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A novel ABA-hypersensitive mutant in Arabidopsis defines a genetic locus that confers tolerance to xerothermic stress.

Hot and dry air (harmattan or xerothermic climate) greatly inhibits plant growth, particularly flowering and seed setting of crops. Little is known about the mechanism of plant response to this extreme environmental stress due to the lack of valuable genetic resource. Here, we report the isolation and characteristics of a unique Arabidopsis mutant, hat1 (harmattan tolerant 1), which shows high tolerance to hot and dry air. Under normal growth conditions, the mutant does not differ in morphology and soil drought tolerance compared to the wild type. When subjected to high temperature (42 degrees C) and low humidity (10-15%), however, it could survive up to 6 days, while the wild type (Col-0) died after 24 h. The hat1 mutant also exhibits enhanced tolerance to soil drought, but only under xerothermic conditions. Mutant plants tightly close their stomata to retain water under xerothermic stress, and are more tolerant to high salinity at all developmental stages, accumulating less Na+ and more K+ than wild-type plants during NaCl treatment. Interestingly, hat1 plants are also ABA-hypersensitive. Genetic analysis revealed that the hat1 phenotype is caused by a dominant mutation at a single nuclear locus. Mapping studies indicate that Hat1 is located at an interval of 168 kb on chromosome 5 in which 21 genes are known to be regulated by diverse abiotic stresses. A mutant of this kind, to our knowledge, has not been previously reported. Thus, this report serves as a starting point in the genetic dissection of the plant response to xerothermic stress, and provides physiological and genetic evidence of the existence of a novel abiotic stress response pathway that is also ABA-dependent.

Abscisic Acid↗

The barley ERF-type transcription factor HvRAF confers enhanced pathogen resistance and salt tolerance in Arabidopsis.

We isolated HvRAF (Hordeum vulgare root abundant factor), a cDNA encoding a novel ethylene response factor (ERF)-type transcription factor, from young seedlings of barley. In addition to the most highly conserved APETALA2/ERF DNA-binding domain, the encoded protein contained an N-terminal MCGGAIL signature sequence, a putative nuclear localization sequence, and a C-terminal acidic transcription activation domain containing a novel mammalian hemopexin domain signature-like sequence. Their homologous sequences were found in AAK92635 from rice and RAP2.2 from Arabidopsis; the ERF proteins most closely related to HvRAF, reflecting their functional importance. RNA blot analyses revealed that HvRAF transcripts were more abundant in roots than in leaves. HvRAF expression was induced in barley seedlings by various treatment regimes such as salicylic acid, ethephon, methyl jasmonate, cellulase, and methyl viologen. In a subcellular localization assay, the HvRAF-GFP fusion protein was targeted to the nucleus. The fusion protein of HvRAF with the GAL4 DNA-binding domain strongly activated transcription in yeast. Various deletion mutants of HvRAF indicated that the transactivating activity was localized to the acidic domain of the C-terminal region, and that the hemopexin domain signature-like sequence was important for the activity. Overexpression of the HvRAF gene in Arabidopsis plants induced the activation of various stress-responsive genes, including PDF1.2, JR3, PR1, PR5, KIN2, and GSH1. Furthermore, the transgenic Arabidopsis plants showed enhanced resistance to Ralstonia solanacearum strain GMI1000, as well as seed germination and root growth tolerance to high salinity. These results collectively indicate that HvRAF is a transcription factor that plays dual regulatory roles in response to biotic and abiotic stresses in plants.

Adaptation, Physiological↗

Long term catheterization of the lumbar epidural space in rats.

This note describes a simple technique for the administration of drug solutions into the lumbar epidural space of the unanesthetized and freely moving rat. A poly-ethylene (PE-10) catheter is inserted into the epidural space through a hole in the third lumbar vertebra, and subcutaneously tunneled towards the neck region. The implantation procedure is well-tolerated, and repeated saline injections over a prolonged period of time were found not to exert any deleterious effect on the animals' apparent health or gross behavior. The viability of the technique is demonstrated in an experiment on the effect of injection volume on the analgesic action of epidurally administered fentanyl.

Analgesia↗

Evaluation of the antidiabetic activity of DPP IV resistant N-terminally modified versus mid-chain acylated analogues of glucose-dependent insulinotropic polypeptide.

Glucose dependent insulinotropic polypeptide (GIP) is a gastrointestinal hormone with therapeutic potential for type 2 diabetes due to its insulin-releasing and antihyperglycaemic actions. However, development of GIP-based therapies is limited by N-terminal degradation by DPP IV resulting in a very short circulating half-life. Numerous GIP analogues have now been generated exhibiting DPP IV resistance and extended bioactivity profiles. In this study, we report a direct comparison of the long-term antidiabetic actions of three such GIP molecules, N-AcGIP, GIP(Lys(37)PAL) and N-AcGIP(Lys(37)PAL) in obese diabetic (ob/ob) mice. An extended duration of action of each GIP analogue was demonstrated prior to examining the effects of once daily injections (25nmolkg(-1) body weight) over a 14-day period. Administration of either N-AcGIP, GIP(Lys(37)PAL) or N-AcGIP(Lys(37)PAL) significantly decreased non-fasting plasma glucose and improved glucose tolerance compared to saline treated controls. All three analogues significantly enhanced glucose and nutrient-induced insulin release, and improved insulin sensitivity. The metabolic and insulin secretory responses to native GIP were also enhanced in 14-day analogue treated mice, revealing no evidence of GIP-receptor desensitization. These effects were accompanied by significantly enhanced pancreatic insulin following N-AcGIP(Lys(37)PAL) and increased islet number and islet size in all three groups. Body weight, food intake and circulating glucagon were unchanged. These data demonstrate the therapeutic potential of once daily injection of enzyme resistant GIP analogues and indicate that N-AcGIP is equally as effective as related palmitate derivatised analogues of GIP.

Animals↗

Contrast nephropathy: review focusing on prevention.

Contrast nephropathy (CN) accounts for significant morbidity and mortality. Patients with pre-existing renal insufficiency, especially those with diabetic nephropathy, are at particular risk. Medullary hypoxia due to decreased renal blood flow and direct cytotoxicity contribute to the pathogenesis. Contrast nephropathy is usually defined as an increase in serum creatinine concentration >0.5 mg/dl or 25% above the baseline level within 48 h. Intravenous hydration (saline 0.45%, if tolerated 0.9% at a rate of 1 ml/kg/h) 12 h before and after contrast exposure and the use of low or iso-osmolality contrast agents are advisable. The benefit of low-dose dopamine as well as the selective dopamine-1 receptor agonist fenoldopam is unproven. Studies on the effectiveness of the adenosine antagonist theophylline have led to conflicting results. Because theophylline has a narrow therapeutic range and may be associated with adverse effects, it is not a prophylactic agent of first choice. The administration of N-acetylcysteine (NAC) has been evaluated in several trials with inconsistent results. Newer data suggest a benefit of high-dose NAC (1,200 mg twice daily) for patients receiving high doses (>140 ml) of contrast agent, or those with advanced renal insufficiency (creatinine >2.5 mg/dl). Whereas prophylactic hemodialysis does not prevent CN, a recent study demonstrated a marked benefit of prophylactic hemofiltration.

Acute Kidney Injury↗

Nicotine sensitization increases dendritic length and spine density in the nucleus accumbens and cingulate cortex.

This study investigated the effects of repeated administrations of nicotine (0.7 mg/kg) on dendritic morphology in the nucleus accumbens (NAcc), prefrontal cortex (Cg 3), and parietal cortex (Par 1). Animals were habituated for 3 days to a locomotor box, and after habituation, every second day for 5 weeks rats were placed into the locomotor chamber immediately after a subcutaneous injection of nicotine or saline. Rats demonstrated tolerance to an initial hypoactive response after each nicotine injection, and this was followed by an increase in activity after each injection (behavioral sensitization). This increase in activity was still present on a nicotine challenge after a 2-week abstinence period. One week after the nicotine challenge day, all rats were perfused and brains were removed. These brains we stained using Golgi-Cox procedures, and dendrites from the nucleus accumbens (N Acc), medial frontal cortex (Cg 3) and parietal cortex (Par 1) were analyzed using the camera lucida procedure. Results showed that rats receiving nicotine demonstrated an increase in dendritic length and spine density relative to controls in the NAcc and Cg3 brain areas, but not Par 1. The increase observed in the NAcc was significantly greater than what has been found with amphetamine or cocaine, and possible underlying mechanisms were discussed.

Animals↗

Reduction of perchlorate and nitrate by salt tolerant bacteria.

Spent regenerant brine from ion-exchange technology for the removal of perchlorate and nitrate produces a high salt waste stream, which requires remediation before disposal. Bioremediation is an attractive treatment option. In this study, we enriched for salt tolerant bacteria from sediments from Cargill salt evaporation facility (California, USA), the Salton Sea (California, USA), and a high density hydrocarbon oxidizing bacterial cocktail. The bacterial cocktail enrichment culture reduced ClO4- from 500 to 260 mg 1 in 4 weeks. Salt tolerant bacterial isolates from the enrichment cultures and two denitrifying salt tolerant bacteria, Haloferax denitrificans and Parococcus halodenitricans, substantially reduced perchlorate. The highest rate of perchlorate removal was recorded with the isolate, Citrobacter sp.: 32% reduction in 1 week. This bacterium substantially reduced perchlorate in 0-5% NaCl solutions and maximally at 30 degrees C and at an initial pH 7.5. In simulated brines containing 7.5% total solids, the Citrobacter sp. significantly reduced both perchlorate and nitrate with 34.9 and 15.6% reduction, respectively, in 1 week. Coculture of a potent perchlorate reducing, non-salt tolerant (non-saline) bacterium, perclace and the Citrobacter sp. proved most effective for perchlorate removal in the brine (46.4% in 1 week). This study demonstrates that both anions can be reduced in treatment of brines from ion exchange systems.

Biodegradation, Environmental↗

Salt stress enhances choline uptake in the halotolerant cyanobacterium Aphanothece halophytica.

The uptake of [14C]choline by a suspension of exponential-phase Aphanothece halophytica under various conditions has been studied. Salt stress was found to enhance the uptake of choline. The kinetics of choline transport followed the Michaelis-Menten relationship with apparent K(m) values of 272 and 286 microM, maximum rates of transport (V(max)) of 18 and 37 nmol/min/mg protein for unstressed and salt-stressed cells, respectively. Choline uptake under salt stress was significantly reduced in chloramphenicol-treated cells, suggesting that the activation by salt stress occurred via an inducible transport system. This was corroborated by the existence of the periplasmic choline binding protein, whose content was higher in cells grown under salt-stress condition. Exogenously provided choline significantly increased the growth rate of cells grown under salt stress, although less efficiently than glycine betaine. The presence of 1 mM choline in the growth medium conferred tolerance to high salinity on A. halophytica with the maintenance of high growth up to 1.5 M NaCl. The uptake of choline was Na(+)-dependent, sensitive to various metabolic inhibitors as well as thiol-reactive agents. The results of competition studies suggested that N-methyl on one end of molecule and on the other end either an aldehyde, an alcohol or a neutral group were important features for substrate recognition.

Betaine↗

Preconditioning during percutaneous transluminal coronary angioplasty by endogenous and exogenous adenosine.

BACKGROUND: The purpose of this study was to assess whether pharmacologic preconditioning by exogenous or endogenous adenosine prevents the deterioration of hemodynamic function and left ventricular performance during percutaneous transluminal coronary angioplasty (PTCA). Ischemic preconditioning renders the heart more resistant to subsequent ischemia. Adenosine plays a key role in its pathogenesis. Coronary angioplasty is a suitable model for the induction of myocardial ischemia. METHODS AND RESULTS: We investigated 30 patients receiving PTCA of the left anterior descending coronary. Patients were randomly allocated to either dipyridamole, leading to the liberation of endogenous adenosine (0.5 mg/kg body weight, intracoronary), exogenous adenosine (20 mg intracoronary), or an equal amount of saline. Chest pain, tolerated inflation time, and ST-segment shift were registered. Left ventricular hemodynamics, isovolumetric phase indexes, indexes of volume, ejection fraction, and indexes of diastolic dysfunction were analyzed. Patients receiving endogenous or exogenous adenosine tolerated longer balloon inflation times (dipyridamole, 208 +/- 23 seconds; adenosine, 188 +/- 41 seconds; control, 153 +/- 36 seconds; P <.05). Deterioration of left ventricular ejection fraction was less severe after adenosine (72% +/- 5% before PTCA vs 64% +/- 6% during angioplasty; P =.11) and could be prevented by intracoronary dipyridamole (69% +/- 12% before PTCA vs 68% +/- 11% after PTCA; P <. 01) compared with the control group (71% +/- 7% before PTCA vs 60% +/- 7% during angioplasty). CONCLUSIONS: Intracoronary application of exogenous adenosine and liberation of endogenous adenosine increase the tolerance to ischemia and prevent deterioration of left ventricular function during ischemia. These findings can be attributed to ischemic preconditioning. However, endogenous adenosine exceeds the protective effects of exogenous adenosine.

Adenosine↗

Oxidant and SDS-stable alkaline protease from a halo-tolerant Bacillus clausii I-52: enhanced production and simple purification.

AIMS: An investigation was carried out on the enhancement of protease production and simple purification of an oxidant and SDS-stable alkaline protease produced by Bacillus clausii I-52 of industrial significance. METHODS AND RESULTS: The supplementation with 0.4% (w/v) NaCl and 0.05% (w/v) FeSO4.7H2O in a culture medium caused an increase in the protease production. The enzyme was purified to homogeneity with overall recovery of 79% and 10-fold purification from culture supernatant using Diaion HPA75, phenyl-Sepharose and DEAE-Sepharose column chromatographies. The protease was a halo-tolerant enzyme with apparent molecular mass of 28 kDa, and the Km and kcat values for N-Succinyl-Ala-Ala-Pro-Phe-pNA at 45 degrees C and pH 11.0 were determined to be 83.9 micromol l(-1) and 238.6 s(-1) respectively. CONCLUSIONS: Bacillus clausii I-52 was identified as a halo-tolerant bacterium, and the extracellular alkaline protease produced by B. clausii I-52 also showed extreme halo-tolerance. The enzyme stability towards SDS and H2O2 could be increased by adding NaCl or propylene glycol to the enzyme solution. SIGNIFICANCE AND IMPACT OF THE STUDY: The alkaline protease secreted by B. clausii I-52 is significant from an industrial perspective because of its stability against surfactants and oxidants as well as its tolerance towards high salinity. These enzymatic properties suggest its suitable application for industrial purposes.

Bacillus↗

Preparation and pharmaceutical evaluation of liposomes entrapping salicylic acid/gamma-cyclodextrin conjugate.

To evaluate the potential use of a drug/cyclodextrin (CyD) conjugate for efficient entrapment in liposomes and prolonged residence of a drug in tissues, we synthesized a salicylic acid (SA) conjugate bound covalently with gamma-cyclodextrin (SA/gamma-CyD conjugate), a model drug/CyD conjugate, and then liposomes entrapping the conjugate (conjugate-in-liposome) were prepared by a freezing-thawing method. The chemical and physicochemical properties of the SA/gamma-CyD conjugate in solution and solid state were investigated and then the physicochemical properties of conjugate-in-liposome, in vitro cellular uptake/release and in vivo disposition of SA/gamma-CyD conjugate after intravenous administration of aqueous suspension containing conjugate-in-liposome in rats, were evaluated, comparing with those of the liposome-entrapped SA alone (SA-in-liposome) or the liposome-entrapped noncovalent SA/gamma-CyD complex (complex-in-liposome). As a result, it was found that the conjugate was amorphous powder and the release of SA from the conjugate in phosphate-buffered saline (PBS) was tolerated to chemical and enzymatic degradation. Meanwhile, the particle sizes and stability of these liposomes were almost identical, and the entrapment ratio of SA/gamma-CyD conjugate in liposomes was higher than those of SA alone and SA/gamma-CyD complex. The cellular uptake of these liposomes was almost equivalent, but the release of SA/gamma-CyD conjugate from RAW264.7 cells was markedly slower, compared with that of SA from cells following cellular uptake of the SA-in-liposome and complex-in-liposome. The disposition of SA or SA/gamma-CyD conjugate following intravenous administration of aqueous suspensions containing each liposome system in rats was comparable, but the residence time of the conjugate in tissues significantly prolonged, compared with that of the SA-in-liposome and complex-in-liposome systems. These results suggest the potential use of SA/gamma-CyD conjugate for efficient entrapment in liposomes as well as of liposomes containing SA/gamma-CyD conjugates for prolonged residence of drugs in tissues.

Animals↗

Isolation of Vibrio and Pseudomonas from brown shrimp (Penaeus californiensis Holmes) intestine.

Bacteria of the genera Vibrio, Pseudomonas and Aeromonas were isolated from the intestine of apparently healthy brown shrimp (Penaeus californiensis Holmes) cultured in a tidal pond. Species from these genera of bacteria have been reported as shrimp pathogens and have been involved in human gastrointestinal disorders related to seafood consumption. Isolation was done first in Marine broth, then in selective media (TCBS, Cetrimide and MacConkey). The oxidase negative strains were discarded as insignificant to shrimp culture. The identification of oxidase positive strains was based in morphological and colonial characteristics, biochemical capabilities, and both salinity and temperature tolerance. API 20E system and fatty acid analysis were also included. Three potentially pathogenic bacteria, Vibrio parahaemolyticus, Vibrio furnissii and Pseudomonas putida were isolated and identified from healthy shrimp intestine.

Animals↗

The Microcystis cyanobacteria bloom in the Swan River--February 2000.

In February 2000 the Swan-Canning estuary in Western Australia experienced a record bloom of the toxic cyanobacteria Microcystis aeruginosa. At its height, concentrations of M. aeruginosa reached integrated water column cell counts of 15,000/ml and formed bright green scums in sheltered bays, where counts of 130 million cells/ml were recorded. Due to public health concerns parts of the river were closed from 10 to 22 February 2000. Two unseasonably large summer rain events in early and late January 2000 created conditions for the bloom. Freshwater runoff, estimated at 270 GL, was enough to fill the Swan-Canning estuary five times over and brought with it high levels of nutrients, mainly nitrogen (> 2.0 mg/L TN) and phosphorus (> 0.15 mg/L TP). A number of methods to reduce bloom accumulations were tried, including an attempt to increase the salinity of the surface water above the critical 10 ppt level for Microcystis; using a bentonite clay and poly-aluminium chloride mixture to flocculate and sink the algae; and sucking up scums using oil spill equipment. Over 900 tonnes of M. aeruginosa were removed and safely disposed using sewage treatment facilities. The bloom collapsed when the freshwater flush subsided and seawater intrusion from the Indian Ocean re-established itself, raising the salinities above the tolerance of Microcystis.

Environmental Monitoring↗

Feasibility of substituting sodium for potassium in crop plants for advanced life support systems.

Recycling of nutrients, air, and water is an integral feature of life support systems designed for long-term space missions. Plants can play a major role in supplying the basic life support requirements, which include providing the crew's food, clean water, and air, and recycling their wastes. The nutrient flux through the plant and human systems needs to be matched in order for nutrients to recycle between humans and plants without an excessive buildup in any one section of the system. Sodium, which is essential at the macronutrient level for human metabolism, has only been shown to be a micronutrient for some plants, with only very limited uptake in most plants. Thus, when Na is added from the outside to meet the human demand in these closed life support systems it will accumulate someplace in the overall system. In simple systems such as these, without a complete biogeological cycle, the buildup of Na could occur in the nutrient solution of the plant system. Various concepts related to the substitution of sodium for potassium in crop plants are currently being investigated by NASA. Results to date suggest that Na concentrations up to 100 g kg-1 dry weight may be achievable in the edible portions of Na-tolerant crops (e.g., red beet and chard). A flow path for nutrient solution high in Na wastes has been suggested for optimizing Na and nitrogen incorporation and utilization from such solutions. Options for further improvements include selecting plant genotypes tolerant to high salinity, which are efficient in Na uptake. This should also be combined with environmental manipulations to maximize Na uptake by crop plants.

Chenopodiaceae↗

Current treatment options for chronic constipation.

Various agents are used for the medical management of chronic constipation but few have been carefully studied. This review examines available data concerning several bulk and fiber products, lubricating agents, stimulants, and osmotic laxatives, alone and in combination. Popular therapeutic options for initial treatment of chronic constipation are dietary fiber and medicinal bulk. Subsequent treatments if fiber is not successful or tolerated would include saline osmotic laxatives, lactulose, or stimulants like senna or bisacodyl. Recent data demonstrate polyethylene glycol laxative to be safe and effective as an initial or second-line agent for chronic constipation. Indications and use of surgery and biofeedback are also discussed.

Biofeedback, Psychology↗

[Constipation: Initial evaluation and diagnostic approach].

Constipation is a common gastrointestinal symptom and affects about 20% of the general population. This symptom can reflect a vast array of problems, from inadequate fiber intake to colonic dismotility function. Identifying chronic constipation subtypes on underlying physiology guides the therapeutic choices. Chronic constipation subtypes includes: slow-transit constipation, functional constipation, irritable bowel syndrome with constipation predominance and pelvic floor dysfunction. The most useful tests for the evaluation of those patients are the colonic transit time with radiopaque markers and anorrectal manometry with balloon expulsion test. Patients with both normal tests have either functional constipation or irritable bowel syndrome with constipation. Subjects with a delayed colonic transit and normal anorectal manometry maybe classified as colonic inertia. Pelvic floor dysfunction maybe suspected if the patient had an abnormal manometry with failure to expulse de balloon. Initial treatments of chronic constipation are dietary fiber and medicinal bulk. Subsequent treatments if fiber is not successful or tolerated would include saline osmotic laxatives such as lactulose and polyethylene glycol, or stimulants like senna or bisacodyl. For patients with pelvic floor dyssynergia biofeedback therapy is the first therapeutic option. In this article we present an overview of current diagnostic tools for patients with chronic constipation.

Algorithms↗

Ocular pharmacokinetics of subconjunctivally versus intravenously administered 6-mercaptopurine.

The levels of 6-mercaptopurine (6-MP) were measured in the anterior chamber aqueous, the vitreous and the serum of rabbits 0.5, 1, 2, 4, 8 and 12 hours following subconjunctival or intravenous injection of 10 mg/kg in 0.5 mL of saline, the maximum tolerated dose as determined experimentally. The mean peak concentrations of 6-MP in the aqueous and the vitreous respectively of the subconjunctivally injected eyes were 15 and 10 times those achieved in all the eyes following intravenous administration. The bioavailability of the drug over 12 hours was 21.67 micrograms/h in the aqueous and 22.22 micrograms/h in the vitreous following subconjunctival administration but only 1.47 and 3.50 micrograms/h respectively following intravenous administration. The serum concentration of 6-MP following subconjunctival injection was about half that following intravenous administration.

Absorption↗

Effects of salinity on protein and lipopolysaccharide pattern in a salt-tolerant strain of Mesorhizobium ciceri.

AIMS: To characterize the physiological and metabolic responses of Mesorhizobium ciceri strain ch-191 to salt stress, investigating the changes induced by salinity in protein and lipopolysaccharide profiles, as well as determining the accumulation of amino acids, glutamate and proline. METHODS AND RESULTS: Strain ch-191 of M. ciceri was grown with different NaCl concentrations. Protein and lipopolysaccharide patterns were determined by electrophoresis. The strain ch-191 tolerated up to 200 mmol l-1 NaCl, although higher salt dosages limited its growth and induced changes in the protein profile. The most noteworthy change in the LPS-I pattern was the decrease in the slowest band and the appearance of an intermediate mobility band. The accumulation of proline in response to salt stress surpassed that of glutamate. CONCLUSION: The protein profile showed major alterations at salinity levels which inhibited growth. However, the alterations in the LPS profile and accumulation of compatible solutes were evident from the lowest levels, suggesting that these changes may constitute adaptative responses to salt, allowing normal growth. SIGNIFICANCE AND IMPACT OF THE STUDY: The selection and characterization of salt-tolerant strains, which also show efficient symbiotic performance under salinity, may constitute a strategy for improving Cicer arietinum-Mesorhizobium ciceri symbiosis in adverse environments.

Bacterial Proteins↗