RESUMO
The aim of this study was to evaluate if Hyperventilation (HV) could avoid the Intracranial Pressure (ICP) peak that occurs during Fiberoptic Bronchoscopy (FB) in severely head injured patients. A Cerebral Perfusion Pressure (CPP) > 75 mmHg was maintained in 34 patients, with a subgroup randomized to receive controlled HV during FB. Measurements were done before the procedure, during maximum ICP values and 30 minutes after FB. The HV group had minor ICP values after FB, without differences in CPP and ICP peak values.
Assuntos
Broncoscopia/métodos , Traumatismos Craniocerebrais/cirurgia , Hemodinâmica/fisiologia , Hiperventilação/fisiopatologia , Pressão Intracraniana/fisiologia , Terapia Respiratória/métodos , Adulto , Pressão Sanguínea , Dióxido de Carbono/sangue , Traumatismos Craniocerebrais/complicações , Feminino , Tecnologia de Fibra Óptica , Humanos , Cuidados Intraoperatórios , Masculino , Monitorização Intraoperatória , Oxigênio/sangueRESUMO
Four groups of plant homeodomain proteins contain a dimerization motif closely linked to the homeodomain. We here show that two sunflower homeodomain proteins, Hahb-4 and HAHR1, which belong to the Hd-Zip I and GL2/Hd-Zip IV groups, respectively, show different binding preferences at a defined position of a pseudopalindromic DNA-binding site used as a target. HAHR1 shows a preference for the sequence 5'-CATT(A/T)AATG-3', rather than 5'-CAAT(A/T)ATTG-3', recognized by Hahb-4. To analyze the molecular basis of this behavior, we have constructed a set of mutants with exchanged residues (Phe-->Ile and Ile-->Phe) at position 47 of the homeodomain, together with chimeric proteins between HAHR1 and Hahb-4. The results obtained indicate that Phe47, but not Ile47, allows binding to 5'-CATT(A/T)AATG-3'. However, the preference for this sequence is determined, in addition, by amino acids located C-terminal to residue 53 of the HAHR1 homeodomain. A double mutant of Hahb-4 (Ile47-->Phe/Ala54-->Thr) shows the same binding behavior as HAHR1, suggesting that combinatorial interactions of amino acid residues at positions 47 and 54 of the homeodomain are involved in establishing the affinity and selectivity of plant dimeric homeodomain proteins with different DNA target sequences.
Assuntos
DNA de Plantas/metabolismo , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Alanina/genética , Alanina/metabolismo , Sequência de Aminoácidos , Pareamento de Bases , Sequência de Bases , Sítios de Ligação , DNA de Plantas/química , Dimerização , Proteínas de Homeodomínio/genética , Modelos Biológicos , Dados de Sequência Molecular , Mutação , Proteínas de Plantas/genética , Proteínas Recombinantes de Fusão/metabolismo , Treonina/genética , Treonina/metabolismoRESUMO
Plant homeodomain-leucine zipper proteins, unlike most animal homeodomains, bind DNA efficiently only as dimers. In the present work, we report that the deletion of the homeodomain N-terminal arm (first nine residues) of the homeodomain-leucine zipper protein Hahb-4 dramatically affects its DNA-binding affinity, causing a 70-fold increase in dissociation constant. The addition of the N-terminal arm of Drosophila Antennapedia to the truncated form restores the DNA-binding affinity of dimers to values similar to those of the native form. However, the Antennapedia N-terminal arm is not able to confer increased binding affinity to monomers of Hahb-4 lacking the leucine zipper motif, indicating that the inefficient binding of monomers must be due to structural differences in other parts of the molecule. The construction of proteins with modifications at residues 5 to 7 of the homeodomain suggests strongly that positively charged amino acids at these positions play essential roles in determining the DNA-binding affinity. However, the effect of mutations at positions 6 and 7 can be counteracted by introducing a stretch of positively charged residues at positions 1 to 3 of the homeodomain. Sequence comparisons indicate that all homeodomain-leucine zipper proteins might use contacts of the N-terminal arm with DNA for efficient binding. The occurrence of a homeodomain with a DNA-interacting N-terminal arm must then be an ancient acquisition in evolution, earlier than the separation of lines leading to metazoa, fungi and plants.
Assuntos
DNA/metabolismo , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/metabolismo , Zíper de Leucina , Proteínas Nucleares , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Fatores de Transcrição , Sequência de Aminoácidos , Animais , Proteína do Homeodomínio de Antennapedia , Sequência de Bases , DNA/genética , Dimerização , Proteínas de Drosophila , Drosophila melanogaster , Evolução Molecular , Proteínas de Homeodomínio/genética , Dados de Sequência Molecular , Proteínas de Plantas/genética , Ligação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Deleção de Sequência/genética , Eletricidade Estática , TermodinâmicaRESUMO
We have used RNA in situ hybridization to analyze the expression of transcripts encoding cytochrome c in different tissues and organs of sunflower (Helianthus annuus). Although northern-blot hybridization experiments indicate that the relative abundance of transcripts does not vary greatly, we have detected important changes in localization during flower development. Enhanced expression is observed in floral meristems as soon as they are discernible from the central portion of the capitulum containing the inflorescence meristem. As flowers develop, labeling is observed in all developing floral organ primordia. Later in development, expression in petals is reduced, and only the central portion of the flower becomes labeled. During the process of stamen formation, hybridization signals were obtained mainly in anthers. Less developed flowers at this stage showed expression through the archesporial tissue. During meiosis, the label was observed mainly in tapetal cells. Specific expression patterns, similar to those obtained for sunflower, were observed when Arabidopsis flowers were analyzed with a homologous cytochrome c probe. Specific patterns of expression were also observed in young sunflower roots. In this case, enhanced expression was detected in developing endodermis and pericycle and in protoxylem initials. We conclude that cell-specific mechanisms operate to regulate the abundance of cytochrome c encoding transcripts in different plant tissues. The overlap between the expression patterns of the nuclear encoded cytochrome c gene and some mitochondrial genes suggests the existence of coordinated mechanisms of expression.
Assuntos
Grupo dos Citocromos c/genética , Regulação da Expressão Gênica de Plantas , Helianthus/genética , Transcrição Gênica , Regulação da Expressão Gênica no Desenvolvimento , Helianthus/citologia , Helianthus/crescimento & desenvolvimento , Hibridização In Situ , Raízes de Plantas/citologia , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Caules de Planta/citologia , Caules de Planta/crescimento & desenvolvimento , Caules de Planta/metabolismo , RNA Mensageiro/genéticaRESUMO
The expression of the Chlamydomonas reinhardtii cytochrome c gene was studied at the steady-state mRNA level. The inclusion of acetate under illumination produced a marked increase in cytochrome c transcripts. This effect was not affected by two inhibitors of mitochondrial energy metabolism. Three different obligate photoautotrophic mutants with defective mitochondria showed normal levels of induction, suggesting that utilization of acetate for respiration is not required for this process. Light, in the presence or absence of acetate, also promoted an increase in cytochrome c transcript levels. This effect could be abolished by treatment of the cells with an inhibitor of the photosynthetic electron transport chain, suggesting that light acts through photosynthesis to promote the induction. In addition, a genomic clone encompassing the Chlamydomonas cytochrome c gene has been isolated and analyzed. The gene contains three introns, two of which are located at positions similar to those in the rice and Arabidopsis cytochrome c genes, indicating the existence of an evolutionary link. It is concluded that the cytochrome c gene from C. reinhardtii is subject to metabolic regulation through a mechanism that responds to the intracellular level of either acetate or a compound derived from its metabolization through a pathway different from mitochondrial respiration.
Assuntos
Chlamydomonas reinhardtii/genética , Grupo dos Citocromos c/genética , Mutação , Sequência de Aminoácidos , Animais , Sequência de Bases , Chlamydomonas reinhardtii/enzimologia , Regulação Enzimológica da Expressão Gênica , Dados de Sequência MolecularRESUMO
The homeobox is a 180 bp consensus DNA sequence present in a number of genes involved in developmental processes. This review focuses on the structure and function of plant homeobox genes and of the proteins they encode. Plant homeobox genes have been identified in studies using mutants, degenerate oligonucleotides deduced from conserved sequences, differential screening or binding to known promoters. According to sequence conservation, plant homeoboxes can be subdivided into different families, each comprising several members. Evolutionary studies indicate that the different families have diverged prior to the separation of the branches leading to animals, plants and fungi. Accordingly, members of different families show characteristic structural and functional properties. As an example, kn1-like genes seem to be involved in different aspects of the control of cell fate determination in the shoot meristem; HD-Zip genes, which encode proteins containing a leucine zipper motif adjacent to the homeodomain, are believed to operate at later stages of development; and gl2-like genes are involved in epidermal cell differentiation. Future studies should be oriented to discern the precise function of the many homeobox genes present in plant genomes, and to evaluate their use as modifiers of plant development.
Assuntos
Genes Homeobox , Genes de Plantas , Plantas/genética , Regulação da Expressão Gênica no Desenvolvimento , Desenvolvimento VegetalRESUMO
Complementary DNA sequences encoding different portions of two sunflower homeodomain proteins were cloned in-frame in the expression vectors pRSET and pGEX-3X. When introduced into competent Escherichia coli cells and induced, the resulting plasmids directed the expression of large amounts (5-10% of total cellular protein) of the encoded polypeptides. As a rule, fusions in pRSET rendered insoluble proteins, while fusions in pGEX were soluble and could be purified in a single step by selective absorption onto glutathione-agarose beads, followed by elution with free glutathione. The purified proteins showed both glutathione S-transferase and DNA-binding activity, indicating that they retain their native conformation. The expression-purification protocol that was employed allowed the isolation of up to 0.7 mg of protein per gram of transformed cells. One of the fusion proteins, RH11 (which is a fusion of the homeodomain protein HAHR1 in pRSET), though insoluble, was able to bind DNA when spotted onto a nitrocellulose filter. This protein could also be simply purified in large amounts by electroelution from sodium dodecyl sulfate-polyacrylamide gels and used to elicit antibodies which recognized both the transgenic fusion and the native protein from sunflower nuclei. Our results clearly show that vector choice is a critical parameter for obtaining large amounts of a desired protein for particular purposes.
Assuntos
Escherichia coli/genética , Helianthus/metabolismo , Proteínas de Homeodomínio/genética , Sequência de Bases , Cromatografia de Afinidade , Clonagem Molecular , Primers do DNA , Eletroforese em Gel de Poliacrilamida , Proteínas de Homeodomínio/isolamento & purificação , Proteínas de Homeodomínio/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismoRESUMO
A 2.5 kb homeobox (HB)-containing cDNA (hahr1) was isolated from a library prepared from rootlets of Helianthus annuus using a polymerase chain reaction (PCR)-based strategy. The putative protein product (77 kDa) contains the homeodomain (HD) and an acidic domain at the N-terminal region (residues 72-155). The deduced amino acid sequence of hahr1 shares a 53% sequence identity with GLABRA2, a HD protein associated with epidermal cell differentiation. Hahr1 expression was primarily found in dry seeds, hypocotyls and roots at stages associated with early developmental events. Expression was completely lacking in leaves and flowers. Evidence for the existence of one related gene expressed in sunflower stems was obtained by the presence of restriction fragment length polymorphism of amplified cDNA products.
Assuntos
Proteínas de Arabidopsis , Regulação da Expressão Gênica de Plantas , Helianthus/genética , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Complementar , Helianthus/crescimento & desenvolvimento , Dados de Sequência Molecular , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Caules de Planta/genética , RNA Mensageiro , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
Sunflower HAHR1 is a homeodomain protein presumably involved in some aspects of root development. In the present work, we have studied the oligomerization properties of HAHR1. A protein containing the entire homeodomain plus adjacent C-terminal sequences (amino acids 86-325) behaves as a dimer in gel filtration experiments. When a fragment C-terminal to the homeodomain (amino acids 151-263) is fused to the N-terminal domain of the lambda phage repressor, it is able to confer binding efficiency to this domain, as judged by protection from lambda superinfection and repression of beta-galactosidase expression under the control of the P(R) promoter. A smaller fragment (amino acids 151-184) confers only conditional repression. GSH transferase fusion proteins containing the entire homeodomain of HAHR1 plus the above-mentioned adjacent sequences bind with similar efficiency a mixture of oligonucleotides selected from a random population. The smaller protein, however, loses its binding capacity when separated from the GSH transferase moiety. Retention of a labelled HAHR1 protein synthesized in vitro by GSH transferase fusions containing different protein fragments adjacent to the homeodomain and bound to GSH agarose suggests that a portion from amino acids 151-263 is required for efficient interaction. The results obtained indicate that HAHR1 interacts with DNA as a dimer and that its dimerization domain is located immediately C-terminal to the homeodomain. We define two regions, the first of which confers non-efficient dimerization; this region would be stabilized by the presence of the second one through putative mutual interactions. A similar motif is present in other related plant homeodomain proteins.
Assuntos
Proteínas de Homeodomínio/genética , Zíper de Leucina/genética , Proteínas de Plantas/genética , Sequência de Aminoácidos , Cromatografia em Gel , Dimerização , Escherichia coli/genética , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/metabolismo , Dados de Sequência Molecular , Proteínas de Plantas/química , Proteínas de Plantas/metabolismoRESUMO
A strategy based on the use of PCR with one degenerate oligonucleotide deduced from conserved sequences and lambda gt10 primers was used to isolate homeobox containing sequences from sunflower stem and root cDNA libraries. Six different partial cDNAs coding for the first 48 amino acids of homeodomains and amino terminal sequences were analyzed and found to be members of the HD-Zip superfamily, which contain a homeobox linked to a leucine zipper coding region. A full-length cDNA clone, Hahb-10, was isolated and characterized. The leucine zipper portions of Hahb-10 and of the previously reported Hahb-1 have been utilized to construct fusions with the N-terminal domain of the lambda repressor. These fusions were tested for their ability to bind to lambda promoters in vivo. The expression of a protein containing an active dimerization domain, but not capable of DNA binding, exerts a dominant negative effect on the ability of repressor-zipper fusions to bind to its target DNA. From these experiments, it was concluded that Hahb-1 and -10, when co-expressed, form preferentially homodimers. Exchange of conserved threonines and leucines at positions a1 and d1 of both zippers reduces dimerization efficiency and allows the formation of heterodimers, suggesting that these residues are, among others, determinants of the specificity of interaction, most likely through changes in hydrophobic packing interactions at the dimer interface. The results imply that a great number of interacting molecular entities compose this protein superfamily which is presumably involved in regulating plant developmental responses.
Assuntos
Helianthus/genética , Proteínas de Homeodomínio/metabolismo , Zíper de Leucina/fisiologia , Proteínas de Plantas/metabolismo , Sequência de Aminoácidos , Sequência de Bases , DNA Complementar/genética , Dimerização , Biblioteca Gênica , Genes de Plantas , Proteínas de Homeodomínio/genética , Dados de Sequência Molecular , Família Multigênica , Proteínas de Plantas/genética , Reação em Cadeia da Polimerase , Ligação Proteica , Proteínas Recombinantes de Fusão/metabolismo , Seleção Genética , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
The analogue (Z)-phosphoenol-3-fluoropyruvate [(Z)-3-fluoro-2-(phosphono-oxy)propenoic acid] was tested as substrate of maize leaf phosphoenolpyruvate carboxylase. Studies with NaH14CO3 indicate that the analogue is carboxylated by the enzyme. However, this reaction accounts for only one-tenth of the activity measured by Pi liberation. The rest of the analogue is merely dephosphorylated. This is the first analogue for which both carboxylation and dephosphorylation have been observed.
Assuntos
Carboxiliases/metabolismo , Fosfoenolpiruvato Carboxilase/metabolismo , Fosfoenolpiruvato/análogos & derivados , Zea mays/enzimologia , Bicarbonatos/metabolismo , Radioisótopos de Carbono , Cromatografia Líquida de Alta Pressão , Hidrazonas/metabolismo , Fosfoenolpiruvato/metabolismo , EspectrofotometriaRESUMO
Phosphoenolpyruvate carboxylase, purified from maize leaves, is rapidly inactivated by the fluorescence probe dansyl chloride. The loss of activity can be ascribed to the covalent modification of an R-NH2 group, presumably the epsilon-NH2 group of lysine. Analysis of the data by the statistical method of Tsou [Sci. Sin. 11, 1535-1558 (1962)] provides clear evidence that a pH 8 eight R-NH2 groups can be modified in the tetrameric form of the enzyme, four of which are essential for catalytic activity. Essential groups are modified about five times more rapidly than the non-essential ones. The enzyme was completely protected against inactivation by Mg2+ plus phosphoenolpyruvate and consequently binding of the modifier to the essential groups is completely abolished. Hence the four essential groups seemed to be located at or near the active site(s). One of the four essential groups was modified with dansyl chloride and the other three progressively with eosin isothiocyanate. In the doubly labeled protein non-radiative single-singlet energy transfer between dansyl chloride (donor) and eosin isothiocyanate (acceptor) was observed. The low variance (+/- 5%) in the efficiency of energy transfer obtained at a particular acceptor stoichiometry (0.8-1.1, 1.9-2.1, 2.9-3.1) in triplicate samples provided confidence that the measured transfer efficiency may be interpreted as transfer between specific sites. The distances calculated from the efficiency of resonance energy transfer revealed two acceptor sites, equally separated, 4.8-5.1 nm from the donor site and third site being 6.4 nm apart from the donor. Under conditions where the tetrameric enzyme dissociates into the monomers, no transfer of resonance energy between the protein-bound dansyl chloride and eosin isothiocyanate was observed. Most likely the four essential lysyl residues in the tetrameric enzyme are located in different subunits of the enzyme, hence each of the subunits would contain a substrate-binding site with one lysyl residue crucial for activity.
Assuntos
Carboxiliases/antagonistas & inibidores , Lisina/análise , Fosfoenolpiruvato Carboxilase/antagonistas & inibidores , Marcadores de Afinidade , Sítios de Ligação , Catálise , Compostos de Dansil/farmacologia , Transferência de Energia , Amarelo de Eosina-(YS)/análogos & derivados , Amarelo de Eosina-(YS)/farmacologia , Concentração de Íons de Hidrogênio , Lisina/fisiologia , Espectroscopia de Ressonância Magnética , Matemática , Espectrometria de Fluorescência , Zea mays/enzimologiaRESUMO
The aim of this work was to investigate the stereoselectivity of maize leaf phosphoenolpyruvate carboxylase with E- and Z-2-phosphoenolbutyrate as inhibitors and substrates. In addition, a procedure is presented for the separation of the isomers of 2-phosphoenolbutyrate. The method is based on the different interaction of those compounds with a strong anion-exchange high-pressure liquid chromatography column using 50 mM potassium phosphate (pH 3) as elution buffer, and allows the obtention of pure E- and Z-P-enolbutyrate with high yield. The same system was used to identify Z-P-enolbutyrate as the product of the phosphorylation of 2-oxobutyrate by rabbit muscle pyruvate kinase. In the presence of 5 mM Mg2+, both isomers of P-enolbutyrate inhibited C4-plant P-enolpyruvate carboxylase; the values of Ki were 15-20 microM and 100-110 microM for Z- and E-P-enolbutyrate, respectively. With 0.5 mM Mn2+, the Z isomer was also effective as inhibitor (Ki = 35-40 microM), while the E isomer produced activation of the carboxylase probably due to its binding at an allosteric site. Both compounds were substrates of the enzyme with similar V/Km values; however, V and Km for the two isomers were significantly different (i.e. Km = 110 microM for Z-P-enolbutyrate and 220 microM for E-P-enolbutyrate). The results indicate the existence of stereoselectivity for the binding of P-enolbutyrate to the active site of P-enolpyruvate carboxylase. However, this fact does not affect the use of the isomers as substrates by the plant carboxylase.
Assuntos
Carboxiliases/metabolismo , Fosfoenolpiruvato Carboxilase/metabolismo , Fosfoenolpiruvato/análogos & derivados , Zea mays/enzimologia , Animais , Sítios de Ligação , Cromatografia Líquida de Alta Pressão , Magnésio/farmacologia , Cloreto de Magnésio , Espectroscopia de Ressonância Magnética , Músculos/enzimologia , Fosfoenolpiruvato/metabolismo , Fosfoenolpiruvato/farmacologia , Fosfoenolpiruvato Carboxilase/antagonistas & inibidores , Coelhos , Estereoisomerismo , Especificidade por SubstratoRESUMO
Phosphoenolpyruvate carboxylase (EC 4.1.1.31) was purified 43-fold from Amaranthus viridis leaves by using a combination of ammonium-sulphate fractionation, chromatography on O-(diethylaminoethyl)-cellulose and hydroxylapatite, and filtration through Sepharose 6B. The purified enzyme had a specific activity of 17.1 µmol·(mg protein)(-1)·min(-1) and migrated as a single band of relative molecular weight 100000 on sodium dodecyl sulphate-polyacrylamide gel electrophoresis. A homotetrameric structure was determined for the native enzyme. Phosphoenolpyruvate carboxylase from Zea mays L. and A. viridis showed partial identity in Ouchterlony two-dimensional diffusion. Isoelectric focusing showed a band at pI 6.2. Km values for phosphoenolpyruvate and bicarbonate were 0.29 and 0.17 mM, respectively, at pH 8.0. The activation constant (Ka) for Mg(2+) was 0.87 mM at the same pH. The carboxylase was activated by glucose-6-phosphate and inhibited by several organic acids of three to five carbon atoms. The kinetic and structural properties of phosphoenolpyruvate carboxylase from A. viridis leaves are similar to those of the enzyme from Zea mays leaves.