Functional characterization of amphipathic α-helix in the osmoregulatory ABC transporter OpuA

The ATP-binding-cassette transporter OpuA from Lactococcus lactis is composed of two ATPase subunits (OpuAA) and two subunits (OpuABC) with the transmembrane domain fused to an extracellular substrate-binding protein. Of the almost 1900 homologues of OpuA known to date, a subset has an amino-terminal amphipathic helix (plus extra transmembrane segment) fused to the core of the transmembrane domain of the OpuABC subunit. FRET measurements indicate that the amphipathic α-helix is located close to the membrane surface, where its hydrophobic face interacts with the transport protein rather than the membrane lipids. Next, we determined the functional role of this accessory region by engineering the amphipathic α-helix. We analyzed the consequence of the mutations in intact cells by monitoring growth and transport of glycine betaine under normal and osmotic stress conditions. More detailed studies were performed in hybrid membrane vesicles, proteoliposomes, and bilayer nanodisks. We show that the amphipathic α-helix of OpuA is necessary for high activity of OpuA but is not critical for the biogenesis of the protein or the ionic regulation of transport.     

Functional Morphology in Paleobiology: Origins of the Method of ‘Paradigms’

From the early nineteenth century, the successful use of fossils in stratigraphy oriented paleontology (and particularly the study of fossil invertebrates) towards geology. The consequent marginalising of biological objectives was countered in the twentieth century by the rise of ‘Paläobiologie’, first in the German cultural area and only later, as ‘paleobiology’, in the anglophone world. Several kinds of paleobiological research flourished internationally after the Second World War, among them the novel field of ‘paleoecology’. Within this field there were attempts to apply functional morphology to the problematical cases of fossil organisms, for which functions cannot be observed directly. This article describes the origins of the kind of functional inference for fossils that I proposed in 1961 as the method of ‘paradigms’ (a year before Thomas Kuhn made that word more widely familiar with a quite different meaning). Here I summarize some of my ‘worked exemplars’, which were intended to show the paradigm method in action. These case-studies were all taken from the paleontologically important phylum of the Brachiopoda, but the method was claimed to have much wider implications for the interpretation of the fossil record in terms of adaptive evolution. This article takes the history of the paradigm method as far as the late 1960s. I hope to trace, in a sequel, its ambivalent fate during the 1970s and beyond, when for example Gould’s critique of ‘the adaptationist programme’ and the rise of computer-based quantitative methods for the evolutionary interpretation of the fossil record led to the relative eclipse of functional morphology in paleontology.     

Functional characterization of mutations in inherited human cPLA₂ deficiency

Group IVA cytosolic phospholipase A(2) (cPLA(2)α) catalyzes the first step in the arachidonic acid cascade leading to the synthesis of important lipid mediators, the prostaglandins and leukotrienes. We previously described a patient deficient in cPLA(2)α activity, which was associated with mutations in both alleles encoding the enzyme. In this paper, we describe the biochemical characterization of each of these mutations. Using saturating concentrations of calcium, we showed that the R485H mutant was nearly devoid of any catalytic activity, that the S111P mutation did not affect the enzyme activity, and that the known K651R polymorphism was associated with activity slightly higher than that of the wild type. Using MDCK cells, we showed that translocation to the Golgi in response to serum activation was impaired for the S111P mutant but not for the other mutants. Using immortalized mouse lung fibroblasts lacking endogenous cPLA(2)α activity, we showed that both mutations S111P and R485H/K651R caused a profound defect in the enzyme catalytic activity in response to cell stimulation with serum. Taken together, our results show that the S111P mutation hampers calcium binding and membrane translocation without affecting the catalytic activity, and that the mutation R485H does not affect membrane translocation but blocks catalytic activity that leads to inactivation of the enzyme. Interestingly, our results show that the common K651R polymorphism confers slightly higher activity to the enzyme, suggesting a role of this residue in favoring a catalytically active conformation of cPLA(2)α. Our results define how the mutations negatively influence cPLA(2)α function and explain the inability of the proband to release arachidonic acid for eicosanoid production.     

The characterization of a protein–polysaccharide isolated from Kurloff cells of the guinea pig

Kurloff cells of guinea pigs increase in number and accumulate in the spleen on oestrogen treatment. Because they contain metachromatic inclusions and are considered to be lymphocytes they were examined as a possible model for mucopolysaccharidoses like Hurler's syndrome, where some lymphocytes are also metachromatic. Oestrogen treatment produced a large increase in a glycosaminoglycan resembling chondroitin 4-sulphate in chemical analysis, chromatographic behaviour and i.r. spectrum but with an additional strong band at 805cm(-1). Material isolated without proteolysis behaved on gel chromatography as a multiple-chain protein-polysaccharide whose molecular size was decreased by proteolysis. It contained xylose and galactose in molar proportions with serine, compatible with the presence of the same linkage region as in cartilage chondroitin 4-sulphate proteins and which likewise underwent alkaline beta-elimination. Kurloff glycosaminoglycan chains were significantly longer than chondroitin sulphate chains of cartilage protein-polysaccharides as assessed by gel chromatography and the molar ratios of galactosamine to xylose or to serine. Kurloff cells thus contain intact rather than partially degraded protein-polysaccharide and hence are not analogous to Hurler cells, and their electron micrographs were also different. The purified Kurloff protein-polysaccharide and glycosaminoglycan isolated here has been shown by Marshall, Swettenham, Vernon-Roberts & Revell (1970) to be toxic specifically to macrophages at extremely low concentrations in vitro, unlike chondroitin sulphate of protein-polysaccharides from cartilage. The toxic constituent may account for the i.r.-absorption band at 805cm(-1). Although active incorporation of [(35)S]sulphate occurs at early stages of Kurloff-cell induction (Marshall et al. 1970), the fully developed Kurloff cell studied here showed very low incorporation in vitro and in vivo, suggesting that the inclusions are specialized for the storage of the toxic material.     

Biochemical characterization of the δ-carbonic anhydrase from the marine diatom Thalassiosira weissflogii,TweCA

Abstract

Diatom genome sequences clearly reveal the presence of different systems for HCO₃^? uptake. Carbon-concentrating mechanisms (CCM) based on HCO₃^? transport and a plastid-localized carbonic anhydrase (CA, EC 4.2.1.1) appear to be more probable than the others because CAs have been identified in the genome of many diatoms. CAs are key enzymes involved in the acquisition of inorganic carbon for photosynthesis in phytoplankton, as they catalyze efficiently the interconversion between carbon dioxide and bicarbonate. Five genetically distinct classes of CAs exist, α-, β-, γ-, δ- and ζ and all of them are metalloenzymes. Recently we investigated for the first time the catalytic activity and inhibition of the δ-class CA from the marine diatom Thalassiosira weissflogii, named TweCA. This enzyme is an efficient catalyst for the CO₂ hydration and its inhibition profile with sulfonamide/sulfamate and anions have also been investigated. Here, we report the detailed biochemical characterization and chemico-physical properties of the δ-CA of T. weissflogii. The δ-CA encoding gene was cloned and expressed in Artic Express cells and the recombinant protein purified to homogeneity. Interesting to note that TweCA has no intrinsic esterase activity with 4-nitrophenyl acetate (pNpA) as substrate although the phylogenetic analysis showed that δ-CAs are closer to the α-CAs than to the other classes of such enzymes.     

Genomic organization of the complex α-gliadin gene loci in wheat

To better understand the molecular evolution of the large -gliadin gene family, a half-million bacterial artificial chromosome (BAC) library clones from tetraploid durum wheat, Triticum turgidum ssp. durum (2n=4x=28, genome AB), were screened for large genomic segments carrying the -gliadin genes of the Gli-2 loci on the group 6 homoeologous chromosomes. The resulting 220 positive BAC clones—each containing between one and four copies of -gliadin sequences—were fingerprinted for contig assembly to produce contiguous chromosomal regions covering the Gli-2 loci. While contigs consisting of as many as 21 BAC clones and containing up to 17 -gliadin genes were formed, many BAC clones remained as singletons. The accuracy of the order of BAC clones in the contigs was verified by Southern hybridization analysis of the BAC fingerprints using an -gliadin probe. These results indicate that -gliadin genes are not evenly dispersed in the Gli-2 locus regions. Hybridization of these BACs with probes for long terminal repeat retrotransposons was used to determine the abundance and distribution of repetitive DNA in this region. Sequencing of BAC ends indicated that 70% of the sequences were significantly similar to different classes of retrotransposons, suggesting that these elements are abundant in this region. Several mechanisms underlying the dynamic evolution of the Gli-2 loci are discussed.     

Isolation and characterization of polymorphic microsatellite loci in Castanopsis chinensis Hance (Fagaceae)

Twelve novel microsatellite loci were isolated and characterized from enriched genomic libraries of Castanopsis chinensis. Four previously reported microsatellites from Castanopsis cuspidata were cross-amplified in C. chinensis. Forty-two sample trees from a wild population were tested for polymorphism using a set of the 16 polymorphic microsatellites. The average allele number of these microsatellites was 4.6 per locus, ranging from 2 to 7. The ranges of observed and expected heterozygosity were 0.262–1.000 and 0.238–0.818, respectively. Significant deviations from Hardy–Weinberg equilibrium were detected at five loci and no linkage disequilibrium was observed.     

Isolation and characterization of 14 polymorphic microsatellite loci in the ark shell Scapharca subcrenata (Bivalvia: Arcidae)

The ark shell Scapharca subcrenata is an important fishery resource, but has been suffering from severe population decline due to the deterioration of the coastal environment and over-exploitation. To investigate the genetic variation and structure among populations of S. subcrenata, 14 polymorphic microsatellite loci were developed. The number of alleles per polymorphic locus ranged from 11 to 29 and the observed and expected heterozygosity varied from 0.400 to 0.923 and from 0.705 to 0.965, respectively. These microsatellites will help advance the investigation of the genetic population structure and genetic diversity in this species.     

Functional role of β domain in the Thermoanaerobacter tengcongensis glucoamylase

Thermoanaerobacter tengcongensis MB4 glucoamylase (TteGA) contains a catalytic domain (CD), which is structurally similar to eukaryotic GA, and a β domain (BD) with ambiguous function. Firstly, BD is found to be essential to TteGA activity because CD alone could not hydrolyze soluble starch. However, starch hydrolysis activity, similar to that of intact TteGA, was restored to CD in the presence of BD. Secondly, BD is found to be an important helper in the correct folding of CD because CD was mainly expressed in the inclusion bodies on its own in Escherichia coli. By contrast, intact TteGA, BD, and CD combined with BD could be expressed as soluble proteins. Additionally, BD is essential to the thermostability of TteGA because CD displayed lower thermostability compared with the intact TteGA and exhibited enhanced thermostability in the presence of BD in vitro. Truncation of TteGA or mutagenesis of the residues that participate in the interdomain interaction at its BD also led to the reduced thermostability of TteGA.     

Functional characterization of orf6 and orf9 genes involved in the biosynthesis of L-oleandrose from Streptomyces antibioticus Tü99

Two genes, orf6 and orf9 located in the L-oleandrose sugar biosynthetic gene cluster of Streptomyces antibioticus Tü99. NovU has been characterized as C-5 methyltrnaferase involved in noviose biosynthetic pathway. We have cloned and heterologously expressed the orf6, orf9, and novU genes in S. venezuelae YJ003-OTBP1. This established the function of orf6 and orf9 as 4-ketoreductase and 3-epimerase, respectively. All of analytical data of the noviosylated 10-deoxymethynolide also is in support of proving their functions. Furthermore biosynthetic pathway 5,5-gem-dimethyl-6-deoxyglucose (TDP-Lnoviose) has been proposed.     

Metal toxicity characterization factors for marine ecosystems—considering the importance of the estuary for freshwater emissions

Purpose

The study develops site-dependent characterization factors (CFs) for marine ecotoxicity of metals emitted to freshwater, taking their passage of the estuary into account. To serve life cycle assessment (LCA) studies where emission location is often unknown, site-generic marine CFs were developed for metal emissions to freshwater and coastal seawater, respectively. The new CFs were applied to calculate endpoint impact scores for the same amount of metal emission to each compartment, to compare the relative ecotoxicity damages in freshwater and marine ecosystems in LCA.

Methods

Site-dependent marine CFs for emission to freshwater were calculated for 64 comparatively independent seas (large marine ecosystems, LMEs). The site-dependent CF was calculated as the product of fate factor (FF), bioavailability factor (BF), and effect factor (EF). USEtox modified with site-dependent parameters was extended with an estuary removal process to calculate FF. BF and EF were taken from Dong et al. Environ Sci Technol 50:269–278 (2016). Site-generic marine CFs were derived from site-dependent marine CFs. Different averaging principles were tested, and the approach representing estuary discharge rate was identified as the best one. Endpoint marine and freshwater metals CFs were developed to calculate endpoint ecotoxicity impact scores.

Results and discussion

Marine ecotoxicity CFs are 1.5 orders of magnitude lower for emission to freshwater than for emission to seawater for Cr, Cu, and Pb, due to notable removal fractions both in freshwater and estuary. For the other metals, the difference is less than half an order of magnitude, mainly due to removal in freshwater. The site-dependent CFs generally vary within two orders of magnitude around the site-generic CF. Compared to USES-LCA 2.0 CFs (egalitarian perspective), the new site-generic marine CFs for emission to seawater are 1–4 orders of magnitude lower except for Pb. The new site-generic marine CFs for emission to freshwater lie within two orders of magnitude difference from USES-LCA 2.0 CFs. The comparative contribution share analysis shows a poor agreement of metal toxicity ranking between both methods.

Conclusions

Accounting for estuary removal particularly influences marine ecotoxicity CFs for emission to freshwater of metals that have a strong tendency to complex-bind to particles. It indicates the importance of including estuary in the characterization modelling when dealing with those metals. The resulting endpoint ecotoxicity impact scores are 1–3 orders of magnitude lower in seawater than in freshwater for most metals except Pb, illustrating the higher sensitivity of freshwater ecosystems to metal emissions, largely due to the higher species density there.

     

Isolation and characterization of microsatellite loci in Paspalum notatum Flüggé (Poaceae)

Paspalum notatum is a forage grass recognized as one of the major constituents of the native grasslands in the New World. The knowledge of the genetic diversity and structure of P. notatum populations is fundamental for the conservation and germplasm management of this species. About 11 microsatellite markers were isolated from P. notatum and characterized in 25 accessions. The average number of alleles per locus was 7.9 and the PIC ranged from 0.36 to 0.89. The data demonstrated that the most of markers are suitable to detect polymorphism and to study the genetic diversity in the P. notatum species. Moreover, the transferability of these microsatellite were tested on other three congeneric species.