Salt-induced cooperativity in ATPase activity of plasma membrane-enriched fractions from cultured Citrus cells: kinetic evidence

ATPase activity was studied in plasma membrane-enriched fractions prepared from cultured Citrus sinensis L. cv. Osbeck cells. In general, properties of the plasma membrane ATPase from cultured cells, such as optimal pH and temperature. V_max and K_m were similar to those already observed in higher plants. The effects of high salt concentrations on ATPase activity were studied in membrane fractions derived from salt-sensitive and salt-tolerant cells grown in the presence or absence of salt. NaCl did not have an in vivo effect on V_max and the apparent K_m value for ATP. However, high concentrations of NaCl, or KCl, added in vitro, induced cooperativity in the enzyme and reduced the affinity of the enzyme for its substrate. Isoosmolar concentrations of sucrose or choline chloride failed to do so. Our results suggest that the plasma membrane ATPase of Citrus cells has more than one substrate-binding site on the native form of the enzyme which interact in the presence of salt and act independently in its absence.     

Genetic Analysis of Hispanic Individuals with Cystic Fibrosis

We have performed molecular genetic analyses of Hispanic individuals with cystic fibrosis (CF) in the southwestern United States. Of 129 CF chromosomes analyzed, only 46% (59/129) carry ΔF508. The G542X mutation was found on 5% (7/129) of CF chromosomes. The 3849+10kbC→T mutation, detected primarily in Ashkenazi Jews, was present on 2% (3/129). R1162X and R334W, mutations identified in Spain and Italy, each occurred on 1.6% (2/129) of CF chromosomes. W1282X and R553X were each detected once. G551D and N1303K were not found. Overall, screening for 22 or more mutations resulted in detection of only 58% of CF transmembrane conductance regulator gene mutations among Hispanic individuals. Analysis of KM19/XV2c haplotypes revealed an unusual distribution. Although the majority of ΔF508 mutations are on chromosomes of B haplotypes, the other CF mutations are on A and C haplotypes at higher-than-expected frequencies. These genetic analyses demonstrate significant differences between Hispanic individuals with CF and those of the general North American population. Assessment of carrier/affected risk in Hispanic CF individuals cannot, therefore, be based on the mutation frequencies found through studies of the general population but must be adjusted to better reflect the genetic makeup of this ethnic group. Further studies are necessary to identify the causative mutation(s) in this population and to better delineate genotype/phenotype correlations. These will enable counselors to provide more accurate genetic counseling.     

The involvement of a vanadate-sensitive ATPase in plasma membranes of a salt tolerant cyanobacterium

Plasma membranes of the marine cyanobacterium Spirulina subsalsa were tested for ATPase activity, and for involvement in salt stress. Transition of cells from saline to hypersaline medium enhances the respiratory activity associated with extrusion of Na⁺ and Cl⁻, and persisting salt stress induces synthesis of respiratory enzymes in the plasma membranes. The membranes possess an ATPase, specific for ATP and Mg²⁺ and sensitive to orthovanadate and dicyclohexylcarbodiimide. Immunoblot analysis of plasma membrane polypeptides from Spirulina subsalsa with anti- Arabidopsis H⁺-ATPase serum identified a single polypeptide of 100 kDa, which cross-reacted with the antibodies. An unusual feature of this ATPase is a specific stimulation by Na⁺ ions. Prolonged adaptation of S. subsals cells to hypersaline conditions induced an increase in ATPase activity in subsequent plasma membrane preparations, as well as a higher content of the 100 kDa polypeptide. It is suggested that the ATPase investigated is an H⁺-pump, which is involved in extrusion of Na⁺ and in conferring resistance to salt stress.     

Molecular characterization of salt-stress-associated protein in citrus: protein and cDNA sequence homology to mammalian glutathione peroxidases

A gene encoding for a citrus salt-stress-associated protein (Cit-SAP) was cloned from Citrus sinensis salt-treated cell suspension. The gene, designated csa, was isolated from a cDNA expression library. The partial amino acid sequence of the protein, as well as that deduced from the nucleotide sequence of csa, revealed a considerable homology to mammalian glutathione peroxidase (GP), and to clone 6P229 from tobacco protoplasts. The increased expression of Cit-SAP in NaCl-treated cultured citrus cells and in citrus plants irrigated with saline water, and its similarity to GP, raise the possibility that one of the effects of salt stress in plants may be the increase of the level of free radicals.     

Drought,heat and salt stress induce the expression of a citrus homologue of an atypical late-embryogenesis Lea5 gene

In a search for genes that are induced in citrus cell suspension in response to salt stress, a cDNA clone with high homology to cotton Lea5 gene was isolated. Data base analysis of the protein deduced from the nucleotide sequence indicates that, like in cotton, the protein from citrus contains regions with significant hydropathic character. The gene, designated C-Lea5, is expressed in citrus leaves as well as cell suspension. The steady-state level of C-Lea5 is increased in cell suspension that is grown in the presence of 0.2 M NaCl. This phenomenon is also observed in leaves of citrus plants irrigated with NaCl and in citrus seedlings which are exposed to drought and heat stress. We suggest that the osmotic stress resulted from elevated level of salt is responsible for the increase in the level of C-Lea5.     

Effect of supplemental oxygen on supramaximal exercise performance and recovery in cystic fibrosis

Shah, Ashish R., Thomas G. Keens, and David Gozal.Effect of supplemental oxygen on supramaximal exercise performance and recovery in cystic fibrosis. J. Appl.Physiol. 83(5): 1641-1647, 1997.The effects ofsupplemental O₂ on recovery fromsupramaximal exercise and subsequent performance remain unknown. Ifrecovery from exercise could be enhanced in individuals with chroniclung disease, subsequent supramaximal exercise performance could also be improved. Recovery from supramaximal exercise and subsequent supramaximal exercise performance were assessed after 10 min of breathing 100% O₂ or room air(RA) in 17 cystic fibrosis (CF) patients [25 ± 10 (SD) yrold, 53% men, forced expired volume in 1 s = 62 ± 21%predicted] and 17 normal subjects (25 ± 8 yr old, 59% men,forced expired volume in 1 s = 112 ± 15% predicted). Supramaximalperformance was assessed as the work of sustained bicycling at a loadof 130% of the maximum load achieved during a graded maximal exercise.Peak minute ventilation(E) andheart rate (HR) were lower in CF patients at the end of eachsupramaximal bout than in controls. In CF patients, single-exponentialtime decay constants indicated faster recovery of HR(_HR = 86 ± 8 and 73 ± 6 s in RA and O₂,respectively, P < 0.01). Similarly, fast and slow time constants of two-exponential equations providing thebest fit for ventilatory recovery were improved in CF patients duringO₂ breathing ( = 132.1 ± 10.5 vs. 82.5 ± 10.4 s; = 880.3 ± 300.1 vs. 368.6 ± 107.1 s,P < 0.01). However, no such improvements occurred in controls. Supramaximal performance after O₂ improved in CF patients (109 ± 6% of the 1st bout after O₂ vs. 94 ± 6% in RA, P < 0.01).O₂ supplementation had no effect on subsequent performance in controls (97 ± 3% inO₂ vs. 93 ± 3% in RA). Weconclude that supplemental O₂after a short bout of supramaximal exercise accelerates recovery andpreserves subsequent supramaximal performance in patients with CF.

     

Elevated α-Linolenic Acid Content in Extra-plastidial Membranes of Tomato Accelerates Wound-Induced Jasmonate Generation and Improves Tolerance to the Herbivorous Insects Heliothis peltigera and Spodoptera littoralis

Journal of Plant Growth Regulation - In tomato, desaturation of linoleic acid (18:2) to α-linolenic acid (18:3) is mediated in the plastidial membranes by the ω-3 fatty acid desaturases 7...     

Platelet-activating factor receptor modulates respiratory adaptation to long-term intermittent hypoxia in mice

During hypoxia, release of platelet-activating factor (PAF) and activation of its cognate receptor (PAFR) regulate neural transmission and are required for full expression of peak hypoxic ventilatory response (pHVR) but not hypercapnic ventilatory response. However, it is unclear whether PAFR underlie components of long-term ventilatory adaptations to hypoxia. To examine this issue, adult male PAFR(+/+) and PAFR(-/-) mice were exposed to intermittent hypoxia (IH) consisting of 90 s 21% O(2) and 90 s 10% O(2) for 30 days, and normoxic and hypoxic ventilatory patterns were assessed using whole body plethysmography. Starting at day 14 of IH, normoxic ventilation in PAFR(-/-) was reduced significantly compared with PAFR(+/+) mice (P < 0.001), the latter exhibiting a prominent long-term ventilatory facilitation (LTVF). However, IH-exposed PAFR(-/-) mice had markedly enhanced pHVR and hypoxic ventilatory decline that became similar to those of IH-exposed PAFR(+/+) mice. Thus we postulate that PAFR expression and/or function underlies critical components of IH-induced LTVF but does not play a role in the potentiation of the hypoxic ventilatory response after IH exposures.