Avaliação da influência de três diferentes configurações de câmaras apicais de implantes zigomáticos na resistência à deformação frente às cargas oblíquas: um estudo comparativo / Influence evaluation of three different configurations of chambers apicals of zigomatic implant in resitance to the deformation with oblique load application: comparative study

A reabilitação protética de pacientes com acentuada reabsorção do rebordo ósseo alveolar no segmento posterior da maxila, é frequentemente um desafio profissional, devido ao insuficiente suporte ósseo protético. O desenvolvimento dos implantes zigomáticos e seu alto índice de sucesso como relatado por Branemark (1999), tem sido um tratamento alternativo, que permite uma ancoragem protética mais rápida e permanente, para o segmento posterior da maxila, eliminando a necessidade de enxertos ósseos adicionais. Uma variedade de implantes tem sido desenvolvidos com este propósito, apresentando diferentes configurações nas câmaras apicais, que são responsáveis por fixar o implante zigomático no osso após a sua colocação. O propósito deste estudo foi avaliar a resistência mecânica de três tipos de câmaras apicais nestes implantes: a) uma câmara de forma elíptica com 1,5mm de diâmetro na sua maior dimensão; b) uma câmara com 1.0 mm de dimensão, e a câmara denominada PCP. Estes implantes foram submetidos às cargas oblíquas em 45 graus. Os resultados do teste de Duncan mostraram que a câmara mais resistente à força aplicada foi a de configuração PCP, posteriormente a de 1,0 mm de diâmetro seguida pela câmara de 1,5 mm.

The prosthetic rehabilitation of patients presenting accentuated alveolar ridge atrophy in the posterior maxillary segments is often challenging due to insufficient posterior prosthetic support. The development of and the high success rates of Zygomatic implants reported by Branemark (1999) has provided a treatment alternative that allows faster and permanent prosthetic anchorage for posterior maxillary segments, eliminating the need of adjunct bone grafting procedures. A variety of implant systems have been developed for this purpose, with different configurations in the apical chamber, which is responsible for locking the implant in the Zygomatic bone following insertion. The aim of this study was to evaluate the mechanical resistance of three types of apical chambers: 1.5 mm oblong chamber, 1.0 mm oblong cham.

Protein-binding partners of the tobacco syntaxin NtSyr1.

Syntaxins and other SNARE (soluble NSF-attachment protein receptor) complex proteins play a key role in the cellular processes of vesicle trafficking, vesicle fusion and secretion. Intriguingly, the SNARE NtSyr1 (=NtSyp121) from Nicotiana tabacum also appears to have a role in signalling evoked by the plant stress hormone abscisic acid. However, partner proteins contributing to its function(s) remain unknown. We used an affinity chromatography approach to identify proteins from tobacco leaf microsomes that directly interact with the hydrophilic (cytosolic) domains of NtSyr1 and report several interacting proteins with sensitivities to the endopeptidase activity of Clostridium botulinum neurotoxins, including one protein that was recognised by alphaAtSNAP33 antiserum, raised against the Arabidopsis SNAP25 homologue. Treatment of microsomal membrane fractions indicated a protein near 55 kDa was sensitive to proteolysis by BotN/A and BotN/E, yielding degradation products of approximately 34 and 23 kDa. Expressed and purified AtSNAP33 also bound directly to the cytosolic domain of NtSyr1 and was sensitive to proteolysis by these toxins, suggesting that NtSyr1, a tobacco homologue of AtSNAP33, and coordinate SNAREs are likely to associate as partners for function in vivo.

Localization and control of expression of Nt-Syr1, a tobacco SNARE protein.

Syntaxins and other SNARE proteins are crucial for intracellular vesicle trafficking, fusion and secretion. Previously, we isolated the syntaxin-related protein Nt-Syr1 from Nicotiana in a screen for ABA-related signalling elements, and demonstrated its role in determining the ABA sensitivity of stomatal guard cells. Because the location and expression of SNAREs are often important clues to their functioning, we have examined the distribution and stimulus-dependent expression of Nt-Syr1 between tissues, as well as its location within the cell, using antisera raised against purified recombinant peptides corresponding to overlapping cytosolic domains of Nt-Syr1. The Nt-Syr1 epitope was strongly represented in roots and to lesser extents in stems, leaves and flowers of well-watered plants. Biochemical analysis and examination of immunogold labelling under the electron microscope indicated Nt-Syr1 to be located primarily at the plasma membrane. Expression of the protein in leaves and to a lesser extent in flowers and stems was transiently enhanced by ABA, but not by auxin, kinetin or gibberellic acid. Expression in leaves was promoted by salt stress and wounding, but not by cold. By contrast, Nt-Syr1 levels in the root were unaffected by ABA. In the leaves, enhanced expression of Nt-Syr1 by salt stress was not observed in aba1 mutant Nicotiana, which is deficient in ABA synthesis, and in plants carrying the Arabidopsis abi1 transgene that suppresses a number of ABA-evoked responses in these plants. However, an enhanced expression in response to wounding was observed, even in the mutant backgrounds. We conclude that Nt-Syr1 expression at the plasma membrane is important for its function and is subject to control by parallel, stress-related signalling pathways, both dependent on and independent of ABA. Nt-Syr1 may be associated with additional functions, especially in the roots, that are unrelated to ABA or stress responses in the plant.

Overexpression of auxin-binding protein enhances the sensitivity of guard cells to auxin.

To explore the role of auxin-binding protein (ABP1) in planta, a number of transgenic tobacco (Nicotiana tabacum) lines were generated. The wild-type KDEL endoplasmic reticulum targeting signal was mutated to HDEL, another common retention sequence in plants, and to KEQL or KDELGL to compromise its activity. The auxin-binding kinetics of these forms of ABP1 were found to be similar to those of ABP1 purified from maize (Zea mays). To test for a physiological response mediated by auxin, intact guard cells of the transgenic plants were impaled with double-barreled microelectrodes, and auxin-dependent changes in K(+) currents were recorded under voltage clamp. Exogenous auxin affected inwardly and outwardly rectifying K(+) currents in a dose-dependent manner. Auxin sensitivity was markedly enhanced in all plants overexpressing ABP1, irrespective of the form present. Immunogold electron microscopy was used to investigate the localization of ABP1 in the transgenic plants. All forms were detected in the endoplasmic reticulum and the KEQL and KDELGL forms passed further across the Golgi stacks than KDEL and HDEL forms. However, neither electron microscopy nor silver-enhanced immunogold epipolarization microscopy revealed differences in cell surface ABP1 abundance for any of the plants, including control plants, which indicated that overexpression of ABP1 alone was sufficient to confer increased sensitivity to added auxin. Jones et al. ([1998] Science 282: 1114-1117) found increased cell expansion in transgenic plants overexpressing wild-type ABP1. Single cell recordings extend this observation, with the demonstration that the auxin sensitivity of guard cell K(+) currents is mediated, at least in part, by ABP1.

Early signalling events in the Avr9/Cf-9-dependent plant defence response.

Abstract Resistance of tomato to the leaf mould fungus Cladosporium fulvum is controlled by the interaction between a plant-encoded resistance gene (Cf-9) and pathogen-encoded avirulence (Avr9) gene. Our objective is to understand the underlying molecular mechanisms that transmit the Cf-9/Avr9-dependent pathogen perception event and activate the plant defence response. Our approach toward the understanding of Cf-function is based on the analysis of early Cf-9/Avr9-mediated responses and signalling events. Because Cf-9 transgenically expressed in tobacco retains its specificity and activity to the Avr9 elicitor, signalling experiments were conducted in the heterologous system using these transgenic lines or derived Cf9 tobacco cell cultures. Among the earliest responses to the Avr9/Cf-9 elicitation event were rapid changes in ion-fluxes, the synthesis of active oxygen species (AOS), probably catalysed by a plant NADPH-oxidase, and the transient activation of two MAP kinases. These kinases were identified as WIPK (wounding-induced protein kinase) and SIPK (salicylic-acid induced kinase) from tobacco. Studies with pharmacological inhibitors suggested that the MAP kinases are located in an independent signalling pathway from the Avr9/Cf-9-dependent synthesis of AOS. SIPK and WIPK were involved in pathogen-related elicitation processes as well as in abiotic stress responses. This indicates that the plant defence is triggered via a signalling network that shares components with pathways originating from abiotic environmental stress stimuli.

K+ channels of Cf-9 transgenic tobacco guard cells as targets for Cladosporium fulvum Avr9 elicitor-dependent signal transduction.

The Cf-9 gene encodes an extracytosolic leucine-rich repeat (LRR) protein that is membrane anchored near its C-terminus. The protein confers resistance in tomato to races of the fungus Cladosporium fulvum expressing the corresponding avirulence gene Avr9. In Nicotiana tabacum the Cf-9 transgene confers sensitivity to the Avr9 elicitor, and leads on elicitation to a subset of defence responses qualitatively similar to those normally seen in the tomato host. One of the earliest responses, both in the native and transgenic hosts, results in K+ salt loss from the infected tissues. However, the mechanism(s) underlying this solute flux and its control is poorly understood. We have explored the actions of Avr9 on Cf-9 transgenic Nicotiana using guard cells as a model. Much detail of guard cell ion channels and their regulation is already known. Measurements were carried out on intact guard cells in epidermal peels, and the currents carried by inward- (IK,in) and outward-rectifying (IK,out) K+ channels were characterized under voltage clamp. Exposures to Avr9-containing extracts resulted in a 2.5- to 3-fold stimulation of IK,out and almost complete suppression of IK,in within 3-5 min. The K+ channel responses were irreversible. They were specific for the Avr9 elicitor, were not observed in guard cells of Nicotiana lacking the Cf-9 transgene and, from kinetic analyses, could be ascribed to changes in channel gating. Both K+ channel responses were found to be saturable functions of Avr9 concentration and were completely blocked in the presence of 0.5 microM staurosporine and 100 microM H7, both broad-range protein kinase antagonists. These results demonstrate the ability of the Cf-9 transgene to couple Avr9 elicitation specifically to a concerted action on two discrete K+ channels and they indicate a role for protein phosphorylation in Avr9/Cf-9 signal transduction leading to transport control.

A tobacco syntaxin with a role in hormonal control of guard cell ion channels.

The plant hormone abscisic acid (ABA) regulates potassium and chloride ion channels at the plasma membrane of guard cells, leading to stomatal closure that reduces transpirational water loss from the leaf. The tobacco Nt-SYR1 gene encodes a syntaxin that is associated with the plasma membrane. Syntaxins and related SNARE proteins aid intracellular vesicle trafficking, fusion, and secretion. Disrupting Nt-Syr1 function by cleavage with Clostridium botulinum type C toxin or competition with a soluble fragment of Nt-Syr1 prevents potassium and chloride ion channel response to ABA in guard cells and implicates Nt-Syr1 in an ABA-signaling cascade.

The role of ABI1 in abscisic acid signal transduction: from gene to cell.

The semi-dominant abi1-1 mutation of Arabidopsis interferes with multiple aspects of abscisic acid signal transduction resulting in reduced seed dormancy and sensitivity of root growth in ABA. Furthermore, the mutant transpires excessively as a result of abnormal stomatal regulation leading to a wilty phenotype. The ABI1 gene has been cloned. The carboxyl-terminal domain of the predicted ABI1 protein is related to the 2C class of serine-threonine phosphatases while no overt homology was found in the extended amino terminus. A combination of in vitro assays and yeast mutant complementation studies confirmed that ABI1 is a functional protein phosphatase 2C. The abi1-1 mutation converts the amino acid glycine180 to aspartic acid, and in the above test systems, causes a partial loss of the phosphatase activity. In transgenic Nicotiana benthamiana guard cells, the abi1-1 gene causes a reduction in the background current of the outward-rectifying potassium channels, and also in the abscisic acid-sensitivity of both the outward- and the inward-rectifying potassium channels in the plasma membrane. However, normal sensitivity of both potassium channels to, and stomatal closure in, abscisic acid was recovered in the presence of H7 and staurosporine, both broad-range protein kinase antagonists. These results suggest the aberrant potassium channel behavior as a major consequence of abi1-1 action and implicate ABI1 as part of a phosphatase/kinase pathway that modulates the sensitivity of guard-cell potassium channels to abscisic acid-evoked signal cascades.

Alteration of anion channel kinetics in wild-type and abi1-1 transgenic Nicotiana benthamiana guard cells by abscisic acid.

The influence of the plant water-stress hormone abscisic acid (ABA) on anion channel activity and its interaction with protein kinase and phosphatase antagonists was examined in stomatal guard cells of wild-type Nicotiana benthamiana L. and of transgenic plants expressing the dominant-negative (mutant) Arabidopsis abi1-1 protein phosphatase. Intact guard cells were impaled with double-barrelled micro-electrodes and membrane current was recorded under voltage clamp in the presence of 15 mM CsCl and 15 mM tetraethylammonium chloride (TEA-Cl) to eliminate K+ channel currents. Under these conditions, the free-running voltage was situated close to 0 mV (+9 +/- 6 mV, n = 18) and the membrane under voltage clamp was dominated by anion channel current (ICl) as indicated from tall current reversal near the expected chloride equilibrium potential, current sensitivity to the anion channel blockers 9-anthracene carboxylic acid and niflumic acid, and by its voltage-dependent kinetics. Pronounced activation of ICl was recorded on stepping from a conditioning voltage of -250 mV to voltages between -30 and +50 mV, and the current deactivated with a voltage-dependent halftime at more negative voltages (tau approximately equal to 0.3 sec at -150 mV). Challenge with 20 microM ABA increased the steady-state current conductance, gCl, near 0 mV by 1.2- to 2.6-fold and at -150 mV by 4.5- to sixfold with a time constant of 40 +/- 4 sec, and it slowed ICl deactivation as much as fourfold at voltages near -50 mV, introducing two additional voltage-sensitive kinetic components to these current relaxations. Neither the steady-state and kinetic characteristics of ICl nor its sensitivity to ABA were influenced by H7 or staurosporine, both broad-range protein kinase antagonists. However, the protein phosphatase 1/2A antagonist calyculin A mimicked the effects of ABA on gCl and current relaxations on its own and exhibited a synergistic interaction with ABA, enhancing ICl sensitivity to ABA three- to four-fold. Quantitatively similar current characteristics were recorded from guard cells of abi1-1 transgenic N. benthamiana, indicating that the abi1-1 protein phosphatase does not influence the anion current or its response to ABA directly. These results demonstrate that ABA stimulates ICl and modulates its voltage sensitivity. Furthermore, they show that ABA promotes ICl, either by introducing additional long-lived states of the channel or by activating a second anion channel with similar permeation characteristics but with a very long dwell time in the open state. Overall, the data are broadly consistent with the view that ABA action engenders coordinate control of ICl together with guard cell K+ channels to effect solute loss and stomatal closure.

Response to treatment with progesterone in a patient with pulmonary lymphangioleiomyomatosis.

Lymphangioleiomyomatosis (LAM) is defined as an abnormal proliferation of smooth muscles around lymphatics, venules, and brochioles. This article describes our experiences treating a 21-year-old, white female who experienced recurrent shortness of breath during air travel last year. Her episode was severe and the patient was transported to the hospital as soon as the airplane landed. Physical exam in the emergency room was significant for absent breath sounds on the right side and the chest X-ray revealed a pneumothorax. She required two chest tubes for complete lung re-expansion. Further evaluation showed an obstructive pattern and air trapping on pulmonary function tests. This patient was treated with Medroxyprogesterone acetate (MPA) for six months and subsequent pulmonary function tests revealed improvement in her condition.

Sensitivity to abscisic acid of guard-cell K+ channels is suppressed by abi1-1, a mutant Arabidopsis gene encoding a putative protein phosphatase.

Abscisic acid (ABA) modulates the activities of three major classes of ion channels--inward- and outward-rectifying K+ channels (IK,in and IK,out, respectively) and anion channels--at the guard-cell plasma membrane to achieve a net efflux of osmotica and stomatal closure. Disruption of ABA sensitivity in wilty abi1-1 mutants of Arabidopsis and evidence that this gene encodes a protein phosphatase suggest that protein (de)-phosphorylation contributes to guard-cell transport control by ABA. To pinpoint the role of ABI1, the abi1-1 dominant mutant allele was stably transformed into Nicotiana benthamiana and its influence on IK,in, IK,out, and the anion channels was monitored in guard cells under voltage clamp. Compared with guard cells from wild-type and vector-transformed control plants, expression of the abi1-1 gene was associated with 2- to 6-fold reductions in IK,out and an insensitivity of both IK,in and IK,out to 20 microM ABA. In contrast, no differences between control and abi1-1 transgenic plants were observed in the anion current or its response to ABA. Parallel measurements of intracellular pH (pHi) using the fluorescent dye 2',7'-bis(2-carboxyethyl)-5-(and -6)-carboxyfluorescein (BCECF) in every case showed a 0.15- to 0.2-pH-unit alkalinization in ABA, demonstrating that the transgene was without effect on the pHi signal that mediates in ABA-evoked K+ channel control. In guard cells from the abi1-1 transformants, normal sensitivity of both K+ channels to and stomatal closure in ABA was recovered in the presence of 100 microM H7 and 0.5 microM staurosporine, both broad-range protein kinase antagonists. These results demonstrate an aberrant K+ channel behavior--including channel insensitivity to ABA-dependent alkalinization of pHi--as a major consequence of abi1-1 action and implicate AB11 as part of a phosphatase/kinase pathway that modulates the sensitivity of guard-cell K+ channels to ABA-evoked signal cascades.

Peptides derived from the auxin binding protein elevate Ca2+ and pH in stomatal guard cells of Vicia faba: a confocal fluorescence ratio imaging study.

Dual-excitation confocal laser scanning microscopy (CLSM) was used to image the pH-indicator, BCECF, iontophoretically microinjected into stomatal guard cells of Vicia faba during challenge with peptides derived from hydrophilic domains of the maize auxin-binding protein. Only the peptide corresponding to the C-terminal end (Pz151-163) caused significant changes in cytosolic pH, stimulating rapid alkalinisation of 0.4 +/- 0.1 pH units. Cytosolic pH was clamped using the permeant weak acid, butyrate, and this treatment buffered the peptide evoked alkalinisation. In concert with the electrical events monitored at the plasma membrane using whole-cell voltage clamp, this provides strong evidence for a role of [H+] as a signal intermediate in the guard cell transduction network. In preliminary experiments using single-wavelength imaging of the calcium-indicator, Fluo-3, Pz151-163 also stimulated rapid, reversible increases in cytosolic calcium, whilst two other peptides tested had no effect.

K+ channels of stomatal guard cells. Characteristics of the inward rectifier and its control by pH.

Intracellular microelectrode recordings and a two-electrode voltage clamp have been used to characterize the current carried by inward rectifying K+ channels of stomatal guard cells from the broadbean, Vicia faba L. Superficially, the current displayed many features common to inward rectifiers of neuromuscular and egg cell membranes. In millimolar external K+ concentrations (Ko+), it activated on hyperpolarization with half-times of 100-200 ms, showed no evidence of time- or voltage-dependent inactivation, and deactivated rapidly (tau approximately 10 ms) on clamping to 0 mV. Steady-state conductance-voltage characteristics indicated an apparent gating charge of 1.3-1.6. Current reversal showed a Nernstian dependence on Ko+ over the range 3-30 mM, and the inward rectifier was found to be highly selective for K+ over other monovalent cations (K+ greater than Rb+ greater than Cs+ much greater than Na+). Unlike the inward rectifiers of animal membranes, the current was blocked by charybdotoxin and alpha-dendrotoxin (Kd much less than 50 nM), as well as by tetraethylammonium chloride (K1/2 = 9.1 mM); gating of the guard cell K+ current was fixed to voltages near -120 mV, independent of Ko+, and the current activated only with supramillimolar K+ outside (EK+ greater than -120 mV). Most striking, however, was inward rectifier sensitivity to [H+] with the K+ current activated reversibly by mild acid external pH. Current through the K+ inward rectifier was found to be largely independent of intracellular pH and the current reversal (equilibrium) potential was unaffected by pHo from 7.4 to 5.5. By contrast, current through the K+ outward rectifier previously characterized in these cells (1988. J. Membr. Biol. 102:235) was largely insensitive to pHo, but was blocked reversibly by acid-going intracellular pH. The action of pHo on the K+ inward rectifier could not be mimicked by extracellular Ca2+ for which changes in activation, deactivation, and conductance were consonant with an effect on surface charge ([Ca2+] less than or equal to 1 mM). Rather, extracellular pH affected activation and deactivation kinetics disproportionately, with acid-going pHo raising the K+ conductance and shifting the conductance-voltage profile positive-going along the voltage axis and into the physiological voltage range. Voltage and pH dependencies for gating were consistent with a single, titratable group (pKa approximately 7 at -200 mV) residing deep within the membrane electric field and accessible from the outside.(ABSTRACT TRUNCATED AT 400 WORDS)

Voltage dependence of the Chara proton pump revealed by current-voltage measurement during rapid metabolic blockade with cyanide.

It is generally agreed that solute transport across the Chara plasma membrane is energized by a proton electrochemical gradient maintained by an H(+)-extruding ATPase. Nonetheless, as deduced from steady-state current-voltage (I-V) measurements, the kinetic and thermodynamic constraints on H(+)-ATPase function remain in dispute. Uncertainties necessarily surround long-term effects of the relatively nonspecific antagonists used in the past; but a second, and potentially more serious problem has sprung from the custom of subtracting, across the voltage spectrum, currents recorded following pump inhibition from currents measured in the control. This practice must fail to yield the true I-V profile for the pump when treatments alter the thermodynamic pressure on transport. We have reviewed these issues, using rapid metabolic blockade with cyanide and fitting the resultant whole-cell I-V and difference-current-voltage (dI-V) relations to a reaction kinetic model for the pump and parallel, ensemble leak. Measurements were carried out after blocking excitation with LaCl3, so that steady-state currents could be recorded under voltage clamp between -400 and +100 mV. Exposures to 1 mM NaCN (CN) and 0.4 mM salicylhydroxamic acid (SHAM) depolarized (positive-going) Chara membrane potentials by 44-112 mV with a mean half time of 5.4 +/- 0.8 sec (n = 13). ATP contents, which were followed in parallel experiments, decayed coincidently with a mean half time of 5.3 +/- 0.9 sec [( ATP]t = 0, 0.74 +/- 0.3 mM; [ATP]t = infinity, 0.23 +/- 0.02 mM). Current-voltage response to metabolic blockade was described quantitatively in context of these changes in ATP content and the consequent reduction in pump turnover rate accompanied by variable declines in ensemble leak conductance. Analyses of dI-V curves (+/- CN + SHAM) as well as of families of I-V curves taken at times during CN + SHAM exposures indicated a stoichiometry for the pump of one charge (H+) transported per ATP hydrolyzed and an equilibrium potential near -420 mV at neutral external pH; under these conditions, the pump accounted for approximately 60-75% of the total membrane conductance near Vm. Complementary results were obtained also in fitting previously published I-V data gathered over the external pH range 4.5-7.5. Kinetic features deduced for the pump were dominated by a slow step preceding H+ unloading outside, and by recycling and loading steps on the inside which were in rapid equilibrium.(ABSTRACT TRUNCATED AT 400 WORDS)