,zinc superoxide dismutase is primarily a cytosolic protein in human cells.
Authors: Crapo J D , Oury T , Rabouille C , Slot J W , Chang L Y
Published: May 2006

The intracellular localization of human copper,zinc superoxide dismutase (Cu,Zn-SOD; superoxide:superoxide oxidoreductase, EC 1.15.1.1) was evaluated by using EM immunocytochemistry and both isolated human cell lines and human tissues. Eight monoclonal antibodies raised against either native or recombinant human Cu,Zn-SOD and two polyclonal antibodies raised against either native or recombinant human Cu,Zn-SOD were used. Fixation with 2% paraformaldehyde/0.2% glutaraldehyde was found necessary to preserve normal distribution of the protein. Monoclonal antibodies were less effective than polyclonal antibodies in recognizing the antigen after adequate fixation of tissue. Cu,Zn-SOD was found widely distributed in the cell cytosol and in the cell nucleus, consistent with it being a soluble cytosolic protein. Mitochondria and secretory compartments did not label for this protein. In human cells, peroxisomes showed a labeling density slightly less than that of cytoplasm.
Glutamate receptors of Drosophila melanogaster: cloning of a kainate-selective subunit expressed in the central nervous system.
Authors: Ultsch A , Schuster C M , Laube B , Schloss P , Schmitt B , Betz H
Published: May 2006

We report the isolation and functional characterization of cDNAs encoding a Drosophila kainate-selective glutamate receptor. The deduced mature 964-residue protein (DGluR-I) is 108,482 Da and exhibits significant homology to mammalian glutamate receptor subunits. Injection of DGluR-I cRNA into Xenopus oocytes generated kainate-operated ion channels which were blocked by the selective non-N-methyl-D-aspartate receptor antagonist 6-cyano-7-nitro-quinoxaline-2,3-dione and philanthotoxin. DGluR-I transcripts are differentially expressed during Drosophila development and, in late embryogenesis, accumulate in the central nervous system.
Sequence and expression of caveolin, a protein component of caveolae plasma membrane domains phosphorylated on tyrosine in Rous sarcoma virus-transformed fibroblasts.
Authors: Glenney J R , Soppet D
Published: May 2006

Caveolae are flask-shaped plasma membrane invaginations abundant in endothelium and muscle but may be present in all cells. They contain a filamentous coat material thought to be important in their structure and function. Recent studies have demonstrated that a 22-kDa protein (caveolin) phosphorylated on tyrosine in Rous sarcoma virus-transformed chicken fibroblasts is a component of the caveolae coat on the inner aspect of the membrane. We now report the deduced protein sequence of chicken caveolin derived from cDNA PCR products and genomic DNA clones. Caveolin is a unique protein of 178 amino acids and displays little sequence similarity to other proteins in the GenBank data base. Hydrophobicity predictions indicate an unusual 40-amino acid hydrophobic region near the C terminus that may be used to anchor the protein to the membrane. When chicken caveolin was expressed in mouse 3T3 cells and detected by immunofluorescence microscopy, the typical caveolae pattern was observed. This includes brightly fluorescent membrane patches in many cases concentrated at the margin of cells and in arrays. Caveolae may be distinct from other membrane domains due at least in part to caveolin.
N-methyl-D-aspartate receptor antagonists disrupt the formation of a mammalian neural map.
Authors: Simon D K , Prusky G T , O'Leary D D , Constantine-Paton M
Published: May 2006

The topographic ordering of retinal connections in the rat superior colliculus emerges during early postnatal life from an initially diffuse projection. Disruption of N-methyl-D-aspartate (NMDA) receptor activity in the superior colliculus during this period interferes with map remodeling. In rats chronically treated with NMDA receptor antagonists during the first two postnatal weeks, aberrant axons remain and arborize at topographically incorrect sites. These results indicate that, at a stage preceding visually evoked activity, normal NMDA receptor function is important for the development of an ordered neural map in the mammalian brain.
Extreme clonal diversity and divergence in populations of a selfing hermaphroditic fish.
Authors: Turner B J , Elder J F , Laughlin T F , Davis W P , Taylor D S
Published: May 2006

Recombination is unknown in natural populations of Rivulus marmoratus, a selfing hermaphrodite, and genetic variation is likely due to mutation alone. DNA fingerprinting with an array of microsatellite [e.g., (CT)9] and minisatellite (e.g., the 33.15 core sequence) probes reveals very high clonal diversity within samples of seven Floridian populations, of which five contain about as many clones as there are individuals. There are 42 clones among 58 individuals surveyed (mean, 1.4 individuals per clone), a level of genetic diversity unprecedented among clonal animals. Moreover, all of the probes recognize the same clones even though, at high hybridization stringencies, there is little overlap in the fingerprint patterns they generate. This suggests that most sympatric clones differ by multiple and independent mutational steps. In one population studied in detail, the average number of mutational steps separating two clones is estimated at 9 or 10 and may be substantially higher. The mutational discontinuities among sympatric clones make it unlikely that they evolved by accumulation of neutral mutations in populations that are otherwise genetically uniform. The data argue that the mixing of unrelated individuals from different local populations occurs to an extent previously unappreciated and/or that divergence of clones is mediated by natural selection. If confirmed, the latter would be a serious challenge to current ideas on the predominant role of recombination in promoting the evolution of biological novelty.
Arabidopsis alternative oxidase sustains Escherichia coli respiration.
Authors: Kumar A M , Söll D
Published: May 2006

Glutamyl-tRNA reductase, encoded by the hemA gene, is the first enzyme in porphyrin biosynthesis in many organisms. Hemes, important porphyrin derivatives, are essential components of redox enzymes, such as cytochromes. Thus a hemA Escherichia coli strain (SASX41B) is deficient in cytochrome-mediated aerobic respiration. Upon complementation of this strain with an Arabidopsis thaliana cDNA library, we isolated a clone which permitted the SASX41B strain to grow aerobically. The clone encodes the gene for Arabidopsis alternative oxidase, whose deduced amino acid sequence was found to have 71% identity with that of the enzyme from the voodoo lily, Sauromatum guttatum. The Arabidopsis protein is expressed as a 31-kDa protein in E. coli and confers on this organism cyanide-resistant growth, which in turn is sensitive to salicylhydroxamate. This implies that a single polypeptide is sufficient for alternative oxidase activity. Based on these observations we propose that a cyanide-insensitive respiratory pathway operates in the transformed E. coli hemA strain. Introduction of this pathway now opens the way to genetic/molecular biological investigations of alternative oxidase and its cofactor.
Vaccinia virus RNA helicase: an essential enzyme related to the DE-H family of RNA-dependent NTPases.
Authors: Shuman S
Published: May 2006

Three distinct nucleic acid-dependent ATPases are packaged within infectious vaccinia virus particles; one of these enzymes (nucleoside triphosphate phosphohydrolase II or NPH-II) is activated by single-stranded RNA. Purified NPH-II is now shown to be an NTP-dependent RNA helicase. RNA unwinding requires a divalent cation and any one of the eight common ribo- or deoxyribonucleoside triphosphates. The enzyme acts catalytically to displace an estimated 10-fold molar excess of duplex RNA under in vitro reaction conditions. NPH-II binds to single-stranded RNA. Turnover of the bound enzyme is stimulated by and coupled to hydrolysis of NTP. Photocrosslinking of radiolabeled RNA to NPH-II results in label transfer to a single 73-kDa polypeptide. The sedimentation properties of the helicase are consistent with NPH-II being a monomer of this protein. Immunoblotting experiments identify NPH-II as the product of the vaccinia virus I8 gene. The I8-encoded protein displays extensive sequence similarity to members of the DE-H family of RNA-dependent NTPases. Mutations in the NPH-II gene [Fathi, Z. & Condit, R.C. (1991) Virology 181, 258-272] define the vaccinia helicase as essential for virus replication in vivo. Encapsidation of NPH-II in the virus particle suggests a role for the enzyme in synthesis of early messenger RNAs by the virion-associated transcription machinery.
Quantum aspects of brain activity and the role of consciousness.
Authors: Beck F , Eccles J C
Published: May 2006

The relationship of brain activity to conscious intentions is considered on the basis of the functional microstructure of the cerebral cortex. Each incoming nerve impulse causes the emission of transmitter molecules by the process of exocytosis. Since exocytosis is a quantal phenomenon of the presynaptic vesicular grid with a probability much less than 1, we present a quantum mechanical model for it based on a tunneling process of the trigger mechanism. Consciousness manifests itself in mental intentions. The consequent voluntary actions become effective by momentary increases of the probability of vesicular emission in the thousands of synapses on each pyramidal cell by quantal selection.
Phage phi 29 regulatory protein p4 stabilizes the binding of the RNA polymerase to the late promoter in a process involving direct protein-protein contacts.
Authors: Nuez B , Rojo F , Salas M
Published: May 2006

Transcription from the late promoter, PA3, of Bacillus subtilis phage phi 29 is activated by the viral regulatory protein p4. A kinetic analysis of the activation process has revealed that the role of protein p4 is to stabilize the binding of RNA polymerase to the promoter as a closed complex without significantly affecting further steps of the initiation process. Electrophoretic band-shift assays performed with a DNA fragment spanning only the protein p4 binding site showed that RNA polymerase could efficiently retard the complex formed by protein p4 bound to the DNA. Similarly, when a DNA fragment containing only the RNA polymerase-binding region of PA3 was used, p4 greatly stimulated the binding of RNA polymerase to the DNA. These results strongly suggest that p4 and RNA polymerase contact each other at the PA3 promoter. In the light of current knowledge of the p4 activation mechanism, we propose that direct contacts between the two proteins participate in the activation process.
Modulation of a transient K+ current in the pleural sensory neurons of Aplysia by serotonin and cAMP: implications for spike broadening.
Authors: Hochner B , Kandel E R
Published: May 2006

To study the contribution of cAMP to the spike broadening produced by serotonin (5-HT) in the pleural sensory neurons of the tail withdrawal reflex, we utilized two phosphodiesterase-resistant cAMP analogs: the Sp diastereomer of cyclic adenosine 3',5'-monophosphothioate (Sp-cAMP[S]), which activates protein kinase A, and the antagonist Rp diastereomer of cyclic adenosine 3',5'-monophosphothioate (Rp-cAMP[S]), agonist Sp-cAMP[S] was injected into the sensory neurons, it caused spike broadening comparable to that induced by 5-HT. In turn, the cAMP antagonist Rp-cAMP[S] blocked approximately 50% of the 5-HT-induced spike broadening. We next examined the K+ currents that are modulated by 5-HT and determined how these currents are affected by cAMP. Confirming Baxter and Byrne [(1989) J. Neurophysiol. 62, 665-679], we found that 5-HT modulated two currents, an S-type K+ current (IKS) as well as a transient and voltage-dependent K+ current (IKV). Rp-cAMP[S] blocked the reduction by 5-HT of the early phase of IKV in parallel with, and to the same degree (60%), as this inhibitor blocked the IKS and spike broadening. These results support the idea that in the pleural sensory neurons cAMP mediates a significant part of the spike broadening that accompanies short-term facilitation produced by 5-HT and that cAMP can produce spike broadening by modulating both IKV and IKS.
Glutathione deficiency increases hepatic ascorbic acid synthesis in adult mice.
Authors: Mårtensson J , Meister A
Published: May 2006

Glutathione deficiency, induced in adult mice by administering buthionine sulfoximine (an inhibitor of glutathione synthesis), led to a rapid and substantial increase in ascorbate in the liver. This effect was apparent 2-4 hr after giving the inhibitor; subsequently, the level of ascorbate decreased and that of dehydroascorbate increased markedly, supporting the conclusion that glutathione functions physiologically to keep ascorbate in its reduced form. In kidney and lung also, ascorbate levels decreased, and dehydroascorbate increased. Increased synthesis of ascorbate in glutathione-deficient adult mice seems to protect against tissue damage. In contrast, newborn rats, which (like guinea pigs and humans) apparently do not synthesize ascorbate, suffer severe damage to liver and other organs; previous studies showed that administration of ascorbate prevents such tissue damage. The findings support the view that the antioxidant actions of glutathione and ascorbate are closely linked and involve a mechanism in which decrease of the glutathione level, perhaps associated with an oxidative event, stimulates ascorbate synthesis.
Genomic polymorphism, recombination, and linkage disequilibrium in human major histocompatibility complex-encoded antigen-processing genes.
Authors: van Endert P M , Lopez M T , Patel S D , Monaco J J , McDevitt H O
Published: May 2006

Recently, two subunits of a large cytosolic protease and two putative peptide transporter proteins were found to be encoded by genes within the class II region of the major histocompatibility complex (MHC). These genes have been suggested to be involved in the processing of antigenic proteins for presentation by MHC class I molecules. Because of the high degree of polymorphism in MHC genes, and previous evidence for both functional and polypeptide sequence polymorphism in the proteins encoded by the antigen-processing genes, we tested DNA from 27 consanguineous human cell lines for genomic polymorphism by restriction fragment length polymorphism (RFLP) analysis. These studies demonstrate a strong linkage disequilibrium between TAP1 and LMP2 RFLPs. Moreover, RFLPs, as well as a polymorphic stop codon in the telomeric TAP2 gene, appear to be in linkage disequilibrium with HLA-DR alleles and RFLPs in the HLA-DO gene. A high rate of recombination, however, seems to occur in the center of the complex, between the TAP1 and TAP2 genes.
Detection of dystrophin in the postsynaptic density of rat brain and deficiency in a mouse model of Duchenne muscular dystrophy.
Authors: Kim T W , Wu K , Xu J L , Black I B
Published: May 2006

Duchenne muscular dystrophy (DMD) is a common, lethal, chromosome X-linked inherited disease. Moderate cognitive impairment is a feature of DMD, but the underlying mechanisms are unknown. DMD is characterized by a defect in a protein, dystrophin, that is located predominantly in muscle but has been detected in brain. We sought to directly localize dystrophin within the complex synaptic structure of the cerebral cortex by focusing on the postsynaptic density (PSD), which appears to be central to synaptic function. We report that a specific anti-dystrophin antibody (anti 6-10) recognizes three distinct proteins in the purified PSD: the 400-kDa dystrophin and two previously unidentified dystrophin-related proteins of 120 and 110 kDa. These proteins exhibited differential regional expression in PSDs from cerebral cortex, cerebellum, and olfactory bulb. In the cortical PSD, the 400-kDa dystrophin was predominant, whereas the 120-kDa protein was the major species in cerebellum and olfactory bulb PSDs. The three proteins were differentially expressed in the PSD during cortical development: the 400-kDa protein exhibited a selective 9-fold increase during postnatal days 7 to 10, suggesting a normal physiological role in synaptic maturation. The PSD from the mdx mouse, a model of human DMD, contained no detectable 400-kDa dystrophin but expressed the two dystrophin-related proteins. Our results indicate that brain dystrophins are localized to the PSD, potentially as three isoforms, and raise the possibility that cognitive abnormalities in DMD are attributable to synaptic dysfunction associated with deficits in brain dystrophin molecules.