RESUMEN

alpha1-Proteinase inhibitor (alpha1-PI) is the main serine proteinase inhibitor in human plasma. Apart from its synthesis in the liver, this anti-inflammatory protein is also synthesized by and excreted from human intestinal epithelial cells. Antiinflammatory actions of alpha1-PI are thought to be of relevance in the pathogenesis of inflammatory bowel disease. To investigate the role of macrophage-derived cytokines on alpha1-PI secretion from intestinal epithelial cells, we cultured Caco-2 cells until differentiation (14 days in culture) on permeable filter supports. Monolayers of differentiated Caco-2 cells were then co-cultured with human peritoneal macrophages, grown on plastic in the basolateral chamber. Under these conditions, alpha1-PI secretion from Caco-2 cells was enhanced by 45%, probably by a direct action of macrophage-derived cytokines on Caco-2 cells. To extend this observation further, we treated differentiated Caco-2 cells with macrophage-derived proinflammatory cytokines (IL-1beta, IL-8, TNF-alpha), as well as with lymphocyte-derived cytokines IL-2, IL-6 and IFN-gamma. As early as after 24h treatment, IL-2 and IL-8 induced a significant and dose-dependent increase of alpha-1-PI secretion into cell culture medium; this effect was completely reversed after immunoneutralization by the antibodies against IL-2 and IL-8 alpha1-PI secretion was only slightly decreased after treatment with IFN-gamma, while IL-1beta, IL-6 and TNF-alpha had no effect. alpha1-PI secretion correlated well with the expression of this protein in differentiated Caco-2 cells after cytokine treatment, as confirmed by Western blot. Our data imply that, in vitro, alpha1-PI secretion in enterocyte-like Caco-2 cells is up-regulated by IL-2 and IL-8. Our results suggest that both lymphocyte- and macrophage-derived cytokines regulate secretion of the anti-inflammatory protein alpha1-PI in intestinal epithelial cells.

Asunto(s)

Citocinas/metabolismo , Mucosa Intestinal/metabolismo , Macrófagos Peritoneales/metabolismo , alfa 1-Antitripsina/metabolismo , Células CACO-2 , Comunicación Celular/inmunología , Técnicas de Cocultivo , Citocinas/inmunología , Citocinas/farmacología , Humanos , Inmunidad Mucosa , Enfermedades Inflamatorias del Intestino/etiología , Enfermedades Inflamatorias del Intestino/inmunología , Mucosa Intestinal/inmunología , Macrófagos Peritoneales/inmunología , alfa 1-Antitripsina/inmunología

RESUMEN

To explore possible pathways for anions to enter the xylem in the root during the transport of salts to the shoot, we used the patch-clamp method on protoplasts prepared from the xylem parenchyma of barley (Hordeum vulgare L.) plants. K(+) currents were suppressed by tetraethylammonium or N-methylglucamine in the solutions in the pipette and the bath, and the permeating anions were Cl(-) or NO(3)(-). We recorded the activities of three distinct anion conductances: (a) an inwardly rectifying anion channel (X-IRAC), characterized by activation at hyperpolarization and open times of up to several seconds; (b) a quickly activating anion conductance (X-QUAC), important for anion efflux at voltages between -50 mV and the equilibrium potential of the prevailing anion; and (c) a slowly activating anion conductance (X-SLAC), activating above -100 mV. Both X-IRAC and X-QUAC were permeable for Cl(-) and NO(3)(-); X-QUAC was also permeable for malate. The occurrence of X-IRAC became more frequent with an increase in cytoplasmic Ca(2+), while the occurrence of X-QUAC decreased. Anion currents through X-SLAC, and particularly through X-QUAC, were estimated to be large enough to account for reported rates of xylem loading, which is in accordance with the notion that xylem loading is a passive process.

Asunto(s)

Aniones/metabolismo , Hordeum/metabolismo , Raíces de Plantas/metabolismo , Sales (Química)/metabolismo , Transporte Biológico , Canales Iónicos/metabolismo , Técnicas de Placa-Clamp , Protoplastos/metabolismo

RESUMEN

We wished to determine whether the capacity of the sugar uptake mechanisms of guard cells of the Argenteum mutant of pea (Pisum sativum L.) sufficed to support a concurrent stomatal opening movement. Sugar uptake by guard cell protoplasts was determined by silicone-oil-filtering centrifugation. The protoplasts took up [(14)C]glucose, [(14)C]fructose, and [(14)C]sucrose (Suc), apparently in symport with protons. Mannose, galactose, and fructose competed with Glc for transport by a presumed hexose carrier. The uptake of Glc saturated with a K(m) of 0.12 mM and a V(max) of 19 fmol cell(-1) h(-1). At external concentrations <1 mM, the uptake of Suc was slower than that of Glc. It exhibited a saturating component with a K(m) varying between 0.25 and 0.8 mM and a V(max) between 1 and 10 fmol cell(-1) h(-1), and at external concentrations >1 mM, a non-saturating component. At apoplastic sugar concentrations below 4 mM, sugar import was estimated to be mainly in the form of hexoses and too slow to support a simultaneous stomatal opening movement. If, however, during times of high photosynthesis and transpiration, the apoplastic Suc concentration rose and entered the range of non-saturating import, absorbed Suc could replace potassium malate as the osmoticum for the maintenance of stomatal opening.

RESUMEN

We report two infants with eosinophilic gastroenteritis (EG). This rare disease can mimic the clinical symptoms and US appearance of idiopathic hypertrophic pyloric stenosis (IHPS). US examination of the antropyloric region with a high-frequency linear transducer can assist in the differentiation of EG from IHPS, which is important because the therapeutic approaches are completely different. Eosinophilic gastroenteritis should be considered in the differential diagnosis of IHPS, especially when there has been an ineffective pyloromyotomy.

Asunto(s)

Eosinofilia/diagnóstico por imagen , Gastroenteritis/diagnóstico por imagen , Estenosis Pilórica/diagnóstico por imagen , Corticoesteroides/uso terapéutico , Diagnóstico Diferencial , Eosinofilia/complicaciones , Eosinofilia/tratamiento farmacológico , Femenino , Gastroenteritis/complicaciones , Gastroenteritis/tratamiento farmacológico , Humanos , Lactante , Recién Nacido , Ultrasonografía , Vómitos/etiología

RESUMEN

Abdominal cysts in girls are frequently observed by abdominal ultrasound (US) and are usually ovarian. In this case a cystic structure located in the right abdomen was seen in a female newborn without symptoms and was initially described as a possible ovarian cyst. Frequent US examinations showed an increase in volume and diameter, and temporary, recurring episodes of hyperbilirubinemia were observed. The US scans showed no relationship to the biliary tree. During a diagnostic laparoscopy, a cystic structure attached to the pyloric region was seen. A laparotomy revealed a cystic duplication of the stomach, which could be resected completely. The finding is discussed emphasizing the importance of clinical findings and diagnostic methods in the diagnosis and management of abdominal cystic masses in females.

Asunto(s)

Abdomen/diagnóstico por imagen , Quistes/diagnóstico por imagen , Hiperbilirrubinemia/complicaciones , Estómago/anomalías , Diagnóstico Diferencial , Femenino , Humanos , Recién Nacido , Estómago/diagnóstico por imagen , Ultrasonografía

RESUMEN

Potato (Solanum tuberosum cv. Désirée) plants expressing yeast invertase directed either to the apoplast, vacuole or cytosol were biochemically and physiologically characterised. All lines of transgenic plants showed similarities to plants growing under water stress. Transformants were retarded in growth, and accumulated hexoses and amino acids, especially proline, to levels up to 40-fold higher than those of the wild types. In all transformants rates of CO2 assimilation and leaf conductance were reduced. From the unchanged intercellular partial pressure of CO2 and apoplastic cis-abscisic acid (ABA) content of transformed leaves it was concluded that the reduced rate of CO2 assimilation was not caused by a limitation in the availability of CO2 for the ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco). In the transformants the amount of Rubisco protein was not reduced, but both activation state and carboxylation efficiency of photosynthesis were lowered. In vacuolar and cytosolic transformants this inhibition of Rubisco might be caused by a changed ratio of organic bound and inorganic phosphate, as indicated by a doubling of phosphorylated intermediates. But in apoplastic transformants the pattern of phosphorylated intermediates resembled that of leaves of water-stressed potato plants, although the cause of inhibition of photosynthesis was not identical. Whereas in water-stressed plants increased contents of the phytohormone ABA are supposed to mediate the adaptation to water stress, no contribution of ABA to reduction of photosynthesis could be detected in invertase transformants.

Asunto(s)

Proteínas Fúngicas/metabolismo , Glicósido Hidrolasas/metabolismo , Fotosíntesis , Ácido Abscísico/metabolismo , Dióxido de Carbono/metabolismo , Clorofila/metabolismo , Citosol , Proteínas Fúngicas/genética , Expresión Génica , Glicósido Hidrolasas/genética , Hexosas/metabolismo , Fenotipo , Fosforilación , Hojas de la Planta/metabolismo , Plantas Modificadas Genéticamente , Prolina/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Ribulosa-Bifosfato Carboxilasa/metabolismo , Solanum tuberosum/crecimiento & desarrollo , Solanum tuberosum/metabolismo , Vacuolas , Agua/metabolismo , Equilibrio Hidroelectrolítico , Levaduras/enzimología , beta-Fructofuranosidasa

RESUMEN

Dahlbäck et al. recently described in vitro resistance to the anticoagulant response of activated protein C (APC), in the majority of cases associated with the Arg506 to Gln point mutation in the factor V gene in thrombophilic patients. To determine to what extent this common gene mutation affects the risk of childhood stroke, its occurrence was prospectively investigated in a population of children with ischaemic stroke. Over a 2-year period the Arg506 to Gln mutation, factor V, protein C, protein S, antithrombin, antiphospholipid antibodies and lipoprotein (a) [Lp(a)] were measured in 14 infants and children with acute ischaemic stroke. Heterozygous factor V Leiden mutation (n = 4), homozygous factor V Leiden mutation (n = 1), protein C deficiency type I (n = 3) and increased Lp(a) (n = 2) were diagnosed in the children investigated. Seven of 14 patients showed an underlying disease and additionally risk factors were present in nine of 14 children. Data of this study indicate that deficiencies in the protein C anticoagulant pathway play an important role in the aetiology of childhood stroke. However, additional triggering factors may promote early manifestation of thromboembolism in children with inherited defects of clotting inhibitors.

Asunto(s)

Arginina/genética , Isquemia Encefálica/genética , Factor V/genética , Glutamina/genética , Mutación Puntual , Adolescente , Arteriopatías Oclusivas/genética , Isquemia Encefálica/sangre , Isquemia Encefálica/fisiopatología , Niño , Preescolar , Activación Enzimática , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Estudios Prospectivos , Proteína C/metabolismo , Factores de Riesgo , Tromboembolia/genética

RESUMEN

Resistance to activated protein C (APCR), in the majority of cases due to the point mutation Arg 506 Gln of the factor V gene, has emerged as the most important hereditary cause of venous thromboembolism. Using an activated thromboplastin time (aPTT) based method in the presence of APC together with a DNA technique based on the polymerase chain reaction, we investigated 37 children with venous (V: n=19) or arterial (A: n=18) thromboembolism and 196 age-matched healthy controls for the presence of this mutation. In the control group 10 children were detected to be heterozygous for the factor V Leiden mutation, indicating a prevalence of 5.1%. 10/19 children (52%) with venous thrombosis and 7/18 (38%) patients with arterial thromboembolism showed the common factor V gene mutation. Additional inherited coagulation disorders were found in 1/10 (V:10%) and 2/7 (A:28%) APC-resistant patients. Inherited coagulation disorders without APCR were diagnosed in 3/9 (V: 33%) and 2/11 (A:18%) children. Furthermore, we diagnosed exogenous risk factors in 6/10 (V: 60%) and 2/7 (A: 28%) children with thrombosis and APCR. These data are evidence that APCR combined with exogenous reasons may play an important role in the early manifestation of thromboembolism during infancy and childhood.

Asunto(s)

Trastornos de las Proteínas Sanguíneas/metabolismo , Embolia y Trombosis Intracraneal/etiología , Proteína C/metabolismo , Tromboembolia/etiología , Adolescente , Trastornos de las Proteínas Sanguíneas/complicaciones , Niño , Preescolar , Femenino , Humanos , Lactante , Recién Nacido , Angiografía por Resonancia Magnética , Masculino , Estudios Prospectivos , Tromboembolia/complicaciones , Tromboembolia/diagnóstico por imagen , Tomografía Computarizada por Rayos X , Ultrasonografía

RESUMEN

Fifty-seven sonograms of 18 children with Hashimoto thyroiditis were retrospectively assessed. Thirty-three examinations (in 12 patients) showed thyroid glands which were more reflective than adjacent strap muscles, 22 studies (in eight patients) demonstrated glands of equal reflectivity and two examinations (in one child) depicted thyroid glands of lower reflectivity than muscle. The most frequent ultrasound appearance consisted of 2-5 mm echopenic lesions generally distributed throughout large poorly-defined hyperreflective glands. In serial examinations no correlation was found between laboratory indexes of thyroid status and sonographic findings.

Asunto(s)

Glándula Tiroides/diagnóstico por imagen , Tiroiditis Autoinmune/diagnóstico por imagen , Niño , Femenino , Humanos , Masculino , Estudios Retrospectivos , Ultrasonografía

RESUMEN

Over the last ten years 62 children were operated for renal tumours. The therapeutic aim in Wilms' tumours is complete macroscopic resection of the primary tumour, assessing resectability as accurately as possible. Overestimating surgical possibilities may lead to intraoperative rupture of the tumour (three cases), while overestimating local tumour extent may result in too much preoperative chemotherapy, which resulted in the life-threatening complication of venous occlusive disease of the hepatic veins (VOD) in three infants. The assessment of resectability may become particularly problematic in bilateral (six cases) or multifocal Wilms' tumours (two cases), and in nephroblastomatosis (two cases), i.e. the persistence of embronal renal tissue, a facultatively precancerous lesion, which requires quarterly sonographic controls and which induced a second metachronous contralateral Wilms' tumour in one child.

Asunto(s)

Neoplasias Renales/cirugía , Nefrectomía , Tumor de Wilms/cirugía , Adolescente , Quimioterapia Adyuvante , Niño , Preescolar , Femenino , Humanos , Lactante , Riñón/patología , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/mortalidad , Neoplasias Renales/patología , Masculino , Estadificación de Neoplasias , Neoplasias Primarias Múltiples/tratamiento farmacológico , Neoplasias Primarias Múltiples/mortalidad , Neoplasias Primarias Múltiples/patología , Neoplasias Primarias Múltiples/cirugía , Lesiones Precancerosas/tratamiento farmacológico , Lesiones Precancerosas/mortalidad , Lesiones Precancerosas/patología , Lesiones Precancerosas/cirugía , Pronóstico , Tasa de Supervivencia , Tumor de Wilms/tratamiento farmacológico , Tumor de Wilms/mortalidad , Tumor de Wilms/patología

RESUMEN

Heterotaxy is a rare congenital disorder characterised by abnormal viscero-vascular situs with either left or right isomerism that usually coincides with complex cardiac malformation. A precise diagnosis must be made for the selection of the most appropriate treatment. The diagnosis and evaluation of these conditions have been dependent on invasive studies such as angiocardiography, although more recently magnetic resonance imaging (MRI) has been advocated. In paediatric patients MRI is far from ideal because of the need for sedation and monitoring during the examination. The authors reviewed the modalities used in the evaluation of heterotaxy with emphasis on the use of ultrasonography, including pulse and colour flow Doppler imaging. A series of 14 children with heterotaxy is presented; ten with left abdominal isomerism (polysplenia) and four with right isomerism (asplenia). Results of abdominal sonography were compared with those of echo- and angiocardiography, surgery and autopsy. Of the ten children with left abdominal isomerism, two had no cardiac abnormalities. All those with right isomerism had a complex vitium. One patient with left abdominal isomerism proved to have right atrial isomerism. Some new reproducible patterns in viscero-vascular derangement are reported. The predictable patterns of left and right isomerism are outlined and atypical features discussed.

Asunto(s)

Anomalías Múltiples/diagnóstico por imagen , Anomalías Múltiples/diagnóstico , Niño , Preescolar , Diagnóstico por Imagen , Femenino , Cardiopatías Congénitas/diagnóstico por imagen , Humanos , Lactante , Recién Nacido , Masculino , Situs Inversus/diagnóstico por imagen , Bazo/anomalías , Síndrome , Ultrasonografía Doppler en Color

RESUMEN

Xylem parenchyma cells are situated around the (apoplastic) xylem vessels and are involved in the control of the composition of the xylem sap by exporting and resorbing solutes. We investigated properties of the K+ inward rectifier in the plasma membrane of these cells by performing patch clamp experiments on protoplasts in the whole-cell configuration. Inward currents were sensitive to the K+ channel blocker TEA+ at a high concentration (20 mM). Barium, another "classical" K+ channel blocker, inhibited K+ currents with a Ki of about 1.3 mM. In contrast to guard cells, the cytosolic Ca2+ level proved to be ineffective in regulating the K+ conductance at hyperpolarization. External Ca2+ blocked currents weakly in a voltage-dependent manner. From instantaneous current-voltage curves, we identified a binding site in the channel pore with an electrical distance of about 0.2 to 0.5. Lanthanum ions reduced the inward current in a voltage-dependent manner and simultaneously displaced the voltage at which half of the channels are in the open state to more positive values. This finding was interpreted as resulting from a sum of two molecular effects, an interaction with the mouth of the channel that causes a reduction of current, and a binding to the voltage sensor, leading to a shielding of surface charges and, subsequently, a modulation of channel gating. A comparison between the K+ inward rectifier in xylem parenchyma cells, guard cells and KAT1 from Arabidopsis leads to the conclusion that these rectifiers form subtypes within one class of ion channels. The ineffectiveness of Ca2+ to control K+ influx in xylem parenchyma cells is interpreted in physiological terms.

Asunto(s)

Raíces de Plantas/metabolismo , Canales de Potasio de Rectificación Interna , Canales de Potasio/metabolismo , Potasio/metabolismo , Bario/farmacología , Calcio/farmacología , Membrana Celular/metabolismo , Electrofisiología , Hordeum , Cinética , Lantano/farmacología , Potenciales de la Membrana/fisiología , Técnicas de Placa-Clamp , Raíces de Plantas/efectos de los fármacos , Bloqueadores de los Canales de Potasio , Protoplastos , Tetraetilamonio , Compuestos de Tetraetilamonio/farmacología

Asunto(s)

Hidrocefalia/cirugía , Complicaciones Posoperatorias/etiología , Infecciones Relacionadas con Prótesis/etiología , Infecciones Estafilocócicas/etiología , Staphylococcus epidermidis , Absceso Subfrénico/etiología , Derivación Ventriculoperitoneal , Femenino , Humanos , Lactante , Complicaciones Posoperatorias/cirugía , Falla de Prótesis , Infecciones Relacionadas con Prótesis/cirugía , Reoperación , Infecciones Estafilocócicas/cirugía , Absceso Subfrénico/cirugía

RESUMEN

To identify mechanisms for the simultaneous release of anions and cations into the xylem sap in roots, we investigated voltage-dependent ion conductances in the plasmalemma of xylem parenchyma cells. We applied the patch-clamp technique to protoplasts isolated from the xylem parenchyma by differential enzymic digestion of steles of barley roots (Hordeum vulgare L. cv Apex). In the whole-cell configuration, three types of cation-selective rectifiers could be identified: (a) one activated at membrane potentials above about -50 mV; (b) a second type of outward current appeared at membrane potentials above +20 to +40 mV; (c) below a membrane potential of approximately -110 mV, an inward rectifier could be distinguished. In addition, an anion-specific conductance manifested itself in single-channel activity in a voltage range extending from about -100 to +30 mV, with remarkably slow gating. In excised patches, K+ channels activated at hyperpolarization as well as at depolarization. We suggest that salt is released from the xylem parenchyma into the xylem apoplast by simultaneous flow of cations and anions through channels, following electrochemical gradients set up by the ion uptake processes in the cortex and, possibly, the release and reabsorption of ions on their way to the xylem.

RESUMEN

Chloroplasts were isolated from ruptured guard-cell protoplasts of the Argenteum mutant of Pisum sativum L. and purified by centrifugation through a Percoll layer. The combined volume of the intact plastids and the uptake of phosphate were determined by silicone oil-filtering centrifugation, using tritiated water and [14C]sorbitol as membrane-permeating and nonpermeating markers and [32P]phosphate as tracer for phosphate. The affinities of the phosphate translocator for organic phosphates were assessed by competition with inorganic phosphate. The affinities for dihydroxyacetone phosphate, 3-phosphoglycerate (PGA), and phosphoenolpyruvate were in the same order as those reported for mesophyll chloroplasts of several species. However, the guard-cell phosphate translocator had an affinity for glucose-6-phosphate that was as high as that for PGA. Guard-cell chloroplasts share this property with amyloplasts from the root of pea (H.W. Heldt, U.I. Flugge, S. Borchert [1991] Plant Physiol 95: 341-343). An ability to import glucose-6-phosphate enables guard-cell chloroplasts to synthesize starch despite the reported absence of a fructose-1,6-bisphosphatase activity in the plastids, which would be required if only C3 phosphates could enter through the translocator.

RESUMEN

Slowly activating anion channel currents were discovered at micromolar 'cytoplasmic' Ca2+ during patch-clamp measurements on guard-cell protoplasts of Vicia faba and Xanthium strumarium. They activated at potentials as low as -200 mV, with time constants between 5 and 60 s, and no inactivation. The broad voltage dependence exhibited a current maximum near -40 mV. The single-channel open time was in the order of seconds, and the unitary conductance was 33 ps, similar to that of the already described 'quick' anion channel of guard cells. Because of its activity at low potentials, the slow anion channel may be essential for the depolarization of the plasmalemma that is required for salt efflux during stomatal closing.

Asunto(s)

Canales Iónicos/fisiología , Fenómenos Fisiológicos de las Plantas , Aniones , Fabaceae/citología , Fabaceae/fisiología , Potenciales de la Membrana , Células Vegetales , Plantas Medicinales , Protoplastos

RESUMEN

In potato plants (Solanum tuberosum), a chimeric yeast-derived invertase gene fused to a 35S cauliflower mosaic virus promoter has been expressed. The protein was targeted to the cell wall by using the signal peptide of proteinase inhibitor II fused to the amino terminus of the yeast invertase. The transformed plants had crinkled leaves, showed a reduced growth rate, and produced fewer tubers. Although in the apoplast of the leaves of the transformed plants the content of glucose and fructose rose by a factor of 20, and that of sucrose declined 20-fold, 98% of the carbohydrate in the phloem sap consisted of sucrose, demonstrating the strong specificity of phloem loading. In the leaf cells of the transformed plants, glucose, fructose, and amino acids, especially proline, were accumulated. Consequently, the osmolality of the cell sap rose from 250 to 350 mosmol/kg. Our results show that the observed 75% decrease of photosynthesis is not caused by a feedback regulation of sucrose synthesis and is accompanied by an increase in the osmotic pressure in the leaf cells. In the transformed plants, not only the amino acid to sucrose ratio in the phloem sap, but also the amino acid and protein contents in the tubers were found to be elevated. In the tubers of the transformed plants, the protein to starch ratio increased.

RESUMEN

Using the patch-clamp technique we discovered that the voltage dependent anion channels in the plasma membrane of guard cells are activated by a rise in cytoplasmic Ca2+ in the presence of nucleotides. Upon activation, these anion channels catalyse anion currents 10-20 times higher than in the inactivated state, thus shifting the plasma membrane from a K+ conducting state to an anion conducting state. Prolonged stimulation by depolarizing voltages results in the inactivation of the anion current (t1/2 = 10-12 s). We suggest that activation of the anion channel by Ca2+ and nucleotides is a key event in the regulation of salt efflux from guard cells during stomatal closure.

Asunto(s)

Adenosina Trifosfato/análogos & derivados , Calcio/farmacología , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Canales Iónicos/fisiología , Fenómenos Fisiológicos de las Plantas , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/farmacología , Ácido Egtácico/farmacología , Fabaceae/fisiología , Canales Iónicos/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Células Vegetales , Plantas Medicinales , Canales de Potasio/efectos de los fármacos , Canales de Potasio/fisiología , Protoplastos/fisiología

RESUMEN

Guard cell protoplasts of Pisum sativum show considerable NADP-dependent malate dehydrogenase (MDH) activity in darkness which can be enhanced severalfold by illumination or treatment with dithiothreitol (DTT). The question arose whether guard cells possess an NADP-MDH different from that present in the chloroplasts of the mesophyll (which is inactive in darkness or in the absence of DTT). MDH activities were determined in extracts of isolated protoplasts from mesophyll and epidermis, and in mechanically prepared epidermal pieces (with guard cells as the only living cells and no interference from proteases originating from the cell wall digesting enzymes). Guard cells possessed NAD-dependent MDHs of high activity and incomplete exclusion of NADP as a coenzyme. This NADP-dependent activity of the NAD-MDH(s) could not be stimulated by DTT or, inferentially, by light. The DTT- (and light-) dependent NADP-MDH represented 0.05% of the total protein of the guard cells and had a specific activity of 0.1 unit per milligram protein; both values are in the same range as the corresponding ones of the mesophyll cells. Agreement was also found in the extent of light activation, in subunit molecular weight, immunological cross-reactions, and in the behavior on an ion exchange column. The activity of the chloroplastic NADP-MDH in guard cells barely suffices to meet the malate requirement for stomatal opening in the light. It is therefore likely that NAD-MDHs residing in other compartments of the guard cells supplement the activity of the chloroplastic NADP-MDH particularly during stomatal opening in darkness.

RESUMEN

We investigated whether the reductive pentose phosphate path in guard cells of Pisum sativum had the capacity to contribute significantly to the production of osmotica during stomatal opening in the light. Amounts of ribulose 1,5-bisphophate carboxylase/oxygenase (Rubisco) were determined by the [(14)C]carboxyarabinitol bisphosphate assay. A guard cell contained about 1.2 and a mesophyll cell about 324 picograms of the enzyme; the ratio was 1:270. The specific activities of Rubisco in guard cells and in mesophyll cells were equal; there was no indication of a specific inhibitor of Rubisco in guard cells. Rubisco activity was 115 femtomol per guard-cell protoplast and hour. This value was different from zero with a probability of 0.99. After exposure of guard-cell protoplasts to (14)CO(2) for 2 seconds in the light, about one-half of the radioactivity was in phosphorylated compounds and <10% in malate. Guard cells in epidermal strips produced a different labelling pattern; in the light, <10% of the label was in phosphorylated compounds and about 60% in malate. The rate of solute accumulation in intact guard cells was estimated to have been 900 femto-osmol per cell and hour. If Rubisco operated at full capacity in guard cells, and hexoses were produced as osmotica, solutes could be supplied at a rate of 19 femto-osmol per cell and hour, which would constitute 2% of the estimated requirement. The capacity of guard-cell Rubisco to meet the solute requirement for stomatal opening in leaves of Pisum sativum is insignificant.