Accumulation of Pb and Zn in Gametophytes and Sporophytes of the Moss Funaria hygrometrica(Funariales)

Accumulation of lead and zinc was studied in the moss Funariahygrometrica Hedw. collected from mine tailings. Heavy metalaccumulation in gametophytes and sporophytes was quantifiedby graphite furnace atomic absorption spectrometry (GFAAS) andinductively coupled plasma-atomic emission spectrometry (ICP-AES).Pb and Zn accumulation in the placental zone was analysed byx-ray scanning electron microscopy (SEM) and transmission electronmicroscopy (TEM) microanalysis. Spectrometry showed that whilemoss gametophytes accumulated considerable concentrations ofheavy metals, sporophytes accumulated only small concentrationsof metals. X-ray SEM and TEM showed that the two metals accumulatedin placental transfer cells on both the gametophytic and sporophyticsides. To investigate the uptake pattern for both metals undercontrolled conditions, F. hygrometrica plants collected froma non-polluted site were treated in the laboratory with separatesolutions of Pb and Zn at two concentrations (10^-2and 10^-4 M)for 24 or 168 h. Metal accumulation was analysed separatelyin gametophytes and sporophytes using GFAAS and ICP–AES.Each generation had a different accumulation quotient for bothmetals, and gametophytes accumulated significantly more metalthan sporophytes. Concentrations of Zn in sporophytes were alwayshigher than concentrations of Pb. The findings are discussedin relation to the role performed by the gametophyte and theplacenta in the accumulation and sequestration of Pb and Zn.Copyright 2001 Annals of Botany Company Atomic spectroscopy, Funaria hygrometrica, gametophyte, Pb and Zn accumulation, sporophyte, x-ray TEM and SEM microanalysis     

Effect of anoxia on the kinetic properties of pyruvate kinase and phosphofructokinase,and on glycogen phosphorylase activity in marine worms and earth worms

Summary The kinetic properties of PK and PFK were studied in aerobic versus 12-hours anoxic marine worms Hedistae(=Nereis) diversicolor and Diopatra neapolitana and earth worms Allolobophora calliginosa and Eisenia foetida. The total glycogen phosphorylase (a+b) activity and the percentage of active a form were also measured in the marine and earth worms under the same conditions. Anoxia exposure did not result in any significant changes of kinetic parameters of PK and total activities of glycogen phosphorylase from marine worms, but it altered the kinetic characteristics of PFK from H. diversicolor. Chromatographical studies showed that PK from both aerobic and anoxic marine worms is eluted from DEAE-cellulose as a single peak at 50 mM KCl. In contrast to marine worms, however, anoxia caused a marked change in kinetic properties of PK from both earth worms, resulting in a reduction of enzyme affinity for its substrate PEP. In addition, the enzyme existed in both earth worms in two distinct variants eluted from DEAE-cellulose column as peak I and peak II at 50 mM and 150 mM KCl, respectively. The ratio of enzyme units (peak I/peak II) was reduced significantly after 12 h of anoxia, indicating that these two peaks are interconvertible. Anoxia also caused a reduction of total glycogen phosphorylase activity in E. foetida and lowered the percentage of active a form of the enzyme by approximately 50% in both earth worms. Kinetic properties of PFK from both earth worms were not significantly affected by anoxia. However, their low Ka values for F-2,6-P₂ imply that this effector may play an important role in PFK control in earth worms under anoxia.Abbreviations F6P fructose-6-phosphate - FBP fructose-1,6-bisphosphate - F-2,6-P fructose-2,6-bisphosphate - PEP phosphoenoylpruvate - PFK 6-phosphofructo-1-kinase (E.C.2.7.1.11) - PK pyruvate kinase (E.C.2.7.1.40) - Pi inorganic phosphate - PMSF phenyl methylsulfonyl fluoride     

Differential exposure of components of cytochromeb−c 1 region in beef heart mitochondria and electron transport particles

The reduction of cyctochromesc +c ₁ by durohydroquinone and ferrocyanide in electron transport particles (ETP) and intact cytochromec-depleted beef heart mitochondria has been studied. At least 94% of the ETP are in an inverted orientation. Durohydroquinone reduces 80% ofc +c ₁ in ETP but less than 20% in mitochondria; sonication of mitochondria allows reduction of cytochromesc +c ₁ (80%). Addition of ferrocyanide (effective redox potential +245 mV) to electron transport particles results in 30% reduction of cytochromesc +c ₁. Addition of ferrocyanide to intact cytochromec-depleted mitochondria does not reduce cytochromec ₁; treatment withN,N,N,N-tetramethylphenylenediamine, Triton X-100, or sonic oscillation results in 30% reduction of cytochromesc +c ₁. TheK _m value of ferrocyanide oxidase for K-ferrocyanide is pH-dependent in ETP only, increasing with increasing pH. The extent of reduction of cytochromec ₁ is also pH-dependent in ETP only, the extent of reduction increasing with decreasing pH. On the basis of these data cytochromec ₁ is exposed to the matrix face and cytochromec is exposed to the cytoplasmic face. No redox center other than cytochromec in the segment between the antimycin site and cytochromec is exposed on the C-side.Abbreviations Used: MES, 2(N-morpholino)-ethanesulfonic acid; EDTA, ethylenediaminetetraacetic acid; TMPD,N,N,N,N-tetramethylphenylenediamine; ETP, electron transport particles; NAD-NADH, nicotinamide adenine dinucleotide; PMS, phenazine methosulfate.     

AMMONIUM ION-INDUCED CHANGES IN FORM AND HYDROXYPROLINE CONTENT OF WALL PROTEIN IN THE LIVERWORT,GYMNOCOLEA INFLATA

Ammonium ion induces phenovariation in Gymnocolea inflata (Huds.) Dum. (Lophoziaceae) similar in nature to that induced in representatives of four other families of leafy liverworts by antagonists of hydroxyproline-protein metabolism. That is, in the presence of ammonium ion, ventral leaves developed and the lateral leaf morphology and the branching pattern of the plants changed. These changes, which reflect altered patterns of morphogenesis, were correlated with statistically significant (P ≥ 0.01) differences in peptidyl-hydroxyproline between normal and NH₄⁺-induced phenovariant plants. The results support the hypothesis that cell wall-associated hydroxyproline-protein plays a morphoregulatory role and that any of a number of factors, genetic, epigenetic or environmental, that could alter the synthesis/distribution of this moiety could cause changes in form.     

Sequential Application of Ligand and Structure Based Modeling Approaches to Index Chemicals for Their hH4R Antagonism

The human histamine H₄ receptor (hH₄R), a member of the G-protein coupled receptors (GPCR) family, is an increasingly attractive drug target. It plays a key role in many cell pathways and many hH₄R ligands are studied for the treatment of several inflammatory, allergic and autoimmune disorders, as well as for analgesic activity. Due to the challenging difficulties in the experimental elucidation of hH₄R structure, virtual screening campaigns are normally run on homology based models. However, a wealth of information about the chemical properties of GPCR ligands has also accumulated over the last few years and an appropriate combination of these ligand-based knowledge with structure-based molecular modeling studies emerges as a promising strategy for computer-assisted drug design. Here, two chemoinformatics techniques, the Intelligent Learning Engine (ILE) and Iterative Stochastic Elimination (ISE) approach, were used to index chemicals for their hH₄R bioactivity. An application of the prediction model on external test set composed of more than 160 hH₄R antagonists picked from the chEMBL database gave enrichment factor of 16.4. A virtual high throughput screening on ZINC database was carried out, picking ∼4000 chemicals highly indexed as H₄R antagonists'' candidates. Next, a series of 3D models of hH₄R were generated by molecular modeling and molecular dynamics simulations performed in fully atomistic lipid membranes. The efficacy of the hH₄R 3D models in discrimination between actives and non-actives were checked and the 3D model with the best performance was chosen for further docking studies performed on the focused library. The output of these docking studies was a consensus library of 11 highly active scored drug candidates. Our findings suggest that a sequential combination of ligand-based chemoinformatics approaches with structure-based ones has the potential to improve the success rate in discovering new biologically active GPCR drugs and increase the enrichment factors in a synergistic manner.     

Seasonal variations of cellular stress response of the gilthead sea bream (Sparus aurata)

The present study aimed to investigate the seasonal cellular stress response in vital organs, like the heart, the liver, the whole blood and the skeletal (red and white) muscles of the Mediterranean fish Sparus aurata during a 1-year acclimatization period in the field, in two examined depths (0–2 m and 10–12 m). Processes studied included heat shock protein expression and protein kinase activation. Molecular responses were addressed through the expression of Hsp70 and Hsp90, the phosphorylation of stress-activated protein kinases and particularly p38 mitogen-activated protein kinase (p38 MAPK), the extracellular signal-regulated kinases (ERK-1/2) and c-Jun N-terminal kinases (JNK1/2/3). The induction of Hsp70 and Hsp90 and the phosphorylation of p38 MAPK, JNKs and ERKs in the examined five tissues of the gilthead sea bream indicated a cellular stress response under the prism of a seasonal pattern which was characterized by distinct tissue specificity. Specifically, Hsp induction and MAPK activation occurred before peak summer water temperatures, with no further increases in their levels despite increases in water temperatures. Moreover, although water temperature did not vary significantly with depth of immersion, significant effects of depth on cellular stress response were observed, probably caused by different light regime. The expression and the activation of these certain proteins can be used as tools to define the extreme thermal limits of the gilthead sea bream.     

Site-specific pyrolysis-induced cleavage at aspartic acid residue in peptides and proteins

A simple and site-specific nonenzymatic method based on pyrolysis has been developed to cleave peptides and proteins. Pyrolytic cleavage was found to be specific and rapid as it induced a cleavage at the C-terminal side of aspartic acid in the temperature range of 220-250 degrees C in 10 s. Electrospray ionization (ESI) mass spectrometry (MS) and tandem-MS (MS/MS) were used to characterize and identify pyrolysis cleavage products, confirming that sequence information is conserved after the pyrolysis process in both peptides and protein tested. This suggests that pyrolysis-induced cleavage at aspartyl residues can be used as a rapid protein digestion procedure for the generation of sequence-specific protein biomarkers.     

Multiple advantageous amino acid variants in the NAT2 gene in human populations

Background

Genetic variation at NAT2 has been long recognized as the cause of differential ability to metabolize a wide variety of drugs of therapeutic use. Here, we explore the pattern of genetic variation in 12 human populations that significantly extend the geographic range and resolution of previous surveys, to test the hypothesis that different dietary regimens and lifestyles may explain inter-population differences in NAT2 variation.

Methodology/Principal Findings

The entire coding region was resequenced in 98 subjects and six polymorphic positions were genotyped in 150 additional subjects. A single previously undescribed variant was found (34T>C; 12Y>H). Several aspects of the data do not fit the expectations of a neutral model, as assessed by coalescent simulations. Tajima''s D is positive in all populations, indicating an excess of intermediate alleles. The level of between-population differentiation is low, and is mainly accounted for by the proportion of fast vs. slow acetylators. However, haplotype frequencies significantly differ across groups of populations with different subsistence.

Conclusions/Significance

Data on the structure of haplotypes and their frequencies are compatible with a model in which slow-causing variants were present in widely dispersed populations before major shifts to pastoralism and/or agriculture. In this model, slow-causing mutations gained a selective advantage in populations shifting from hunting-gathering to pastoralism/agriculture. We suggest the diminished dietary availability of folates resulting from the nutritional shift, as the possible cause of the fitness increase associated to haplotypes carrying mutations that reduce enzymatic activity.