Coenzyme reorientations in the active sites of aspartate aminotransferase isoenzymes studied by linear dichroism method

Cytosolic and mitochondrial pig heart aspartate aminotransferases (cAspAT and mAspAT) and chicken heart cAspAT have been oriented in a compressed slab of polyacrylamide gel and their linear dichroism LD spectra have been recorded. The coenzyme's tilt angles in the active sites of chicken cAspAT and pig mAspAT and their quasisubstrate complexes imitating catalytic intermediates have been computed. The computations are based on reduced linear dichroism values (delta A/A), the known directions of the transition dipole moments in the coenzyme ring and atomic coordinates of the coenzyme obtained by X-ray crystallography. It has been found that formation of the enzyme complex with glutarate and protonation of the internal pyridoxal-lysine aldimine induce reorientations of the coenzyme. As a result of protonation, the coenzyme ring tilts by 27 degrees in cAspAT and 13 degrees in mAspAT. Formation of the external aldimine with 2-methylaspartate is accompanied by tilting of the coenzyme ring by 44 degrees in cAspAT and 39 degrees in mAspAT. For the quinonoid complex with erythro-3-hydroxyaspartate, the tilt angles were found to be 63 degrees in cAspAT and 53 degrees in mAspAT. It is inferred that the basic features of the active site dynamics are similar in the three AspAT's studied. The differences in the coenzyme tilt angles between cAspAT and mAspAT may be linked to catalytic and structural peculiarities of the isoenzymes.     

大鼠脑缺血/再灌注过程中血流量和脑组织含水量的变化趋势*

目的:研究大鼠脑缺血/再灌注过程中血流量及与脑组织水含量变化的趋势。方法:选取5只成年SD雄性大鼠(n=5),参照改良Zea-Longa线栓法制备大鼠大脑中动脉缺血/再灌注模型,2 h后拔出线栓。利用PeriCam PSI血流灌注成像系统实时监测大鼠在缺血前及缺血5 min、30 min、1 h、2 h、再灌注5 min、30 min、1 h、2 h、4 h、6 h及24 h的血流灌注量,记录在ROI(感兴趣区)测量的数值。再选取15只成年SD雄性大鼠,分为Control组、缺血2 h、再灌注30 min、4 h及24 h组(n=3)。正常组不做任何处理,实验组按上述线栓法制备MCAO模型。取新鲜脑组织用干湿重法测定其左、右半球的水含量。结果:栓塞时缺血侧血流量逐渐下降,缺血2 h下降最低(P＜0.05);再灌注早期血流量恢复较大(P＜0.05),30 min时显著下降(P＜0.05),4 h明显上升(P＜0.05),24 h再次上升(P＜0.05)但低于缺血前血流量(P＞0.05)。脑组织水含量测量,缺血2 h组和再灌注30 min组与正常组无明显差异(P＞0.05);再灌4 h组和再灌24 h组明显增高(P＜0.05),且再灌24 h组明显高于再灌4 h组(P＜0.05)。结论:大鼠脑缺血/再灌注过程中血流量和脑组织中水含量的变化存在一定的规律,且脑组织中水含量与再灌注过程中血流量的变化有一定关系。     

Impacts of cage culture on physico-chemical and bacteriological water quality in Lake Volta,Ghana

The effects of cage fish farming on physico-chemical and bacteriological water quality in Lake Volta, Ghana, were investigated in 2013–2014. Farmed and unfarmed (control) areas of the lake were selected for monitoring. Nutrients, temperature, dissolved oxygen, conductivity, turbidity, pH, total coliforms, Pseudomonas and Vibrio spp. in the water were monitored monthly. Analyses of the water samples were carried out according to standard procedures. Physico-chemical quality of the water in both farm and control sites were within ranges typical of minimally impacted water and did not vary significantly between the two contrasting sites. The bacteriological analysis, however, revealed contamination of the lake water by fish farming. The bacterial counts at the farmed sites were significantly higher (p < 0.05) than those of the control sites, with figures at the farmed sites ranging from 132 to 1 708 cfu 100 ml?1 for total coliforms, 514 to 5 170 cfu 100 ml?1 Pseudomonas spp. and 14 to 516 cfu 100 ml?1 for Vibrio spp. The results suggested that cage fish farming has increased bacterial loads in the lake water, but has had minimal impact on its physico-chemical quality.     

Clustering of halophytic species from Cyprus based on ionic contents

This paper presents the work conducted on the chemical constituents of some common and widely distributed halophyte taxa from Cyprus with the aim that these studies will help in the evaluation of halophytes for different economical purposes. The plant species of Crithmum maritimum L., Limbarda crithmoides (L.) Dumort, Atriplex portulacoides L., Salsola kali L., Atriplex halimus L., Limonium oleifolium Mill., L. meyeri (Boiss.) Kuntze; and Tetraena alba (L.f.) Beier & Thulin were collected in the middle of July. The shoot tissue and leaf samples were collected from the natural habitats and left for drying under air circulation followed by placing them in oven at 60 °C for 96 hours. The material was crushed using mortar and pestle and subjected to an analysis of macro- and micro-nutrients and biochemical compounds. K⁺/Na⁺ in the leaf tissues of the dicot species showed relatively high values depicting their behavior as Na+ includes but very low Cl- levels were recorded. Out of the species investigated here in 4 TFAA content was rather high. Values ranging from 0.5% to 1% dry weight were exhibited in one species. However, only 3 species showed very low TFAA values. Later may be due to low nitrogen availability in their environment. The phenetic analyses of eight halophyte species performed on the data matrix using Ntsys-pc program version 2.1 revealed that, cluster analysis of the overall results obtained here leads to 2 clusters. This discrimination appears to be as a result of their different abilities to accumulate either proline or glycine betaine.     

Redescription of a rare deep-sea batfish, <Emphasis Type="Italic">Halieutopsis bathyoreos</Emphasis> (Lophiiformes: Ogcocephalidae)

Halieutopsis bathyoreos Bradbury, 1988 (Lophiiformes: Ogcocephalidae), previously described only on the basis of the holotype (62.6mm in standard length) from the central North Pacific, is redescribed on the basis of the holotype and six additional specimens (41.2–68.7mm in standard length) collected from the western South Pacific, off Papua New Guinea, and the western North Pacific, including the Japanese Archipelago. Halieutopsis bathyoreos is distinguished from its congeners by having a shelflike rostrum extending anteriorly well beyond the mouth, a dorsal escal lobe slightly bisected ventrally, an illicial cavity square in outline and completely visible in ventral view, and lacking tubercles on the ventral surface of the disk. The following characters are newly added to the diagnoses of this species: rostrum width 21–29% of head length, tubercles on the dorsal surface of the disk about half the diameter of those on the lateral margin, and 13–15 large lateral-line scales on the tail.     

Fungi are involved in the effects of litter mixtures on consumption by shredders

1. Decomposition of litter mixtures in both terrestrial and aquatic ecosystems often shows non‐additive diversity effects on decomposition rate, generally interpreted in streams as a result of the feeding activity of macroinvertebrates. The extent to which fungal assemblages on mixed litter may influence consumption by macroinvertebrates remains unknown. 2. We assessed the effect of litter mixing on all possible three‐species combinations drawn from four tree species (Alnus glutinosa, Betula pendula, Juglans regia and Quercus robur) on both fungal assemblages and the rate of litter consumption by a common shredder, Gammarus fossarum. After a 9‐week inoculation in a stream, batches of leaf discs were taken from all leaf species within litter mixture combinations. Ergosterol, an indicator of fungal biomass, and the composition of fungal assemblages, assessed from the conidia released, were determined, and incubated litter offered to G. fossarum in a laboratory‐feeding experiment. 3. Mixing leaf litter species enhanced both the Simpson’s index of the fungal assemblage and the consumption of litter by G. fossarum, but had no clear effect on mycelial biomass. Specifically, consumption rates of J. regia were consistently higher for mixed‐species litter packs than for single‐species litter. In contrast, the consumption rates of B. pendula were not affected by litter mixing, because of the occurrence of both positive and negative litter‐mixing effects in different litter species combinations that counteracted each other. 4. In some litter combinations, the greater development of some fungal species (e.g. Clavariopsis aquatica) as shown by higher sporulation rates coincided with increased leaf consumption, which may have resulted from feeding preferences by G. fossarum for these fungi. 5. Where litter mixture effects on decomposition rate are mediated via shredder feeding, this could be due to indirect effects of the fungal assemblage.     

The coup de grâce for the nested clade phylogeographic analysis?

Nested clade phylogeographic analysis (NCPA) has become a popular method for reconstructing the history of populations across species ranges. Ever since its invention in 1995, criticisms have been formulated, but the method, which has been regularly updated, continues to attract investigators. Molecular Ecology has published a large fraction of the literature on the topic — both pro and con. A recent study by Panchal and Beaumont (2007) finally allows a precise evaluation of the method by developing software that automates the somewhat complicated NCPA procedure. Using simulations of random-mating populations, Panchal and Beaumont find a high frequency of false-positives with their automated NCPA procedure (over 75%). These findings, which echo and amplify earlier warnings, appear serious enough to suggest to researchers to await further evaluation of the method. Although no other all-encompassing method such as the NCAP currently exists to evaluate phylogeographic data sets, researchers have many alternative methods to test ever more refined hypotheses.     

Silencing of EEF2K (eukaryotic elongation factor-2 kinase) reveals AMPK-ULK1-dependent autophagy in colon cancer cells

EEF2K (eukaryotic elongation factor-2 kinase), also known as Ca²⁺/calmodulin-dependent protein kinase III, functions in downregulating peptide chain elongation through inactivation of EEF2 (eukaryotic translation elongation factor 2). Currently, there is a limited amount of information on the promotion of autophagic survival by EEF2K in breast and glioblastoma cell lines. However, the precise role of EEF2K in carcinogenesis as well as the underlying mechanism involved is still poorly understood. In this study, contrary to the reported autophagy-promoting activity of EEF2K in certain cancer cells, EEF2K is shown to negatively regulate autophagy in human colon cancer cells as indicated by the increase of LC3-II levels, the accumulation of LC3 dots per cell, and the promotion of autophagic flux in EEF2K knockdown cells. EEF2K negatively regulates cell viability, clonogenicity, cell proliferation, and cell size in colon cancer cells. Autophagy induced by EEF2K silencing promotes cell survival and does not potentiate the anticancer efficacy of the AKT inhibitor MK-2206. In addition, autophagy induced by silencing of EEF2K is attributed to induction of protein synthesis and activation of the AMPK-ULK1 pathway, independent of the suppression of MTOR activity and ROS generation. Knockdown of AMPK or ULK1 significantly abrogates EEF2K silencing-induced increase of LC3-II levels, accumulation of LC3 dots per cell as well as cell proliferation in colon cancer cells. In conclusion, silencing of EEF2K promotes autophagic survival via activation of the AMPK-ULK1 pathway in colon cancer cells. This finding suggests that upregulation of EEF2K activity may constitute a novel approach for the treatment of human colon cancer.     

Chemoenzymatic labeling of DNA methylation patterns for single-molecule epigenetic mapping

DNA methylation, specifically, methylation of cytosine (C) nucleotides at the 5-carbon position (5-mC), is the most studied and significant epigenetic modification. Here we developed a chemoenzymatic procedure to fluorescently label non-methylated cytosines in CpG context, allowing epigenetic profiling of single DNA molecules spanning hundreds of thousands of base pairs. We used a CpG methyltransferase with a synthetic S-adenosyl-l-methionine cofactor analog to transfer an azide to cytosines instead of the natural methyl group. A fluorophore was then clicked onto the DNA, reporting on the amount and position of non-methylated CpGs. We found that labeling efficiency was increased up to 2-fold by the addition of a nucleosidase, presumably by degrading the inactive by-product of the cofactor after labeling, preventing its inhibitory effect. We used the method to determine the decline in global DNA methylation in a chronic lymphocytic leukemia patient and then performed whole-genome methylation mapping of the model plant Arabidopsis thaliana. Our genome maps show high concordance with published bisulfite sequencing methylation maps. Although mapping resolution is limited by optical detection to 500–1000 bp, the labeled DNA molecules produced by this approach are hundreds of thousands of base pairs long, allowing access to long repetitive and structurally variable genomic regions.