Physiological and biochemical responses of Quercus pubescens to air warming and drought on acidic and calcareous soils.

The drought- and thermo-tolerant Quercus pubescens, a tree species growing on both acidic and calcareous soils in the sub-Mediterranean region, was exposed to soil drought (-60% to -80% soil water content) and air warming (+1.2 °C daytime temperature), singly and in combination. The experiment was conducted on two natural forest soils with similar texture but different pH (acidic and calcareous soils). The physiological (photosynthesis) and biochemical (antioxidant system) responses of Q. pubescens were investigated. On acidic soil, Q. pubescens had a higher reactive oxygen species (ROS) content than on calcareous soil, confirming that this species is better adapted to the latter soil type. A down-regulation of ascorbate-glutathione cycle enzymes suggests that ROS were used as signalling molecules. Air warming stimulated stomatal opening, while soil drought induced stomatal closure in the late afternoon and reduced Rubisco carboxylation efficiency. Photosynthetic performance in the combined treatment was higher than under single drought stress and similar to control and air warming. Q. pubescens biochemical responses depended on soil pH. On acidic soil, Q. pubescens trees exposed to air warming used ROS as signalling molecules. On calcareous soil, these trees were able to balance both soil drought and air warming stress, avoiding ROS toxic effects by increasing antioxidant enzyme activitiy and maintaining a high enzymatic antioxidant defence. When combined, drought and air warming induced either more severe (higher oxidative pressure and impairment of the light-harvesting complex) or different responses (decline of the thermal energy dissipation capacity) relative to the single stressors. Overall, however, Q. pubescens preserved the functionality of the photosynthetic apparatus and controlled the antioxidant system response, thus confirming its drought and thermo-tolerance and therefore its potential to adapt to the ongoing climate change.

The environmental endocrine disruptor, bisphenol A, affects germination, elicits stress response and alters steroid hormone production in kiwifruit pollen.

In vitro toxicity of the endocrine disruptor bisphenol A (BPA) to pollen, the male haploid generation of higher plants, was studied. BPA caused significant inhibition of both tube emergence and elongation of kiwifruit pollen in a dose-dependent manner, beginning at 10 mg · l(-1); morphological changes to tubes were also detected. Despite strong inhibition of pollen tube production and growth, a large percentage of treated cells remained viable. Immunoblotting experiments indicated that levels of BiP and 14-3-3, which are proteins involved in stress response, substantially increased in BPA-treated pollen compared to controls. The increases were dose-dependent in the range 10-50 mg · l(-1) BPA, i.e. even when germination ability was completely blocked. Steroid hormones (17 ß-estradiol, progesterone and testosterone) were detected in kiwifruit pollen, and their levels increased during germination in basal medium. In a BPA treatment of 30 mg · l(-1), larger increases in both estrogen and testosterone concentrations were detected, in particular, a six-fold increase of 17 ß-estradiol over control concentration (30 min). The increased hormone levels were maintained for at least the 90 min incubation. Increasing concentrations of exogenous testosterone and 17 ß-estradiol increasingly inhibited pollen tube emergence and elongation. Current data for BPA-exposed kiwifruit pollen suggest a toxicity mechanism that is at least in part based on a dramatic imbalance of steroid hormone production during tube organisation, emergence and elongation. It may be concluded that BPA, a widespread environmental contaminant, can cause serious adverse effects to essential pollen functions. On a broader scale, this chemical poses a potential risk to the reproductive success of higher plants.

Cyclosporin A inhibits programmed cell death and cytochrome c release induced by fusicoccin in sycamore cells.

Programmed cell death plays a vital role in normal plant development, response to environmental stresses, and defense against pathogen attack. Different types of programmed cell death occur in plants and the involvement of mitochondria is still under investigation. In sycamore (Acer pseudoplatanus L.) cultured cells, the phytotoxin fusicoccin induces cell death that shows apoptotic features, including chromatin condensation, DNA fragmentation, and release of cytochrome c from mitochondria. In this work, we show that cyclosporin A, an inhibitor of the permeability transition pore of animal mitochondria, inhibits the cell death, DNA fragmentation, and cytochrome c release induced by fusicoccin. In addition, we show that fusicoccin induces a change in the shape of mitochondria which is not prevented by cyclosporin A. These results suggest that the release of cytochrome c induced by fusicoccin occurs through a cyclosporin A-sensitive system that is similar to the permeability transition pore of animal mitochondria and they make it tempting to speculate that this release may be involved in the phytotoxin-induced programmed cell death of sycamore cells.

Comparison between the effects of fusicoccin, Tunicamycin, and Brefeldin A on programmed cell death of cultured sycamore (Acer pseudoplatanus L.) cells.

Programmed cell death occurs in plants during several developmental processes and during the expression of resistance to pathogen attack (i.e., the hypersensitive response). An unsolved question of plant programmed cell death is whether a unique signaling pathway or different, possibly convergent pathways exist. This problem was addressed in cultured sycamore (Acer pseudoplatanus L.) cells by comparing the effects of fusicoccin, Tunicamycin and Brefeldin A, inducers of programmed cell death with well-defined molecular and cellular targets, on some of the parameters involved in the regulation of this process. In addition to cell death, the inducers are able to stimulate the production of H2O2, the leakage of cytochrome c from mitochondria, the accumulation of cytosolic 14-3-3 proteins, and changes at the endoplasmic reticulum level, such as accumulation of the molecular chaperone binding protein and modifications in the organelle architecture. Interestingly, no additive effect on any of these parameters is observed when fusicoccin is administered in combination with Tunicamycin or Brefeldin A. Thus, these inducers seem to utilize the same or largely coincident pathways to induce programmed cell death and involvement of the endoplasmic reticulum, in addition to that of mitochondria, appears to be a common step.

Fusicoccin induces in plant cells a programmed cell death showing apoptotic features.

Programmed cell death plays a pivotal role in several developmental processes of plants and it is involved in the response to environmental stresses and in the defense mechanisms against pathogen attack. It has not yet been defined which part of the death signalling mechanism and which molecules involved in programmed cell death are common to animals and plants. In this paper we show that fusicoccin, a well-known phytotoxin, induces a strong acceleration in the appearance of Evans Blue-stainable (dead) cells in sycamore (Acer pseudoplatanus L.) cultures. This fusicoccin-induced cell death shows aspects common to the form of animal programmed cell death termed apoptosis: i.e., cell shrinkage, changes in nucleus morphology, increase in DNA fragmentation detectable by a specific immunological reaction, and presence of oligonucleosomal-size fragments (laddering) in DNA gel electrophoresis. Since fusicoccin has a well-identified molecular target, the plasma membrane H(+)-ATPase, and thoroughly investigated physiological effects, this toxin appears to be a useful tool to study the transduction of death signals leading to programmed cell death in plants.

Tunicamycin and Brefeldin A induce in plant cells a programmed cell death showing apoptotic features.

The recent identification of DAD (defender against apoptotic death) gene in plants suggests that the N-linked glycosylation of proteins could be an important control point of plant programmed cell death. In this paper we describe the effects of Tunicamycin, an inhibitor of N-linked protein glycosylation, and Brefeldin A, an inhibitor of protein trafficking from the Golgi apparatus, on sycamore (Acer pseudoplatanus L.) cell cultures. These two chemicals proved able to induce a strong acceleration of the cell death; changes in cell and nucleus morphology; an increase in DNA fragmentation, detectable by a specific immunological reaction; and the presence of oligonucleosomal-size fragments (laddering) in DNA gel electrophoresis. Moreover, Brefeldin A, but not Tunicamycin, strongly stimulated the production of hydrogen peroxide. These results indicate that also in plants chemicals interfering with the activities of endoplasmic reticulum and of Golgi apparatus strongly induce a form of programmed cell death showing apoptotic features.

A survey of the necessity of the hospitalization day in an Italian teaching hospital.

To assess the extent of inappropriate hospital use in an adult in-patients population we used a modified version of the Appropriateness Evaluation Protocol (A.E.P.) to evaluate retrospectively a cross-section of 273 patient-days in a large teaching hospital in the Greater Milan area. Overall, 41% were judged to represent inappropriate hospital use on the basis of the protocol's criteria. The rate of inappropriate hospital use was significantly associated with admitting specialty, ranging from 12% for surgery, to 20% for cardiology and to about 60% in psychiatric, geriatrics and neurology departments (p less than 0.01). Hospital days of patients with longer stays were more frequently inappropriate: a statistically significant trend of inappropriateness emerged ranging from 30% among patients with total length of stay (LOS) of 1-10 days to 60% among those with LOS greater than 30 days (p less than 0.01). This study confirms that there is a substantial rate of unnecessary use of hospitals but that such inappropriateness does not seem in most cases to be easily modifiable through "simple" organizational changes.

Central nervous system involvement in elderly patients with non-insulin-dependent diabetes mellitus.

We examined cognitive functions and central conduction velocities in 20 patients, over 63-year old, with NIDDM compared with 20 normal, age-matched controls. Mean conduction velocity of median nerve, was significantly lower in diabetics than in controls, but absolute values were within normal range. Neurological examination showed clinical signs of lower limbs neuroperipheral involvement. Only one cognitive subtest performance was abnormal whereas there was no impairment in central conduction velocities. In our study population, although median nerve conduction velocity values may introduce a bias of low peripheral neuropathy incidence, there was no evidence of a massive or progressive specific central nervous system involvement caused by NIDDM.

Inhibitor studies of methionyl-tRNA transformylase of Euglena gracilis.

Inhibitor studies of the only known eukaryotic methionyl-tRNA transformylase (10-formyltetrahydrofolate:L-methionyl-tRNA N-transformylase, EC 2.1.2.9) were carried out. All the natural pteroylglutamic acid derivatives examined, with the exception of pteroylglutamic acid, are inhibitors. The most effective is 5-methyltetrahydrofolate (5-CH3-H4PteGlu) (KI = 3 . 10(-6) M), which is the only noncompetitive inhibitor of the enzyme. All the other derivatives tested are competitive, and H4PteGlu shows a cooperative inhibition. These and other data obtained with pteroylglutamic analogues show that, in contrast to the bacterial enzyme, Euglena transformylase is also inhibited by compounds without a fully reduced pyrazine ring and is very sensitive to compounds with a methyl group in position 5 or 10 of the pteridine ring.

Dependence of mitochondrial protein synthesis initiation on formylation of the initiator methionyl-tRNAf.

The effect of N10-formyl-H4folate on mitochondrial peptide chain initiation has been studied in isolated mitochondria of Saccharomyces cerevisiae. The addition of N10-formyl-H4-folate strongly stimulates the incorporation of amino acids into mitochondrial protein at both 6 and 15 mm Mg2+. Still higher stimulation (up to 10-fold) has been obtained in the production of de novo synthesized initial peptides, measured as peptidyl puromycin derivatives. The maximum effect is observed at 0.1 mM N10-formyl-H4folate. At 5 mM puromycin, the ratio formylated/unformylated peptides is 3, as shown by electrophoretic analysis. At 10 mM puromycin, the ratio is increased to more than 6. This is due to the presence of deformylase and amidohydrolase activities, which are more effective the longer the initial peptide is synthesized; at increasing puromycin concentrations, progressively shorter peptide chains are formed. Chemically synthesized fMet-puromycin and Met-puromycin are virtually stable when incubated with intact or frozen and thawed mitochondria. More careful kinetic analysis shows an early cessation of the initial peptide formation in the samples without N10-formyl-H4-folate. This indicates that the formylation of methionyl-tRNA formylatable species is an absolute requirement for mitochondrial peptide chain initiation.