Palms of the past: can morphometric phytolith analysis inform deep time evolution and palaeoecology of Arecaceae?

BACKGROUND AND AIMS: Palm fossils are often used as evidence for warm and wet palaeoenvironments, reflecting the affinities of most modern palms. However, several extant palm lineages tolerate cool and/or arid climates, making a clear understanding of the taxonomic composition of ancient palm communities important for reliable palaeoenvironmental inference. However, taxonomically identifiable palm fossils are rare and often confined to specific facies. Although the resolution of taxonomic information they provide remains unclear, phytoliths (microscopic silica bodies) provide a possible solution because of their high preservation potential under conditions where other plant fossils are scarce. We thus evaluate the taxonomic and palaeoenvironmental utility of palm phytoliths. METHODS: We quantified phytolith morphology of 97 modern palm and other monocot species. Using this dataset, we tested the ability of five common discriminant methods to identify nine major palm clades. We then compiled a dataset of species' climate preferences and tested if they were correlated with phytolith morphology using a phylogenetic comparative approach. Finally, we reconstructed palm communities and palaeoenvironmental conditions at six fossil sites. KEY RESULTS: Best-performing models correctly identified phytoliths to their clade of origin only 59 % of the time. Although palms were generally distinguished from non-palms, few palm clades were highly distinct, and phytolith morphology was weakly correlated with species' environmental preferences. Reconstructions at all fossil sites suggested that palm communities were dominated by Trachycarpeae and Areceae, with warm, equable climates and high, potentially seasonal rainfall. However, fossil site reconstructions had high uncertainty and often conflicted with other climate proxies. CONCLUSIONS: While phytolith morphology provides some distinction among palm clades, caution is warranted. Unlike prior spatially restricted studies, our geographically and phylogenetically broad study indicates phytolith morphology may not reliably differentiate most palm taxa in deep time. Nevertheless, it reveals distinct clades, including some likely to be palaeoenvironmentally informative.

alpha-Tocopherol increases caspase-3 up-regulation and apoptosis by beta-carotene cleavage products in human neutrophils.

It has been found that beta-carotene cleavage products (CarCP), besides having mutagenic and toxic effects on mitochondria due to their prooxidative properties, also initiate spontaneous apoptosis of human neutrophils. Therefore, it was expected that antioxidants such as alpha-tocopherol would inhibit the stimulation of apoptosis and caspase-3 activity by CarCP. However, we found that alpha-tocopherol increases caspase-3 up-regulation and stimulation of apoptosis of human neutrophils by CarCP. Ascorbic acid does not alter this caspase-3 up-regulating and proapoptotic effect exerted by alpha-tocopherol. Both alpha-tocopherol and ascorbic acid, in the absence of CarCP, decrease intracellular caspase-3 activity and spontaneous apoptosis of neutrophils. Uric acid alone or in combination with CarCP does not exert apparent effects on caspase-3 activity and apoptosis. Up-regulating effect of alpha-tocopherol is not observed in the presence of retinol that markedly stimulates apoptosis by itself, whereas increase of caspase-3 activity is induced by concomitant addition of alpha-tocopherol and beta-ionone, a cyclohexenyl degradation product of beta-carotene with shorter aliphatic chain.

Effects of calmodulin antagonists on calcium pump and cytosolic calcium level in human neutrophils.

The concentration of cytosolic free calcium was monitored in suspensions of intact human neutrophils in phosphate-buffered saline by means of the fluorescent indicator Indo 1 trapped in the cytosol. Trifluoperazine and n-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide markedly reduced the amplitude of the transient increase in cytosolic Ca2+ triggered by CaCl2 as well as by N-formyl-methionyl-leucyl-phenylalanine. The effect of the calmodulin antagonists on the calcium burst observed upon cell activation was much more pronounced in the presence of extracellular free calcium than in EGTA-containing media; it was not inhibited by wortmannin or thapsigargin. Nevertheless, trifluoperazine and n-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide inhibited the plasma-membrane Ca2+ ATPase if added to plasma membrane-enriched fractions of neutrophils. These results suggest that calmodulin antagonists affect calcium ion influx even if they inhibit plasma membrane Ca2+ ATPase.

Succinylpurinemic autism: increased sensitivity of defective adenylosuccinate lyase towards 4-hydroxy-2-nonenal.

We studied the effect of trans-4-hydroxy-2-nonenal on the wild-type human adenylosuccinate lyase and on the enzyme from a patient compound-heterozygous for two missense mutations (P75A/D397Y; McKusick 103050.0003/103050.0004). Both the enzymes were inhibited by 10-50 microM trans-4-hydroxy-2-nonenal in a concentration-dependent manner by means of a mixed-type co-operative mechanism. A significantly stronger inhibition was noticed in the presence of the defective enzyme. Nonanal and trans-2,3-nonenal inhibited the enzymes to a less extent and at about 10-times higher concentrations. Hydroxylamine reversed the inhibition by trans-4-hydroxy-2-nonenal, trans-2,3-nonenal or nonanal in the case of the wild-type enzyme, but it was ineffective to reverse the inhibition by trans-4-hydroxy-2-nonenal on the defective enzyme. Dithiothreitol slightly decreased the inhibition exerted by trans-4-hydroxy-2-nonenal on both the wild-type and the defective adenylosuccinate lyase, while it did not produce practically any change in the presence of trans-2,3-nonenal or nonanal.

Failure of muscle energy metabolism in a patient with adenylosuccinate lyase deficiency. An in vivo study by phosphorus NMR spectroscopy.

31P-nuclear magnetic resonance spectroscopy was used to investigate in vivo the energy metabolism of the calf muscle in a 10-year-old patient with adenylosuccinate lyase deficiency and severe psychomotor retardation. The patient showed a markedly reduced PCr/P(i) molar ratio, known to well represent the cytosolic phosphorylation potential, due to low PCr and high P(i) content in resting muscle. Moreover, intracellular ATP concentration was significantly lower than in the control group both at rest and at the end of post-exercise recovery. The rate of patient's PCr recovery after an exercise in ischaemic conditions was also out of the reference range, suggesting a reduced ability of mitochondria to respond to metabolic needs.

Identification of new mutations in the adenylosuccinate lyase gene associated with impaired enzyme activity in lymphocytes and red blood cells.

We determined the DNA sequence of the adenylosuccinate lyase (ASL) gene from a 13 year-old female, who showed a reduced ASL enzymatic activity in lymphocytes and red blood cells and suffered from severe psychomotor retardation. The patient was the offspring of a non-consanguineous marriage. She was found to be compound heterozygous for two missense-mutations located on different alleles (C300-G and G1266-T): the first mutation replaces Pro75 by Ala, the second mutation replaces Asp397 by Tyr.

Stoichiometry of hemolysis by the polyene antibiotic lucensomycin.

The stoichiometry of hemolysis by the polyene antibiotic lucensomycin was investigated. It appears that hemolysis occurs only when a relatively high fraction (probably between 15 and 40%) of the cholesterol sites in the erythrocyte membrane have combined with the polyene. Also in phospholipid-cholesterol vesicles the increase of permeability requires occupancy of 40-50% of the existing cholesterol sites. As for the possible cooperative effect in the hemolytic process, it is probable that several (at least 9-10) lucensomycin-cholesterol adducts must interact on each side of the membrane to form an aqueous channel; the distribution of these adducts in the erythrocyte membrane occurs, however, apparently at random.

Effect of D-ribose on purine synthesis and neurological symptoms in a patient with adenylosuccinase deficiency.

Oral supplementation of 10 mmol/kg/day of D-ribose to a patient with an inherited deficit of adenylosuccinase, severe psychomotor retardation, and epilepsy caused a marked increase in plasma concentration and urinary excretion of urate, while minor changes in succinylpurine levels were observed. D-Ribose administration was accompanied by a slight improvement of behaviour and a progressive reduction of seizure frequency, which increased dramatically upon two attempts to withdraw the drug. Substitution of D-ribose with an equivalent amount of D-glucose did not result in an increase of seizure frequency.

Inhibition of defective adenylosuccinate lyase by HNE: a neurological disease that may be affected by oxidative stress.

Adenylosuccinate lyase is an enzyme of fumarase superfamily that participates in the purine biosynthetic pathway, catalysing the nonhydrolytic cleavage of succinyl groups from SAICA ribotide and adenylosuccinate. Enzyme defects are associated with a human inherited disease, which arises from single point mutations to the gene and results in mild to severe psychomotor retardation, epilepsy, muscle wasting, and autistic features. Adenylosuccinate lyase activity is lost to a different extent in the patients. Diminished levels of enzyme have been attributed to loss of catalytic activity, protein instability, or environmental factors. P100A/D422Y mutation represents a feasible model for studying the effect of cell milieu on the activity of the impaired enzyme. The defective enzyme is inhibited by micromolar concentrations of trans-4-hydroxy-2-nonenal (HNE), a major product of membrane peroxidation that has been found to accumulate in brain tissues of patients with neurodegenerative disorders. It is suggested that inactivation of defective adenylosuccinate lyase by HNE and other membrane peroxidation products may account, at least in part, for the impairment of neurological functions and recurrent worsening of the symptoms.

Inhibition of ion transport ATPases by HNE.

4-Hydroxy-2,3-trans-nonenal (HNE), a major lipid peroxidation product, has been shown to react with specific amino acid residues of proteins and alter their function. In vitro exposure of erythrocyte ghosts and neutrophil membranes to HNE results in the inhibition of ion transport ATPases. Neutrophil membrane Ca2+-ATPase is strongly inhibited by micromolar concentrations of HNE, while HNE is considerably less effective against neutrophil Mg2+-ATPase and the erythrocyte ghost enzymes.

Effect of carotenoid oxidation products on neutrophil viability and function.

Human neutrophils are short-lived cells that play important roles in host defense and acute inflammation by releasing hydrolytic and cytotoxic proteins and reactive oxygen derivatives. Apoptosis, a physiological mechanism for cell death, regulates both production and survival of neutrophils, representing a basic biological mechanism for this type of cells. Carotenoids may react with toxic oxygen metabolites released by neutrophils to form a multitude of carotenoid cleavage products that exert, in turn, relevant prooxidative biological effects. Recent data suggest that carotenoid oxidation products may affect neutrophil viability and function by exerting proapoptotic activity and interfering with superoxide production by activated cells. The prooxidant and proapoptotic activities of carotenoid oxidation products could account, at least in some cases, for the procancerogenic properties of carotenoid rich diet.

Combined effects exerted by amphotericin B and lucensomycin on cation permeability in a unilamellar vesicle system.

The permeability of artificial unilamellar vesicles and of plasma membrane vesicles from homogenized yeast in aqueous solutions of polyene antibiotics (amphotericin B and lucensomycin) was studied by measuring proton leakage by a pH-stat method. Micromolar concentrations of amphotericin B induced a remarkable proton efflux from the vesicles. Lucensomycin exerted similar effects only at 100 times higher concentrations. The latter antibiotic, at concentrations one order of magnitude lower than those necessary to induce a detectable proton efflux, seemed to protect the vesicles from the subsequent permeabilizing action of amphotericin B.

Interaction of lucensomycin with membranes: the role of non-steroidal components.

The binding of lucensomycin to unilamellar phospholipid/cholesterol vesicles and to colloidal emulsions of cholesterol in aqueous solutions was studied by monitoring the changes in the electronic absorption spectra of the polyene antibiotic. The total extent of the absorption variations was a direct function of cholesterol concentration and quite independent of the nature of the emulsion. The rate of binding, relatively slow in the colloidal systems, was greatly enhanced when cholesterol was included in phospholipid-containing membranes. The rate of lucensomycin binding to colloidal cholesterol increased with increasing cholesterol concentrations and/or stirring the heterogeneous suspension. The time course of lucensomycin binding to vesicles appeared to be independent of the concentrations of phospholipids and cholesterol.

The interaction of a new anti-tumour drug, KAR-2 with calmodulin.

1. KAR-2 (3"-(beta-chloroethyl)-2",4"-dioxo-3,5" -spiro-oxazolidino-4-deacetoxy-vinblastine) is a semisynthetic bis-indol derivative, with high anti-microtubular and anti-tumour activities but with low toxicity. KAR-2, in contrast to other biologically active bis-indols (e.g. vinblastine) did not show anti-calmodulin activity in vitro (enzyme kinetic, fluorescence anisotropy and immunological tests). 2. Direct binding studies (fluorescence resonance energy transfer, circular dichroism) provided evidence for the binding of KAR-2 to calmodulin. The binding affinity of KAR-2 to calmodulin (dissociation constant was about 5 microM) in the presence of Ca2+ was comparable to that of vinblastine. 3. KAR-2 was able to interact with apo-calmodulin as well; in the absence of Ca2+ the binding was of cooperative nature. 4. The effect of drugs on Ca2+ homeostasis in human neutrophil cells was investigated by means of a specific fluorescent probe. Trifluoperazine extensively inhibited the elevation of intracellular Ca2+ level, vinblastine did not appreciably affect it, KAR-2 stimulated the Ca2+ influx and after a transient enhancement the Ca2+ concentration reached a new steady-state level. 5. Comparison of the data obtained with KAR-2 and bis-indols used in chemotherapy suggests that the lack of anti-calmodulin potency resides on the spiro-oxazolidino portion of KAR-2. This character of KAR-2 manifested itself in various systems and might result in its low in vivo toxicity, established in an anti-tumour test.

Polyene antibiotics inhibit superoxide-producing NADPH oxidase in a polymorphonuclear cell-free system.

We studied the effect of polyene macrolide antibiotics on the NADPH-dependent superoxide production induced by arachidonic acid in a cell-free system consisting of the membrane and cytosolic fractions obtained from bovine polymorphonuclear leukocytes. Preincubation of the membrane fraction with polyenes before addition of the soluble components of the reaction mixture resulted in a dose-dependent inhibition of superoxide production.

Inhibition of NADPH oxidase-mediated superoxide radical formation in PMA-stimulated human neutrophils by 4-hydroxynonenal--binding to -SH and -NH2 groups.

4-Hydroxynonenal (HNE), a major lipid peroxidation product, effectively inhibits the superoxide radical formation by NADPH oxidase of phorbol myristate acetate (PMA)--stimulated human PMNL. The I50 value for the inhibition of NADPH oxidase-mediated superoxide radical formation by 4-hydroxynonenal was found to be 19 microM. The HNE inhibition involves the reaction with both -SH and -NH2 groups. Superoxide formation as final result of the NADPH oxidase cascade was almost completely restored by addition of dithiothreitol. In presence of hydroxylamine only a minor restoration of superoxide radical formation was found. A combination of dithiothreitol and hydroxylamine yielded the greatest recovery. Two other aldehydes with the same chain length as HNE but different binding to lysine, histidine and cysteine residues, trans-2,3-nonenal and nonanal, gave I50 values for the inhibition of NADPH oxidase-mediated superoxide formation rate of 110 microM or > 300 microM, respectively.

S-adenosylhomocysteine hydrolase and adenosine deaminase activities in human red cell ageing.

The enzyme activities of S-adenosylhomocysteine hydrolase, adenosine deaminase and pyruvate kinase were determined in normal human erythrocytes subpopulations of different ages separated by centrifugation on a discontinuous Percoll:NaCl density gradient. The levels of S-adenosylhomocysteine hydrolase activity were found to undergo a sharp decrease with red cell ageing. Adenosine deaminase activities were, however, less critically dependent on erythrocyte age.

Octopamine plasma levels and hepatic encephalopathy: a re-appraisal of the problem.

An investigation on the blood levels of octopamine was carried out on 70 adult individuals. There was a statistically significant correlation between the levels of octopamine and hepatic encephalopathy. Normal subjects had values below 1 ng/ml, while patients with grade 3 or grade 4 encephalopathy constantly showed values above 3.2 ng/ml. In these two groups the distribution was fairly homogeneous. Through the differences between cirrhotics without neurologic involvement and those with grade 1 or 2 hepatic encephalopathy displayed statistical significance, distribution of values in these groups was rather non-homogeneous. Octopamine levels paralleled variations in mental state in 3 out 4 cases. No difference was found between venous and arterial values. The reaction of transmethylation used in the assay of octopamine was constantly found to be inhibited by the presence of plasma. This inhibition is probably due to the presence of one or more beta-hydroxyphenylethanolamines other than octopamine.

High sensitivity of plasma membrane ion transport ATPases from human neutrophils towards 4-hydroxy-2,3-trans-nonenal.

Lipid peroxidation results in release of 4-hydroxy-2,3-trans-nonenal (HNE), which is known to conjugate to specific amino acids of proteins and may alter their function. The effect of HNE on the activities of Na(+)/K(+)-ATPase, Mg(2+)-ATPase, Ca(2+)-ATPase, and calmodulin-stimulated Ca(2+)-ATPase has been studied both in erythrocyte ghosts and in neutrophil membrane preparations. Neutrophil Ca(2+)-ATPase was strongly inhibited by micromolar concentrations of HNE (IC(50) = 12 microM), that means in the range of pathophysiologically relevant HNE levels. The IC(50) value for neutrophil Na(+)/K(+)-ATPase was about 40 microM. HNE was considerably less effective against neutrophil Mg(2+)-ATPase and the erythrocyte ghost enzymes (IC(50) values range from 91 to 240 microM). The data suggest that HNE may play a specific role in the regulation of neutrophil calcium homeostasis in response to oxidative stress.

Interaction of lucensomycin with cholesterol in membranes: kinetic and structural studies.

The variations of optical density and fluorescence of lucensomycin are good indices of the binding of this polyenic antibiotic to membranes. The former parameter reflects more generally the binding to any site present in the membrane, while the latter is more specific for binding to cholesterol. Equilibrium titration experiments performed in the presence of an excess of membrane-bound cholesterol suggest that lucensomycin self-associates and that the binding and polymerization sites of the antibiotic are identical or quasi-identical; hence polymerization leads to a loss of binding sites and vice versa. The non-steroidal components of the membrane (such as proteins and lipids) seem to affect the rate of the individual reaction leading to the formation of the cholesterol-lucensomycin complexes, rather than the ratio among these heterologous aggregates at equilibrium. Polyene concentrations, which induce detectable proton release from unilamellar vesicles, are at least two orders of magnitude higher than those necessary to perform a spectroscopic titration of membrane cholesterol.