INCIPIENT SPECIATION OF SEA STAR POPULATIONS BY ADAPTIVE GAMETE RECOGNITION COEVOLUTION

Reproductive isolation—the key event in speciation—can evolve when sexual conflict causes selection favoring different combinations of male and female adaptations in different populations. Likely targets of such selection include genes that encode proteins on the surfaces of sperm and eggs, but no previous study has demonstrated intraspecific coevolution of interacting gamete recognition genes under selection. Here, we show that selection drives coevolution between an egg receptor for sperm (OBi1) and a sperm acrosomal protein (bindin) in diverging populations of a sea star (Patiria miniata). We found positive selection on OBi1 in an exon encoding part of its predicted substrate‐binding protein domain, the ligand for which is found in bindin. Gene flow was zero for the parts of bindin and OBi1 in which selection for high rates of amino acid substitution was detected; higher gene flow for other parts of the genome indicated selection against immigrant alleles at bindin and OBi1. Populations differed in allele frequencies at two key positively selected sites (one in each gene), and differences at those sites predicted fertilization rate variation among male–female pairs. These patterns suggest adaptively evolving loci that influence reproductive isolation between populations.     

Effects of different dietary fatty acids profiles on the growth performance and body composition of juvenile tench (<Emphasis Type="Italic">Tinca tinca</Emphasis> (L.))

Juvenile tench (initial weight of about 57 g) were fed feed supplemented with fish oil (group FO), linseed oil (group LO), peanut oil (group PO), or rapeseed oil (group RO) containing 47% protein and 12% fat for 55 days. The inclusion of the tested oils was 50 g kg⁻¹ (42% total crude lipids in diets). No significant differences were noted in the fish growth performance. The proximate composition of the whole fish bodies and the viscera (water, protein, fat, ash) was similar in all the dietary treatments (P > 0.05). Differences were noted only with regard to the ash content of the fillets (P < 0.05). The analysis of the fatty acids profiles of tench (whole fish) indicated there were significant differences in the total content of monoenoic and polyenoic (PUFA) acids. Significant differences were also noted with regard to n-3 PUFA and n-6 PUFA. Consequently, the ratio of n-3/n-6 acids ranged from 1.6 (group PO) to 2.08 (group LO; P < 0.05). The feed applied was not confirmed to have had an impact on the fatty acids profile of the tench fillets. There was a statistically significant intergroup difference in the content of saturated fatty acids (SFA) in tench viscera. In the fish fed vegetable oils supplemented diets, the level of SFA was lower (P < 0.05).     

Glycoprotein production for structure analysis with stable, glycosylation mutant CHO cell lines established by fluorescence-activated cell sorting

Stable mammalian cell lines are excellent tools for the expression of secreted and membrane glycoproteins. However, structural analysis of these molecules is generally hampered by the complexity of N‐linked carbohydrate side chains. Cell lines with mutations are available that result in shorter and more homogenous carbohydrate chains. Here, we use preparative fluorescence‐activated cell sorting (FACS) and site‐specific gene excision to establish high‐yield glycoprotein expression for structural studies with stable clones derived from the well‐established Lec3.2.8.1 glycosylation mutant of the Chinese hamster ovary (CHO) cell line. We exemplify the strategy by describing novel clones expressing single‐chain hepatocyte growth factor/scatter factor (HGF/SF, a secreted glycoprotein) and a domain of lysosome‐associated membrane protein 3 (LAMP3d). In both cases, stable GFP‐expressing cell lines were established by transfection with a genetic construct including a GFP marker and two rounds of cell sorting after 1 and 2 weeks. The GFP marker was subsequently removed by heterologous expression of Flp recombinase. Production of HGF/SF and LAMP3d was stable over several months. 1.2 mg HGF/SF and 0.9 mg LAMP3d were purified per litre of culture, respectively. Homogenous glycoprotein preparations were amenable to enzymatic deglycosylation under native conditions. Purified and deglycosylated LAMP3d protein was readily crystallized. The combination of FACS and gene excision described here constitutes a robust and fast procedure for maximizing the yield of glycoproteins for structural analysis from glycosylation mutant cell lines.     

Modeling Rett syndrome with stem cells

The discovery that somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) raised the exciting possibility of modeling diseases with patient-specific cells. Marchetto et?al. (2010) now use iPSC technology to generate, characterize, and treat an in?vitro model for the autism spectrum disorder Rett syndrome.     

Selective Condensation Drives Partitioning and Sequential Secretion of Cyst Wall Proteins in Differentiating Giardia lamblia

Controlled secretion of a protective extracellular matrix is required for transmission of the infective stage of a large number of protozoan and metazoan parasites. Differentiating trophozoites of the highly minimized protozoan parasite Giardia lamblia secrete the proteinaceous portion of the cyst wall material (CWM) consisting of three paralogous cyst wall proteins (CWP1–3) via organelles termed encystation-specific vesicles (ESVs). Phylogenetic and molecular data indicate that Diplomonads have lost a classical Golgi during reductive evolution. However, neogenesis of ESVs in encysting Giardia trophozoites transiently provides basic Golgi functions by accumulating presorted CWM exported from the ER for maturation. Based on this “minimal Golgi” hypothesis we predicted maturation of ESVs to a trans Golgi-like stage, which would manifest as a sorting event before regulated secretion of the CWM. Here we show that proteolytic processing of pro-CWP2 in maturing ESVs coincides with partitioning of CWM into two fractions, which are sorted and secreted sequentially with different kinetics. This novel sorting function leads to rapid assembly of a structurally defined outer cyst wall, followed by slow secretion of the remaining components. Using live cell microscopy we find direct evidence for condensed core formation in maturing ESVs. Core formation suggests that a mechanism controlled by phase transitions of the CWM from fluid to condensed and back likely drives CWM partitioning and makes sorting and sequential secretion possible. Blocking of CWP2 processing by a protease inhibitor leads to mis-sorting of a CWP2 reporter. Nevertheless, partitioning and sequential secretion of two portions of the CWM are unaffected in these cells. Although these cysts have a normal appearance they are not water resistant and therefore not infective. Our findings suggest that sequential assembly is a basic architectural principle of protective wall formation and requires minimal Golgi sorting functions.     

Evidence for a glucose 1-phosphate translocator in storage tissue amyloplasts of potato (Solanum tuberosum) suspension-cultured cells

Transport of glucose 1-phosphate (G1P) and highly purified triose phosphate into storage tissue amyloplasts was studied. Isolated amyloplasts from potato ( Solanum tuberosum L., dihaploid stock, HH 258) were transport-functional and metabolically active in starch synthesis. Fourty percent of the amyloplasts were intact and there was only a small degree (0–1.6%) of contamination by other cellular compartments. G1P showed a clear uptake pattern paralleled by starch synthesis. Uptake of triose phosphates was virtually nil. Uptake of GIP was pH dependent with a sharp maximum at pH 5.7 and showed Michaelis-Menten kinetics with an apparent K_m of 0.5 m M . Temperature influenced the rate of uptake, the highest rate being at 25°C. Fructose l-phosphate, ADP-glucose, glucose, and inorganic phosphate inhibited the uptake of G1P. Uptake was also inhibited by DIDS (1–25 μ M ) and by Phloretin (45–750 μW). It is therefore concluded that the transport of GIP across the inner amyloplast membrane is mediated by a hexose phosphate translocator selective for phosphate and glucose moieties of the molecule. Considering the low pH maximum for G1P uptake it is possible that the uptake of G1P, and eventually starch synthesis, is regulated by an acidification of the intermembrane space by proton pumps of the inner amyloplast membrane.     

Comparing the Environmental Performance of Fluorescent Whitening Agents with Peroxide Bleaching of Mechanical Pulp

The use of fluorescent whitening agents (FWAs) instead of oxidative bleaching agents such as peroxide is an alternative for the bleaching of mechanical pulp. By this approach, the chromophores of the wood components in the pulp are not destroyed chemically but the brightness of the pulp is achieved by increased re-emission of blue light in the range of 400–500 nm. In this study, a typical FWA and peroxide bleaching chemicals are compared with respect to both production and application in the pulp mill. The life-cycle inventory shows that, on the one hand, the production of the FWA leads to higher releases of salts and adsorbable organically bound halogens (AOX) to surface waters and that, on the other hand, significantly less FWA is required in the application step in order to reach the same pulp brightness. The life-cycle impact assessment of the production step is presented in terms of Eco-indicator 95. These results, however, do not cover the environmental fate of various chemicals released to the aquatic environment in the course of the bleaching/whitening step. Therefore, this part is assessed by means of a more detailed investigation of the chemicals' environmental fate in rivers and their aquatic toxicity.     

Secreted PCSK9 downregulates low density lipoprotein receptor through receptor-mediated endocytosis

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a protease that regulates low density lipoprotein receptor (LDLR) protein levels. The mechanisms of this action, however, remain to be defined. We show here that recombinant human PCSK9 expressed in HEK293 cells was readily secreted into the medium, with the prosegment associated with the C-terminal domain. Secreted PCSK9 mediated cell surface LDLR degradation in a concentration- and time-dependent manner when added to HEK293 cells. Accordingly, cellular LDL uptake was significantly reduced as well. When infused directly into C57B6 mice, purified human PCSK9 substantially reduced hepatic LDLR protein levels and resulted in increased plasma LDL cholesterol. When added to culture medium, fluorescently labeled PCSK9 was endocytosed and displayed endosomal-lysosomal intracellular localization in HepG2 cells, as was demonstrated by colocalization with DiI-LDL. PCSK9 endocytosis was mediated by LDLR as LDLR deficiency (hepatocytes from LDLR null mice), or RNA interference-mediated knockdown of LDLR markedly reduced PCSK9 endocytosis. In addition, RNA interference knockdown of the autosomal recessive hypercholesterolemia (ARH) gene product also significantly reduced PCSK9 endocytosis. Biochemical analysis revealed that the LDLR extracellular domain interacted directly with secreted PCSK9; thus, overexpression of the LDLR extracellular domain was able to attenuate the reduction of cell surface LDLR levels by secreted PCSK9. Together, these results reveal that secreted PCSK9 retains biological activity, is able to bind directly to the LDLR extracellular domain, and undergoes LDLR-ARH-mediated endocytosis, leading to accelerated intracellular degradation of the LDLR.