Statolith shape and microstructure in studies of systematics, age and growth in planktonic paralarvae of gonatid squids (Cephalopoda, Oegopsida) from the western Bering Sea

The microstructure, morphology and ontogenetic development ofstatoliths and age and growth of 405 planktonic paralarvae and117 juveniles belonging to 10 species of gonatid squids (Cephalopoda,Oegopsida) were studied in the region of the continental slopein the western part of the Bering Sea (57°00'–61°30'N,163°00'E–179°20'W). The statolith microstructureof all species was characterized by the presence of a largedroplet-shaped nucleus and bipartite postnuclear zone dividedinto two by the first stress check, except for Berryteuthismagister which had only one stress check and an undivided postnuclearzone. In Gonatus spp., completion of development of the postnuclearzone coincided with full development of the central hook onthe tentacular club. Daily periodicity of statolith growth incrementswas validated by maintaining 13 paralarvae of the four mostabundant species in captivity. All species might be subdividedinto two groups based on statolith microstructure, i.e. specieswith a central position of the nucleus within the first statolithcheck (Gonatopsis spp., Egonatus tinro and B.magister) and specieswith the nucleus shifted to the inner side of the first statolithcheck (Gonatus spp.). Comparative analysis of statolith morphologyshowed that paralarval statoliths have species-specific charactersthat allowed the construction of keys to identify species ofgonatid paralarvae based on their statoliths. Analysis of paralarvalgrowth using statoliths revealed that these cold-water planktonicgonatid paralarvae have fast growth rates, attaining a mantlelength of 7–10 mm at 15–20 days and 20–25mm at 35–70 days.     

Synthesis and maturation of green fluorescent protein in a cell-free translation system

Summary A cell-free translation system producing mature green fluorescent protein (GFP) can be a useful tool for studying the mechanism and kinetics of GFP chromophore formation, as well as for fast protein engineering. We report here that the mature GFP can be formed in the cell-free translation system from E.coli. The synthesis of GFP in the cell-free system reaches a plateau in 30 to 40 min whereas its maturation is completed in 4 h from the beginning of translation. The delay between the GFP synthesis and the chromophore formation in the cell-free system provides the possibility to isolate and to analyse maturation intermediates for elucidation of the modification pathway.     

The photoproduction of superoxide radicals and the superoxide dismutase activity of Photosystem II. The possible involvement of cytochrome b559

In the present study the light induced formation of superoxide and intrinsic superoxide dismutase (SOD) activity in PS II membrane fragments and D1/D2/Cytb559-complexes from spinach have been analyzed by the use of ferricytochrome c (cyt c(III)) reduction and xanthine/xanthine oxidase as assay systems. The following results were obtained: 1.) Photoreduction of Cyt c (III) by PS II membrane fragments is induced by addition of sodium azide, tetracyane ethylene (TCNE) or carbonylcyanide-p-trifluoromethoxy-phenylhydrazone (FCCP) and after removal of the extrinsic polypeptides by a 1M CaCl₂-treatment. This activity which is absent in control samples becomes completely inhibited by the addition of exogenous SOD. 2.) The TCNE induced cyt c(III) photoreduction by PS II membrane fragments was found to be characterized by a half maximal concentration of c_1/2=10 M TCNE. Simultaneously, TCNE inhibits the oxygen evolution rate of PS II membrane fragments with c_1/2 3 M. 3.) The photoproduction of O₂ ^– is coupled with H⁺-uptake. This effect is diminished by the addition of the O₂ ^–-trap cyt c(III). 4.) D1/D2/Cytb559-complexes and PS II membrane fragments deprived of the extrinsic proteins and manganese exhibit no SOD-activity but are capable of producing O₂ ^– in the light if a PS II electron donor is added.Based on these results the site(s) of light induced superoxide formation in PS II is (are) inferred to be located at the acceptor side. A part of the PS II donor side and Cyt b559 in its HP-form are proposed to provide an intrinsic superoxide dismutase (SOD) activity.Abbreviations ADRY acceleration of the deactivation reactions of the water-splitting system Y - ANT-2p 2-(3-chloro-4-trifluoromethyl)anilino-3,5-dinitrothiophene - BCP bromocresol purple - cyt cytochrome - Cyt c cytochrome c - DCIP 2,6-dichlorophenol-indophenol - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DEDTC Diethyldithiocarbamate - DMBQ 2,5-dimethyl-p-benzoquinone - DPC 1,5-diphenylcarbazide - FCCP carbonylcyanide-p-trifluoro/methoxy-phenylhydrazone - HP high potential - LP low potential - MES 2-(N-morpholino)ethanesulfonic acid - NADP nicotinamide adenine dinucleotide phosphate - SOD superoxide dismutase - TCNE tetracyane ethylene - TEMED N,N,N,N-tetramethylethylenediamine     

The retinal pigment epithelium as a gateway for monocyte trafficking into the eye

The choroid plexus epithelium within the brain ventricles orchestrates blood‐derived monocyte entry to the central nervous system under injurious conditions, including when the primary injury site is remote from the brain. Here, we hypothesized that the retinal pigment epithelium (RPE) serves a parallel role, as a gateway for monocyte trafficking to the retina following direct or remote injury. We found elevated expression of genes encoding leukocyte trafficking determinants in mouse RPE as a consequence of retinal glutamate intoxication or optic nerve crush (ONC). Blocking VCAM‐1 after ONC interfered with monocyte infiltration into the retina and resulted in a local pro‐inflammatory cytokine bias. Live imaging of the injured eye showed monocyte accumulation first in the RPE, and subsequently in the retina, and peripheral leukocytes formed close contact with the RPE. Our findings further implied that the ocular milieu can confer monocytes a phenotype advantageous for neuroprotection. These results suggest that the eye utilizes a mechanism of crosstalk with the immune system similar to that of the brain, whereby epithelial barriers serve as gateways for leukocyte entry.     

Responses of hepatic biochemical markers in bream <Emphasis Type="Italic">Abramis brama</Emphasis> L. to dietary administered polychlorinated biphenyls

An experimental study of the effects of dietary administered polychlorinated biphenyls (PCBs) on bream (Abramis brama L.) is reported in the present article. Responses of xenobiotic biotransformation system (ethoxyresorufin-O-deethylase and glutathione-S-transferase activities), antioxidant system (superoxide dismutase and catalase activities), and lipid peroxidation system (levels of conjugated dienes and malonic dialdehyde) are investigated. A PCB dose of 2 mg/kg feed does not cause irreversible physiological transformations in bream after 14 days of administration. The defense systems appear to efficiently suppress the effects of the xenobiotic and maintain stable and low intensity of the destructive processes at the exposure conditions used.     

Effects of Metarhizium robertsii on the bloodsucking mosquito Aedes flavescens and non‐target predatory insects (Odonata)

The effects of different concentrations and methods of treatment with Metarhizium robertsii Bisch., Rehner & Humber conidia on the non‐target aquatic dragonfly larvae Lestes sponsa Hansemann, Lestes dryas Kirby and Aeshna affinis Vander Linden and on the target bloodsucking mosquito larvae Aedes (O.) flavescens (Muller) were analysed. We found that dragonflies are significantly less susceptible than mosquitoes to the fungus. Larvae of L. sponsa larvae were more susceptible to wet conidia than dry conidia. However, the mortality of the air‐breathing larvae of A. affinis was significantly higher after treatment with dry conidia relative to aqueous suspension. The results help to minimize the negative effects of entomopathogenic fungi on non‐target predator insects under the control of mosquito larvae.     

Potentiation and Block of ASIC1a by Memantine

Acid-sensing ion channels (ASICs) are modulated by various classes of ligands, including the recently described hydrophobic monoamines, which inhibit and potentiate ASICs in a subunit-specific manner. In particular, memantine inhibits ASIC1a and potentiates ASIC2a homomers. The aim of the present work was to characterize action mechanism of memantine on recombinant ASIC1a expressed in CHO (Chinese hamster ovary) cells. We have demonstrated that effect of memantine on ASIC1a strongly depends on membrane voltage, conditioning pH value and application protocol. When applied simultaneously with activating acidification at hyperpolarized voltages, memantine caused the strongest inhibition. Surprisingly, application of memantine between ASIC1a activations at zero voltage caused significant potentiation. Analysis of the data suggests that memantine produces two separate effects, voltage-dependent open-channel block and shift of steady-state desensitization curve to more acidic values. Putative binding sites are discussed based on the computer docking of memantine to the acidic pocket and the pore region.     

Diversity of Trypanosomatids in Cockroaches and the Description of Herpetomonas tarakana sp. n.

In this study, we surveyed six species of cockroaches, two synanthropic (i.e. ecologically associated with humans) and four wild, for intestinal trypanosomatid infections. Only the wild cockroach species were found to be infected, with flagellates of the genus Herpetomonas. Two distinct genotypes were documented, one of which was described as a new species, Herpetomonas tarakana sp. n. We also propose a revision of the genus Herpetomonas and creation of a new subfamily, Phytomonadinae, to include Herpetomonas, Phytomonas, and a newly described genus Lafontella n. gen. (type species Lafontella mariadeanei comb. n.), which can be distinguished from others by morphological and molecular traits.     

Production of a Brassica napus Low-Molecular Mass Acyl-Coenzyme A-Binding Protein in Arabidopsis Alters the Acyl-Coenzyme A Pool and Acyl Composition of Oil in Seeds

Low-molecular mass (10 kD) cytosolic acyl-coenzyme A-binding protein (ACBP) has a substantial influence over fatty acid (FA) composition in oilseeds, possibly via an effect on the partitioning of acyl groups between elongation and desaturation pathways. Previously, we demonstrated that the expression of a Brassica napus ACBP (BnACBP) complementary DNA in the developing seeds of Arabidopsis (Arabidopsis thaliana) resulted in increased levels of polyunsaturated FAs at the expense of eicosenoic acid (20:1^cisΔ11) and saturated FAs in seed oil. In this study, we investigated whether alterations in the FA composition of seed oil at maturity were correlated with changes in the acyl-coenzyme A (CoA) pool in developing seeds of transgenic Arabidopsis expressing BnACBP. Our results indicated that both the acyl-CoA pool and seed oil of transgenic Arabidopsis lines expressing cytosolic BnACBP exhibited relative increases in linoleic acid (18:2^cisΔ9,12; 17.9%–44.4% and 7%–13.2%, respectively) and decreases in 20:1^cisΔ11 (38.7%–60.7% and 13.8%–16.3%, respectively). However, alterations in the FA composition of the acyl-CoA pool did not always correlate with those seen in the seed oil. In addition, we found that targeting of BnACBP to the endoplasmic reticulum resulted in FA compositional changes that were similar to those seen in lines expressing cytosolic BnACBP, with the most prominent exception being a relative reduction in α-linolenic acid (18:3^cisΔ9,12,15) in both the acyl-CoA pool and seed oil of the former (48.4%–48.9% and 5.3%–10.4%, respectively). Overall, these data support the role of ACBP in acyl trafficking in developing seeds and validate its use as a biotechnological tool for modifying the FA composition of seed oil.Cytosolic low-molecular mass (approximately 10 kD) acyl-coenzyme A-binding protein (ACBP) consists of a four-α-helix domain capable of binding acyl-CoAs with high affinity in a wide range of eukaryotic organisms (Faergeman et al., 2007). It is believed to serve a housekeeping function of maintaining free acyl-CoA concentrations at low nanomolar levels and, thus, prevents micelle formation and the partitioning of acyl-CoA into membranes (Knudsen et al., 1999). This protein is also considered to contribute to another facet of acyl-CoA pool maintenance via its role in the intracellular transport of acyl-CoAs in the aqueous environment of the cytosol (Rasmussen et al., 1994). Moreover, it has also been shown to exhibit more specialized functions in metabolic processes in which acyl-CoA is actively involved, depending on the tissue and physiological state (Guerrero et al., 2006; Xiao and Chye 2011; Yurchenko and Weselake, 2011).In the developing seeds of oleaginous plants, fatty acids (FAs) are synthesized de novo in plastids and are activated to acyl-CoAs upon their transfer to the cytosol, after which time they can undergo additional modifications (e.g. elongation and desaturation) on the membranes of the endoplasmic reticulum (ER; for review, see Rawsthorne, 2002). While FA elongation is performed on the acyl-CoA substrate, the introduction of the second and third double bonds requires the acyl group to be esterified to phosphatidylcholine (PC; Jaworski, 1987). The composition of the acyl-CoA pool, therefore, is highly dynamic and represents a net result of both de novo synthesis and acyl-editing processes (Bates et al., 2009).The acyl-CoA pool provides substrate for acyltransferases involved in the biosynthesis of triacylglycerol (TAG), which is a major component of seed oil (Weselake et al., 2009). More specifically, TAG synthesis typically occurs via a series of acyl-CoA-dependent acylations of a glycerol backbone derived from sn-glycerol-3-phosphate in a pathway known as the sn-glycerol-3-phosphate or Kennedy pathway (for review, see Snyder et al., 2009; Weselake et al., 2009), although acyl-CoA-independent reactions can also be involved in the production of TAG (Stobart et al., 1997; Banaś et al., 2000; Dahlqvist et al., 2000) and thus contribute to its final composition. Low-molecular mass ACBPs have been demonstrated to modulate the activities of Kennedy pathway acyltransferases in a manner dependent upon the ratio of ACBP to acyl-CoA, stimulating TAG biosynthesis under conditions of acyl-CoA excess and inhibiting acyltransferase activities when relative amounts of acyl-CoA are low compared with ACBP, thus regulating the size of the acyl-CoA pool (for review, see Yurchenko and Weselake, 2011).The acyl-CoA pool in seeds is also influenced through a distinct route involving lysophosphatidylcholine acyltransferase (LPCAT), which catalyzes the acyl-CoA-dependent acylation of lysophosphatidylcholine at the sn-2 position to form PC (Ichihara et al., 1995). Acyl groups esterified to PC become substrates for FA desaturation and other modifications (Miquel and Browse, 1992; Broun et al., 1998) and can then be returned back to the acyl-CoA pool or channeled into TAG through acyl-CoA-independent mechanisms (Stymne and Stobart, 1984; Weselake, 2005; Lager et al., 2013). The efficiency of this acyl group channeling to and from PC is an important determinant of the overall composition of FAs in the acyl-CoA pool and, subsequently, in seed oil.Previously, we demonstrated that the expression of the Brassica napus low-molecular mass ACBP (hereafter referred to as BnACBP) in the presence of Arabidopsis (Arabidopsis thaliana) LPCAT isoforms in an in vitro system enhanced the incorporation of oleic acid (18:1^cisΔ9; hereafter referred to as 18:1) into PC and the release of linoleic acid (18:2^cisΔ9,12; hereafter referred to as 18:2) from PC into acyl-CoA (Yurchenko et al., 2009). In line with these results, the expression of BnACBP complementary DNA (cDNA) in Arabidopsis developing seeds was also shown to result in elevated levels of the polyunsaturated fatty acids (PUFAs) 18:2 and α-linolenic acid (18:3^cisΔ9,12,15; hereafter referred to as 18:3) in seed oil, mainly at the expense of eicosenoic acid (20:1^cisΔ11; hereafter referred to as 20:1) and saturated fatty acids (SFAs; Yurchenko et al., 2009). Based on these findings, BnACBP was proposed to be involved in acyl exchange between acyl-CoA and PC pools, which may affect the rate of FA modifications and, ultimately, the FA composition of seed oil (Yurchenko et al., 2009).In this study, we endeavored to provide further evidence that low-molecular mass ACBP functions in acyl trafficking by investigating whether changes in the FA composition of TAG in Arabidopsis seeds expressing BnACBP were correlated with modifications in the composition of the acyl-CoA pool. In addition, since FA modifications such as elongation and desaturation as well as TAG synthesis occur on ER membranes, we also examined the effect of changing the subcellular localization of BnACBP (from the cytosol to the ER) on the acyl composition of TAG and the acyl-CoA pool in transgenic Arabidopsis. Consequently, we generated localized pools of acyl-CoAs that could be readily accessed by acyltransferases involved in seed oil biosynthesis. Taken together, our findings provide insight into the role of low-molecular mass ACBP in seed oil metabolism and suggest that ACBP (either in its native cytosolic form or as an ER-targeted fusion protein) may serve as a useful tool in biotechnological modifications of FA composition in oil crops.     

Lifespan Extension Conferred by Endoplasmic Reticulum Secretory Pathway Deficiency Requires Induction of the Unfolded Protein Response

Cells respond to accumulation of misfolded proteins in the endoplasmic reticulum (ER) by activating the unfolded protein response (UPR) signaling pathway. The UPR restores ER homeostasis by degrading misfolded proteins, inhibiting translation, and increasing expression of chaperones that enhance ER protein folding capacity. Although ER stress and protein aggregation have been implicated in aging, the role of UPR signaling in regulating lifespan remains unknown. Here we show that deletion of several UPR target genes significantly increases replicative lifespan in yeast. This extended lifespan depends on a functional ER stress sensor protein, Ire1p, and is associated with constitutive activation of upstream UPR signaling. We applied ribosome profiling coupled with next generation sequencing to quantitatively examine translational changes associated with increased UPR activity and identified a set of stress response factors up-regulated in the long-lived mutants. Besides known UPR targets, we uncovered up-regulation of components of the cell wall and genes involved in cell wall biogenesis that confer resistance to multiple stresses. These findings demonstrate that the UPR is an important determinant of lifespan that governs ER stress and identify a signaling network that couples stress resistance to longevity.