Optimizing cerebral perfusion pressure during fiberoptic bronchoscopy in severe head injury: effect of hyperventilation.

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.

Combinatorial interactions of two amino acids with a single base pair define target site specificity in plant dimeric homeodomain proteins.

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.

Positively charged residues at the N-terminal arm of the homeodomain are required for efficient DNA binding by homeodomain-leucine zipper proteins.

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.

Cell-type-specific expression of plant cytochrome c mRNA in developing flowers and roots.

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.

The cytochrome c gene from the green alga Chlamydomonas reinhardtii. Structure and expression in wild-type cells and in obligate photoautotrophic (dk) mutants.

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.

A monomer-dimer equilibrium modulates the interaction of the sunflower homeodomain leucine-zipper protein Hahb-4 with DNA.

We have analysed the interaction of the sunflower homeodomain leucine-zipper (Hd-Zip) protein Hahb-4 with DNA. The complete Hd-Zip domain from Hahb-4 was able to select specific sequences from a random oligonucleotide mixture that contained a 9-bp core with four fixed and five degenerate positions. Analysis of the binding of some of the selected sequences suggests that Hahb-4 preferentially binds the dyad-symmetrical sequence CAAT(A/T)ATTG. Single-nucleotide replacements at positions 1, 5 or 9 of this sequence produced a decrease in binding of 2-4-fold. DNA binding as a function of protein concentration was non-hyperbolic. This behaviour could be explained by an equation in which dimer formation is a pre-requisite for DNA binding. A global dissociation constant (Kd) of 1.31x10(-14) M2 could be calculated. The removal of the leucine zipper promoted a change in specificity and a decrease in binding affinity (Kd=5. 03x10(-5) M). Mutation of Phe-20 of the homeodomain into Leu completely abolished DNA binding. The mutant protein, however, was able to inhibit DNA binding by the non-mutant form, presumably through the formation of heterodimers. The analysis of this inhibitory effect at different mutant concentrations allowed the estimation of the Kd for the dimer-monomer equilibrium [about (2-4)x10(-6) M]; from this, a Kd of 3-6x10(-9) M for the dimer-DNA complex could be estimated. The results obtained indicate that the formation of dimers is the main factor influencing the interaction of Hahb-4 with DNA. It is proposed that shifts in a dimer-monomer equilibrium could be used within the cell to modulate the interaction of this protein with target genes.

Homeoboxes in plant development.

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.

Expression of sunflower homeodomain containing proteins in Escherichia coli: purification and functional studies.

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.

Isolation and expression pattern of hahr1, a homeobox-containing cDNA from Helianthus annuus.

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.

A novel type of dimerization motif, related to leucine zippers, is present in plant homeodomain proteins.

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.

Interaction between proteins containing homeodomains associated to leucine zippers from sunflower.

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.

A gene involved in the biogenesis of c-type cytochromes is co-transcribed with a ribosomal protein gene in wheat mitochondria [corrected].

Sequence analysis of a transcribed region of the wheat mitochondrial (mt) genome revealed two open reading frames (orfs) coding for proteins of 589 and 174 amino acids. Both genes are co-transcribed in a 2.6-kb RNA. The largest orf codes for a hydrophobic protein which bears similarity to a bacterial protein involved in the biogenesis of c-type cytochromes. Its corresponding RNA sequence is fully edited at 34 positions. The second orf encodes a protein homologous to the amino-terminal third of E. coli ribosomal protein S1, corresponding to the ribosome-binding domain of this protein. Its RNA sequence is edited at four positions, one of the edits creating a stop codon. The presence of both proteins in wheat mitochondria was demonstrated using specific antibodies raised against fusion proteins obtained in E. coli from the corresponding cDNAs.

Carboxylation and dephosphorylation of phosphoenol-3-fluoropyruvate by maize leaf phosphoenolpyruvate carboxylase.

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.

Proximity between fluorescent probes attached to four essential lysyl residues in phosphoenolpyruvate carboxylase. A resonance energy transfer study.

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.

Stereoselectivity of the interaction of E- and Z-2-phosphoenolbutyrate with maize leaf phosphoenolpyruvate carboxylase.

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.

Changes in the quaternary structure of phosphoenolpyruvate carboxylase induced by ionic strength affect its catalytic activity.

Phosphoenolpyruvate carboxylase from maize leaves dissociated into dimers and/or monomers when exposed to increasing ionic strength (e.g. 200-400 mM NaCl) as indicated by gel filtration experiments. Changes in the oligomerization state were dependent on pH, time of preincubation with salt and protein concentration. A dissociation into dimers and monomers was observed at pH 8, while at pH 7 dissociation into the dimeric form only was observed. Exposure of the enzyme to higher ionic strength decreased the activity in a time-dependent manner. Turnover conditions and glucose 6-phosphate protected the carboxylase from the decay in activity, which was faster at pH 7 than at pH 8. The results suggest that changes in activity of the enzyme, following exposure to high ionic strength, are the consequence of dissociation. Tetrameric and dimeric forms of the phosphoenolpyruvate carboxylase seemingly reveal different catalytic properties. We suggest that the distinct catalytic properties of the different oligomeric species of phosphoenolpyruvate carboxylase and changes in the equilibrium between them could be the molecular basis for an effective regulation of metabolite levels by this key enzyme of C4 plants.

Interaction of acetyl phosphate and carbamyl phosphate with plant phosphoenolpyruvate carboxylase.

Acetyl phosphate produced an increase in the maximum velocity (Vmax. for the carboxylation of phosphoenolpyruvate catalysed by phosphoenolpyruvate carboxylase. The limiting Vmax. was 22.2 mumol X min-1 X mg-1 (185% of the value without acetyl phosphate). This compound also decreased the Km for phosphoenolpyruvate to 0.18 mM. The apparent activation constants for acetyl phosphate were 1.6 mM and 0.62 mM in the presence of 0.5 and 4 mM-phosphoenolpyruvate respectively. Carbamyl phosphate produced an increase in Vmax. and Km for phosphoenolpyruvate. The variation of Vmax./Km with carbamyl phosphate concentration could be described by a model in which this compound interacts with the carboxylase at two different types of sites: an allosteric activator site(s) and the substrate-binding site(s). Carbamyl phosphate was hydrolysed by the action of phosphoenolpyruvate carboxylase. The hydrolysis produced Pi and NH4+ in a 1:1 relationship. Values of Vmax. and Km were 0.11 +/- 0.01 mumol of Pi X min-1 X mg-1 and 1.4 +/- 0.1 mM, respectively, in the presence of 10 mM-NaHCO3. If HCO3- was not added, these values were 0.075 +/- 0.014 mumol of Pi X min-1 X mg-1 and 0.76 +/- 0.06 mM. Vmax./Km showed no variation between pH 6.5 and 8.5. The reaction required Mg2+; the activation constants were 0.77 and 0.31 mM at pH 6.5 and 8.5 respectively. Presumably, carbamyl phosphate is hydrolysed by phosphoenolpyruvate carboxylase by a reaction the mechanism of which is related to that of the carboxylation of phosphoenolpyruvate.

Active-site-directed inhibition of phosphoenolpyruvate carboxylase from maize leaves by bromopyruvate.

Bromopyruvate is a competitive inhibitor of maize leaf phosphoenolpyruvate carboxylase with respect to phosphoenolpyruvate (Ki: 2.3 mM at pH 8). Relatively low concentrations of this compound completely and irreversibly inactivated the enzyme. The inactivation followed pseudo-first-order kinetics. The haloacid combines first with the carboxylase to give a reversible enzyme-bromopyruvate complex and then alkylates the enzyme. The maximum inactivation rate constant was 0.27 min-1 at pH 7.2 and 30 degrees C and the concentration of bromopyruvate giving half-maximum rate of inactivation was 1.8 mM. The inactivation was prevented by the substrate phosphoenolpyruvate, in the absence or presence of MgCl2, and by the competitive inhibitor P-glycolate. Malate afforded protection at pH 7 but not at pH 8. MgCl2 enhanced the inactivation when it was carried out at pH 7; its effect was mainly due to a decrease in the dissociation constant of the complex between bromopyruvate and the enzyme from 2 to 1.4 mM. This behavior was not observed at pH 8. Analysis of the inactivation at different pH suggests that a group of pKa near 7.5 is important for the binding of the reagent to the carboxylase. Determination of the number of sulfhydryl groups of the native and modified enzyme with [3H]-N-ethylmaleimide suggests that the inactivation correlates with the modification of thiol groups in the enzyme. The substrate prevented the modification of these groups. The results suggest that the alkylating reagent modifies cysteinyl residues at the phosphoenolpyruvate binding site of the carboxylase.