Soil Carbon, Nitrogen and Phosphorus Dynamics as Affected by Solarization Alone or Combined with Organic Amendment

Soil solarization, alone or combined with organic amendment, is an increasingly attractive approach for managing soil-borne plant pathogens in agricultural soils. Even though it consists in a relatively mild heating treatment, the increased soil temperature may strongly affect soil microbial processes and nutrients dynamics. This study aimed to investigate the impact of solarization, either with or without addition of farmyard manure, in soil dynamics of various C, N and P pools. Changes in total C, N and P contents and in some functionally-related labile pools (soil microbial biomass C and N, K₂SO₄-extractable C and N, basal respiration, KCl-exchangeable ammonium and nitrate, and water-soluble P) were followed across a 72-day field soil solarization experiment carried out during a summer period on a clay loam soil in Southern Italy. Soil physico-chemical properties (temperature, moisture content and pH) were also monitored. The average soil temperature at 8-cm depth in solarized soils approached 55 °C as compared to 35 °C found in nonsolarized soil. Two-way ANOVA (solarization×organic amendment) showed that both factors significantly affected most of the above variables, being the highest influence exerted by the organic amendment. With no manure addition, solarization did not significantly affect soil total C, N and P pools. Whereas soil pH, microbial biomass and, at a greater extent, K₂SO₄-extractable N and KCl-exchangeable ammonium were greatly affected. An increased release of water-soluble P was also found in solarized soils. Yet, solarization altered the quality of soluble organic residues released in soil as it lowered the C-to-N ratio of both soil microbial biomass and K₂SO₄-extractable organic substrates. Additionally, in solarized soils the metabolic quotient (qCO₂) significantly increased while the microbial biomass C-to-total organic C ratio (microbial quotient) decreased over the whole time course. We argued that soil solarization promoted the mineralization of readily decomposable pools of the native soil organic matter (e.g. the microbial biomass) thus rendering larger, at least over a short-term, the available fraction of some soil mineral nutrients, namely N and P forms. However, over a longer prospective solarization may lead to an over-exploitation of labile organic resources in agricultural soils. Manure addition greatly increased the levels of both total and labile C, N and P pools. Thus, addition of organic amendments could represent an important strategy to protect agricultural lands from excessive soil resources exploitation and to maintain soil fertility while enhancing pest control.     

A 50 Hz sinusoidal magnetic field does not damage MG-63 three-dimensional tumor spheroids but induces changes in their invasive properties

The possibility that a sinusoidal 50 Hz magnetic field with a magnetic flux density of 1 mT can damage MG-63 osteosarcoma spheroids and induce variations in the invasive properties of these three-dimensional model systems after 2 days of exposure was investigated. Specifically, possible damage induced by these fields was examined by determining changes in spheroid surface morphology (light microscopy), growth (spheroid diameter and protein content determination), lactate dehydrogenase release, and reduced glutathione amount. Possible changes in the invasive properties were studied by invasion chambers. The results show no induction of cell damage by ELF fields while invasion chamber assays demonstrate a significant increase in the invasive potential of exposed spheroids. In order to determine if the fibronectin or hyaluronan receptors are involved, Western blot analysis was conducted on these two proteins. No significant variations were observed in either receptor in MG-63 multicellular tumor spheroids.     

Use of an inhibitor to identify members of the hormone-sensitive lipase family

Hormone-sensitive lipase (HSL) contributes importantly to the mobilization of fatty acids from the triacylglycerols stored in adipocytes, which provide the main source of energy in mammals. On the basis of amino acid sequence alignments and three-dimensional structures, this enzyme was previously found to be a suitable template for defining a family of serine carboxylester hydrolases. In this study, the HSL family members are characterized rather on the basis of their inhibition by 5-methoxy-3-(4-phenoxyphenyl)-3H-[1,3,4]oxadiazol-2-one (compound 7600). This compound inhibits mammalian HSL as well as other HSL family members, such as EST2 from the thermophilic eubacterium Alicyclobacillus acidocaldarius and AFEST from the hyperthermophilic archaeon Archaeoglobus fulgidus. Various carboxylester hydrolases that are not members of the HSL family were found not to be inhibited by compound 7600 under the same experimental conditions. These include nonlipolytic hydrolases such as Torpedo californica acetylcholinesterase and pig liver esterase, as well as lipolytic hydrolases such as human pancreatic lipase, dog gastric lipase, Thermomyces lanuginosus lipase, and Bacillus subtilis LipA. When vinyl esters were used as substrates, the residual activity of HSL, AFEST, and EST2 decreased with an increase in compound 7600 concentration in the incubation mixture. The inhibitor concentration at which the enzyme activity decreased to 50% after incubation for 5 min was 70, 20, and 15 nM with HSL, AFEST, and EST2, respectively. Treating EST2 and AFEST with the inhibitor resulted in an increase in the molecular mass, as established by performing matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis. This increase in the molecular mass, which corresponds approximately to the molecular mass of the inhibitor, indicates that a covalent enzyme-inhibitor complex has been formed. Surface-enhanced laser desorption ionization time-of-flight mass spectrometry analysis of a trypsin digest of AFEST treated with the inhibitor or not treated showed the occurrence of an increase in the molecular masses of the "GESAGG"-containing peptide, which is compatible with the formation of a covalent complex with the inhibitor.     

Phylogenetic conservation of cytostatic factor related genes in the ascidian Ciona intestinalis

In all vertebrates, mature oocytes arrest at the metaphase of the II meiotic division, while some invertebrates arrest at metaphase-I, others at prophase-I. Fertilization induces completion of meiosis and entry into the first mitotic division. Several experimental models have been considered from both vertebrates and invertebrates in order to shed light on the peculiar aspects of meiotic division, such as the regulation of the cytostatic factor (CSF) and the maturation promoting factor (MPF) in metaphase I or II. Recently, we proposed the oocytes of ascidian Ciona intestinalis as a new model to study the meiotic division. Here, taking advantage of the recent publication of the C. intestinalis genome, we presented a phylogenetic analysis of key molecular components of the CSF-related machinery. We showed that the Mos/MAP kinase pathway is perfectly conserved in ascidians. We demonstrated the presence of a CSF-like activity in metaphase-I arrested C. intestinalis oocytes able to block cell division in two-cell embryos. We further investigated the regulation of CSF by demonstrating that both CSF and MPF inactivation, at the exit of metaphase-I, are independent from protein synthesis, indicating the absence of short-lived factors that regulate metaphase stability, as in other invertebrate species. The results obtained suggest that meiotic regulation in C. intestinalis resembles that of vertebrates, such as Xenopus accordingly to the position of this organism in the evolutionary tree.     

Fasting and differential chemotherapy protection in patients

Chronic calorie restriction has been known for decades to prevent or retard cancer growth, but its weight-loss effect and the potential problems associated with combining it with chemotherapy have prevented its clinical application. Based on the discovery in model organisms that short term starvation (STS or fasting) causes a rapid switch of cells to a protected mode, we described a fasting-based intervention that causes remarkable changes in the levels of glucose, IGF-I and many other proteins and molecules and is capable of protecting mammalian cells and mice from various toxins, including chemotherapy. Because oncogenes prevent the cellular switch to this stress resistance mode, starvation for 48 hours or longer protects normal yeast and mammalian cells and mice but not cancer cells from chemotherapy, an effect we termed Differential Stress Resistance (DSR). In a recent article, ten patients who fasted in combination with chemotherapy, reported that fasting was not only feasible and safe but caused a reduction in a wide range of side effects accompanied by an apparently normal and possibly augmented chemotherapy efficacy. Together with the remarkable results observed in animals, these data provide preliminary evidence in support of the human application of this fundamental biogerontology finding, particularly for terminal patients receiving chemotherapy. Here we briefly discuss the basic, pre-clinical and clinical studies on fasting and cancer therapy.Key words: fasting, cancer, chemotherapy, calorie restriction, stress resistanceAfter decades of slow progress in the identification of treatments effective on a wide range of malignancies, cancer treatment is now turning to personalized therapies based in part on pharmacogenomics. By contrast, aging research is moving in the opposite direction by searching for common ways to prevent, postpone and treat a wide range of age-related diseases, based on the modulation of genetic pathways that are conserved from yeast to mammals.¹ In fact, it may be a solid evolutionary and comparative biology-foundation, which makes this ambitious goal of biogerontologists a realistic or at least a promising one. On the other hand, the progress of biogerontology is viewed by many clinicians as too fundamental and far from translational applications. In most cases, it is not clear how aging research will be translated into FDA approved drugs or treatments that have effects that are superior to those already available or being developed. For example, it is not clear how the long-term 20–30% reduction in calorie intake (dietary restriction, DR) that we and many others before us have shown to be effective in extending the life span of model organisms will make humans live longer or healthier.^1–3 Furthermore, despite the fact that long-term DR was confirmed to reduce cancer and cardiovascular disease in monkeys⁴ and to be effective in preventing obesity, type 2 diabetes, inflammation, hypertension and atherosclerosis, as indicated by the early results in humans studies,⁵ it is highly unlikely to be adopted in its more extreme and effective version by even a small portion of the population. For example, the 20 to 40% chronic reduction in daily calorie intake shown to be effective in retarding cancer growth in mice would not be feasible for cancer therapy for multiple reasons: (1) the effects of chronic DR in patients with a clinically evident tumor is expected to delay but not stop the progression of the disease^6–8 and this delay may only occur for a portion of the malignancies,⁹ (2) although weight loss and cachexia in the early stages of treatment are less prevalent than commonly thought,^10–12 the ∼15% loss of BMI and ∼30% long-term loss of body fat caused by a moderate (20%) calorie restriction¹³ may be tolerated by only a very small portion of cancer patients receiving treatment, (3) Because this long-term restriction is accompanied by delayed wound healing and immunologic impairment in rodents,^1,14,15 it is not clear what risks it may impose on cancer patients receiving treatment.¹⁶ Our studies of DSR, which were triggered by our fundamental findings that switching yeast cells to water protected them against a wide range of toxins, started as a way to address these concerns but also as an attempt to achieve a much more potent therapeutic effect than that achieved by DR.^17,18 Because starvation-induced protection can increase many fold when combined with modulation of pro-aging pathways and since it is in principle blocked by the expression of any oncogene, it has the potential to provide a method to allow common chemotherapy to selectively kill cancer cells, independently of the type of cancer.^19–21 The DSR experiments in mammals were also based on our hypothesis that stress resistance and aging regulatory pathways were conserved from yeast to mammals.We found that fasting for 48 or more hours or in vitro starvation conditions that mimic fasting protected mice and/or normal cells but not cancer cells from various chemotherapy drugs and other deleterious agents.²¹ This effect was shown to depend in part on the reduction of circulating IGF-I and glucose levels.^21,22 Although a differential regulation of cell division in normal and cancer cells^23,24 is likely to contribute to DSR, much of it appears to be dependent on protective systems which are normally maintained in an inactive or low activity state even in non-dividing cells.^1,25 In fact, in non-dividing yeast and mice, deficiencies in glucose or IGF-I signaling that match those observed after starvation promote resistance to doxorubicin, a chemotherapy drug that specifically targets muscle cells in the heart.^21,22As expected, many clinicians were skeptical of our hypothesis that cancer treatment could be improved not by a “magic bullet” but by a “not so magic DSR shield” as underlined by Leonard Saltz, an oncologist at Memorial Sloan-Kettering Cancer Center: “Would I be enthusiastic about enrolling my patients in a trial where they''re asked not to eat for 2.5 days? No.”²⁶ However, ten oncologists did allow their patients, suffering from malignancies ranging from stage II breast cancer to stage IV esophageal, prostate and lung malignancies to undergo a 48–140 hours pre-chemotherapy and a 5–56 hours post chemotherapy water-only fast. The six patients who received chemotherapy with or without fasting reported a reduction in fatigue, weakness and gastrointestinal side effects while fasting²⁷ (Fig. 1). A trend for a reduction of many additional side effects was also reported by the group of patients who always fasted before chemotherapy.²⁷ In those patients whose cancer progression was assessed, chemotherapy was effective and in some cases it was highly effective.²⁷ A clinical trial sponsored by the V-Foundation for Cancer Research, aimed at testing the safety and efficacy of a 24 hour fast in combination with chemotherapy, is in its safety stage. Because it was originally limited to patients diagnosed with bladder cancer the clinical trial progressed slowly. However, its recent expansion to include patients receiving platinum-based chemotherapy (breast, ovarian, lung cancer), is expected to expedite it. Conclusive results for the effect of a 3–4 day fast on chemotherapy-dependent side effects and possibly therapeutic index are not expected to become available for several years. Even if a more modest effect than the 1,000-fold differential protection against oxidative stress and chemotherapy observed in normal and cancer-like yeast cells was achieved in humans, this method could result in long-term survival for many patients with metastatic cancers, particularly those in which malignant cells have not acquired multidrug resistance.Open in a separate windowFigure 1Average self-reported severity of symptoms in patients that have received chemotherapy with or without fasting.     

Glycoalkaloid Profile in Potato Haploids Derived from Solanum tuberosum–S. bulbocastanum Somatic Hybrids

Cultivated and wild potato species synthesize a wide variety of steroidal glycoalkaloids (GA) that may affect either human health or biotic stress resistance. Therefore, GA composition must be a major criterion in the evaluation of breeding products when species genomes are merged and/or manipulated. This work reports the results of GA analysis performed on unique haploid (2n=2x=24) plants obtained from tetraploid (2n=4x=48) Solanum bulbocastanum–S. tuberosum hybrids through in vitro anther culture. Glycoalkaloids were extracted from tubers and analyzed by HPLC. Haploids generally showed the occurrence of parental GA. However, in several cases loss of parental GA and gain of new GA lacking in the parents was observed. It may be hypothesized that new GA profiles of our haploids is the result of either genetic recombination or combinatorial biochemistry events. To highlight differences between haploids and parents, soluble proteins and antioxidant activities were also determined. Both were always higher in haploids compared to their parents. The nature of the newly formed GAs will be further investigated, because they may represent new metabolites that can be used against pest and diseases, or are useful for human health.     

Pea powdery mildew <Emphasis Type="Italic">er1</Emphasis> resistance is associated to loss-of-function mutations at a <Emphasis Type="Italic">MLO</Emphasis> homologous locus

The powdery mildew disease affects several crop species and is also one of the major threats for pea (Pisum sativum L.) cultivation all over the world. The recessive gene er1, first described over 60 years ago, is well known in pea breeding, as it still maintains its efficiency as a powdery mildew resistance source. Genetic and phytopathological features of er1 resistance are similar to those of barley, Arabidopsis, and tomato mlo powdery mildew resistance, which is caused by the loss of function of specific members of the MLO gene family. Here, we describe the obtainment of a novel er1 resistant line by experimental mutagenesis with the alkylating agent diethyl sulfate. This line was found to carry a single nucleotide polymorphism in the PsMLO1 gene sequence, predicted to result in premature termination of translation and a non-functional protein. A cleaved amplified polymorphic sequence (CAPS) marker was developed on the mutation site and shown to be fully co-segregating with resistance in F₂ individuals. Sequencing of PsMLO1 from three powdery mildew resistant cultivars also revealed the presence of loss-of-function mutations. Taken together, results reported in this study strongly indicate the identity between er1 and mlo resistances and are expected to be of great breeding importance for the development of resistant cultivars via marker-assisted selection.     

Backbone dynamics of alamethicin bound to lipid membranes: spin-echo electron paramagnetic resonance of TOAC-spin labels

Alamethicin F50/5 is a hydrophobic peptide that is devoid of charged residues and that induces voltage-dependent ion channels in lipid membranes. The peptide backbone is likely to be involved in the ion conduction pathway. Electron spin-echo spectroscopy of alamethicin F50/5 analogs in which a selected Aib residue (at position n = 1, 8, or 16) is replaced by the TOAC amino-acid spin label was used to study torsional dynamics of the peptide backbone in association with phosphatidylcholine bilayer membranes. Rapid librational motions of limited angular amplitude were observed at each of the three TOAC sites by recording echo-detected spectra as a function of echo delay time, 2τ. Simulation of the time-resolved spectra, combined with conventional EPR measurements of the librational amplitude, shows that torsional fluctuations of the peptide backbone take place on the subnanosecond to nanosecond timescale, with little temperature dependence. Associated fluctuations in polar fields from the peptide could facilitate ion permeation.     

Guanine nucleotide- and muscarinic agonist-dependent phosphoinositide metabolism in synaptoneurosomes from cerebral cortex of immature rats

Guanine nucleotide-, neurotransmitter-, and fluoride-stimulated accumulation of [³H]inositol phosphates ([³H]InsPs) was measured in [³H]inositol-labeled synaptoneurosomes from cerebral cortex of immature (7-day-old) and adult rats, in order to clarify the role of GTP-binding proteins (G-proteins) in modulating phosphoinositide (PtdIns) metabolism during brain development. GTP(S) [Guanosine 5-O-(3-thio)triphosphate] time- and concentration-dependently stimulated PtdIns hydrolysis. Its effect was potentiated by full (carbachol, metacholine) and partial (oxotremorine) cholinergic agonists through activation of muscarinic receptors. The presence of deoxycholate was required to demonstrate agonist protentiation of the guanine nucleotide effect. The response to GTP(S) was higher in adult than in immature rats, while the effect of cholinergic agonists was similar at the two ages examined. At both ages, histamine potentiated the effect of GTP(S), while norepinephrine was ineffective. At both ages, guanosine 5-O-(2-thio)diphosphate [GDP(S)] and pertussis toxin significantly decreased GTP(S)-induced [³H]InsPs formation. The phorbol ester phorbol 12-myristate 13-acetate (PMA), on the other hand, did not inhibit the guanine nucleotide response in synaptoneurosomes from immature rats. NaF mimicked the action of GTP(S) in stimulating PtdIns hydrolysis. Its effect was not affected by carbachol and was highly synergistic with that of AlCl₃, according to the concept that fluoroaluminate (AlF₄ ^–) is the active stimulatory species. No quantitative differences were found in the response to these salts between immature and adult animals. These results provide evidence that, in both the immature and adult rat brain, neuroreceptor activation is coupled to PtdIns hydrolysis through modulatory G-proteins.