Hemolymph cholinesterase activity in the Mussel <Emphasis Type="Italic">Crenomytilus grayanus</Emphasis> (Dunker, 1853) that was exposed to adverse natural and anthropogenic conditions

The influence of habitat conditions on the activity, the structure of the substrate specificity (the ratio of the substrate hydrolysis rates), and the kinetic parameters of substrate hydrolysis due to the effect of hemolymph cholinesterase of the mussel Crenomytilus grayanus was studied. Mussels were collected from areas that are influenced by seasonal and stationary upwelling, as well as from a polluted area. Upwelling and anthropogenic pressure were shown to alter the structure of hemolymph cholinesterase substrate specificity in mussels, up to complete loss of the ability to catalyze the hydrolysis of propionyland butyrylthiocholine. It was established that during the seasonal upwelling the efficiency of the cholinergic process in mussels is provided by a wide range of effective concentrations of the substrates and by decreasing their affinity to the enzyme. Under the conditions of chronic anthropogenic pollution, the cholinesterase of the mussel hemolymph loses its ability to hydrolyze substrates other than acetylthiocholine.     

High‐level production of soluble pyrroloquinoline quinone‐dependent glucose dehydrogenase in Escherichia coli

Pyrroloquinoline quinone‐dependent glucose dehydrogenase (EC1.1.5.2, PQQGDH) has attracted progressive attention due to its application in glucose detection in clinic diagnosis and industrial bioprocess controls. To satisfy its increasing demand, improvement of PQQGDH production derived from Acinetobacter calcoaceticus L.M.D. 79.41 in recombinant Escherichia coli is necessary and is therefore the focus of the current study. Different carbon sources as well as induction conditions were investigated for overexpression of soluble PQQGDH. The results indicate that the target protein was optimally produced with 20 g/L glucose as the substrate. Moreover, the highest expression level (1530 kU/L) was achieved by a novel two‐temperature cultivation strategy in the 10‐L fermentor. This presents a sixfold improvement over previously reported values. After Ni‐NTA affinity chromatography purification, high‐purity enzyme with the specific activity of 5811 U/mg was obtained with a purification yield of 55%. The purified recombinant PQQGDH showed thermal stability and substrate specificity as the native enzyme. In summary, this work provides an alternative production process to overexpress PQQGDH and shows high applicability for large‐scale production of this important glucose dehydrogenase.     

Morphological Adaptations of the Mussel Crenomitilus grayanus (Bivalvia) to Attached Life

The morphology of the mussel Crenomytilus grayanus from different biotopes manifesting the adaptation of this mollusk to attached life on the bottom surface was studied. It was established that colonization of rocky coastal areas with active hydrodynamics by mussels is related to retardation of linear growth rates, the formation of convex shells, and vigorous development of retractor muscles of the foot and byssus. An environment protected from the effect of surfs on silted bottoms facilitated the fast growth of mussels, which resulted in the formation of an elegant flat shell. In that biotope mussels are specified by a rather poor development of the muscles responsible for its attachment to the substrate.     

Effect of copper ions on spatial density of NO synthase-positive cells in intestine of the mussel <Emphasis Type="Italic">Crenomytilus grayanus</Emphasis> (mollusca: bivalvia: mytilidae). A histochemical study

By the histochemical method of detection of NADPH-diaphorase (NADPH-d) (EC1.6.99.1) [1] the state of nitroxidergic enteric nervous system of the mussel Crenomytilus grayanus was studied under conditions of an increased copper concentration in water. Under the action of copper ions the density of distribution of NADPH-d-positive cells has been established to be changed as compared with control throughout 28 days. A sharp rise of proportion of the labeled cells and their enzyme activity was noted after one day of the experiment. The labeled bipolar cells were of dark blue color and were located within the epithelium. There were revealed numerous nerve fibers penetrating the intestinal epithelium throughout its entire length as well as bipolar nerve cells in epithelium of the minor typhlosole and of crystalline style sac; in control molluscs the NADPH-d-positive cells in these parts were absent. After 7 days the difference between control and experimental decreased and remained at this level after 14 days, while after 21 days of exposition the proportion of labeled cells in the experimental mussels was lower than in control, but increased again after 28 days. It is suggested that nitric oxide is an important protective factor of the intestinal epithelium of the mussel C. grayanus and participates in adaptation of this mollusc to action of the elevated concentration of copper ions in water.     

Two binding proteins of the ABC transporter that confers growth of Bifidobacterium animalis subsp. lactis ATCC27673 on β‐mannan possess distinct manno‐oligosaccharide‐binding profiles

Human gut bifidobacteria rely on ATP‐binding cassette (ABC) transporters for oligosaccharide uptake. Multiple oligosaccharide‐specific solute‐binding protein (SBP) genes are occasionally associated with a single ABC transporter, but the significance of this multiplicity remains unclear. Here, we characterize BlMnBP1 and BlMnBP2, the two SBPs associated to the β‐manno‐oligosaccharide (MnOS) ABC transporter in Bifidobacterium animalis subsp. lactis. Despite similar overall specificity and preference to mannotriose (K_d≈80 nM), affinity of BlMnBP1 is up to 2570‐fold higher for disaccharides than BlMnBP2. Structural analysis revealed a substitution of an asparagine that recognizes the mannosyl at position 2 in BlMnBP1, by a glycine in BlMnBP2, which affects substrate affinity. Both substitution types occur in bifidobacterial SBPs, but BlMnBP1‐like variants prevail in human gut isolates. B. animalis subsp. lactis ATCC27673 showed growth on gluco and galactomannans and was able to outcompete a mannan‐degrading Bacteroides ovatus strain in co‐cultures, attesting the efficiency of this ABC uptake system. By contrast, a strain that lacks this transporter failed to grow on mannan. This study highlights SBP diversification as a possible strategy to modulate oligosaccharide uptake preferences of bifidobacterial ABC‐transporters during adaptation to specific ecological niches. Efficient metabolism of galactomannan by distinct bifidobacteria, merits evaluating this plant glycan as a potential prebiotic.     

Polymorphism and physiology of arsenate tolerance in Holcus lanatus L. from an uncontaminated site

The polymorphism of arsenate tolerance in a Holcus lanatus L. population from an uncontaminated soil was investigated and a high percentage of tolerant individuals (65%) was found in the population studied. Influx of arsenate was highly correlated to arsenate tolerance within the population, with the most tolerant individuals having the lowest rates of arsenate influx. Isotherms for the high affinity arsenate uptake systems were determined in six tolerant and six non-tolerant genotypes. Tolerant plants had the lowest rates of arsenate influx. This was achieved by adaptation of the V_max of arsenate influx with the V_max of the high affinity uptake system saturating at lower substrate concentrations in the tolerant plants. The polymorphism is discussed with relation to adaptation to the extreme environments to which the plants are subjected on mine-spoil soils.     

An improved fluorogenic substrate for the detection of alkaline phosphatase activity

We designed a new alkaline phosphatase (ALP)-sensitive fluorogenic probe in which a self-immolative spacer group, p-hydroxybenzyl alcohol, is linked to a profluorogenic compound to improve substrate specificity. Enzymatic hydrolysis converts the fluorogenic substrate 1 to a highly fluorescent reporter 3, thus allowing for the fast and quantitative analysis of ALP activity with greatly increased affinity for the enzyme.     

Posttranslational oxidative modification of (R)‐2‐(2,4‐dichlorophenoxy)propionate/α‐ketoglutarate‐dependent dioxygenases (RdpA) leads to improved degradation of 2,4‐dichlorophenoxyacetate (2,4‐D)

Microbial activities and the versatility gained through adaptation to xenobiotic compounds are the main biological forces to counteract environmental pollution. The current results present a new adaptive mechanism that is mediated through posttranslational modifications. Strains of Delftia acidovorans incapable of growing autochthonously on 2,4‐dichlorophenoxyacetate (2,4‐D) were cultivated in a chemostat on 2,4‐D in the presence of (R)‐2‐(2,4‐dichlorophenoxy)propionate. Long‐term cultivation led to enhanced 2,4‐D degradation, as demonstrated by improved values of the Michaelis–Menten constant K_m for 2,4‐D and the catalytic efficiency k_cat/K_m of the initial degradative key enzyme (R)‐2‐(2,4‐dichlorophenoxy)propionate/α‐ketoglutarate‐dependent dioxygenases (RdpA). Analyses of the rdpA gene did not reveal any mutations, indicating a nongenetic mechanism of adaptation. 2‐DE of enzyme preparations, however, showed a series of RdpA forms varying in their pI. During adaptation increased numbers of RdpA variants were observed. Subsequent immunoassays of the RdpA variants showed a specific reaction with 2,4‐dinitrophenylhydrazine (DNPH), characteristic of carbonylation modifications. Together these results indicate that posttranslational carbonylation modified the substrate specificity of RdpA. A model was implemented explaining the segregation of clones with improved degradative activity within the chemostat. The process described is capable of quickly responding to environmental conditions by reversibly adapting the degradative potential to various phenoxyalkanoate herbicides.     

快速展示糖苷水解酶活性架构单点突变导致结合力变化的电泳技术

酶分子在长期进化过程中形成一系列氨基酸残基组成的活性架构,参与底物的识别、结合与催化过程,而活性架构中相应氨基酸残基是如何影响酶分子结合底物的能力,进而影响酶分子的催化效率,一直是酶分子理性改造研究的热点.利用亲和电泳技术,可以快速展示内切纤维素酶Tr Cel12A和木聚糖酶Tl Xyn A活性架构中不同突变体的催化活性及其迁移率的变化,进而通过在不同底物浓度凝胶中蛋白质相对迁移率变化程度的定量回归分析,发现由氨基酸单点突变导致蛋白质迁移率的相对变化,可以定量表征酶分子突变前后结合底物能力的变化.亲和电泳测定的有效阻滞常数Kb值与等温滴定量热法和荧光光谱法测定的相关参数比较具有明显相关性.由于亲和电泳技术在测定酶分子与底物的结合能力时具有简便、快速、灵敏的特点,因而可作为常规生化实验室常规普筛技术来检测突变文库中系列突变体导致结合力的变化.     

A historical perspective of the genus Mytilus (Bivalvia: Mollusca) in New Zealand: multivariate morphometric analyses of fossil, midden and contemporary blue mussels

The taxonomic status of smooth shelled blue mussels of the genus Mytilus has received considerable attention in the last 25 years. Despite this, the situation in the southern hemisphere remains uncertain and is in need of clarification. Recent work suggests that contemporary New Zealand mussels from two cool/cold temperate locations are M. galloprovincialis. However, the distribution of Mytilus in New Zealand ranges from 35 ° to 52 ° south (～ 1800 km), meaning that large areas of the subtropical/warm temperate north and the subantarctic south remain unsampled, an important consideration when species of this genus exhibit pronounced macrogeographical differences in their distributions which are associated with environmental variables such as water temperature, salinity, wave action and ice cover. This study employed multivariate morphometric analyses of one fossil, 83 valves from middens, and 92 contemporary valves from sites spanning the distributional range of blue mussels to determine a historical and contemporary perspective of the taxonomic status of Mytilus in New Zealand. The findings indicated that all fossil and midden mussels are best regarded as M. galloprovincialis and confirmed that contemporary mussels, with one possible regional exception, are also best regarded as M. galloprovincialis. Contemporary mussels from the Bay of Islands (warm temperate/subtropical) exhibited much greater affinity to M. edulis than they did to M. galloprovincialis, indicating that mussels from this area require detailed genetic examination to determine their taxonomic status. The analyses revealed a significant difference between the fossil/midden mussels and the contemporary mussels, consistent with levels of present day differentiation among intraspecific populations and not thought to reflect any substantive temporal change between mussels of the two groups. The continuous distribution of M. galloprovincialis in New Zealand from the warm north to the subantarctic south indicates that the physiology of this species is adapted to a wide range of water temperature conditions. Therefore, the distribution of this species on a worldwide scale is unlikely to be restricted by its adaptation to warm water alone, as has previously been widely assumed. © 2004 The Linnean Society of London, Biological Journal of the Linnean Society, 2004, 82 , 329–344.     

A nuclear‐localized rice glyoxalase I enzyme,OsGLYI‐8, functions in the detoxification of methylglyoxal in the nucleus

The cellular levels of methylglyoxal (MG), a toxic byproduct of glycolysis, rise under various abiotic stresses in plants. Detoxification of MG is primarily through the glyoxalase pathway. The first enzyme of the pathway, glyoxalase I (GLYI), is a cytosolic metalloenzyme requiring either Ni²⁺ or Zn²⁺ for its activity. Plants possess multiple GLYI genes, of which only some have been partially characterized; hence, the precise molecular mechanism, subcellular localization and physiological relevance of these diverse isoforms remain enigmatic. Here, we report the biochemical properties and physiological role of a putative chloroplast‐localized GLYI enzyme, OsGLYI‐8, from rice, which is strikingly different from all hitherto studied GLYI enzymes in terms of its intracellular localization, metal dependency and kinetics. In contrast to its predicted localization, OsGLYI‐8 was found to localize in the nucleus along with its substrate, MG. Further, OsGLYI‐8 does not show a strict requirement for metal ions for its activity, is functional as a dimer and exhibits unusual biphasic steady‐state kinetics with a low‐affinity and a high‐affinity substrate‐binding component. Loss of AtGLYI‐2, the closest Arabidopsis ortholog of OsGLYI‐8, results in severe germination defects in the presence of MG and growth retardation under salinity stress conditions. These defects were rescued upon complementation with AtGLYI‐2 or OsGLYI‐8. Our findings thus provide evidence for the presence of a GLYI enzyme and MG detoxification in the nucleus.     

Directed evolution and rational approaches to improving <Emphasis Type="Italic">Streptomyces clavuligerus</Emphasis> deacetoxycephalosporin C synthase for cephalosporin production

It is approximately 60 years since the discovery of cephalosporin C in Cephalosporium acremonium. Streptomycetes have since been found to produce the structurally related cephamycin C. Studies on the biosynthetic pathways of these two compounds revealed a common pathway including a step governed by deacetoxycephalosporin C synthase which catalyses the ring-expansion of penicillin N to deacetoxycephalosporin C. Because of the therapeutic importance of cephalosporins, this enzyme has been extensively studied for its ability to produce these antibiotics. Although, on the basis of earlier studies, its substrate specificity was believed to be extremely narrow, relentless efforts in optimizing the in-vitro enzyme assay conditions showed that it is able to convert a wide range of penicillin substrates differing in their side chains. It is a member of 2-oxoglutarate-dependent dioxygenase protein family, which requires the iron(II) ion as a co-factor and 2-oxoglutarate and molecular oxygen as co-substrates. It has highly conserved HXDX _n H and RXS motifs to bind the co-factor and co-substrate, respectively. With advances in technology, the genes encoding this enzyme from various sources have been cloned and heterologously expressed for comparative analyses and mutagenesis studies. A high level of recombinant protein expression has also enabled crystallization of this enzyme for structure determination. This review will summarize some of the earlier biochemical characterization and describe the mechanistic action of this enzyme revealed by recent structural studies. This review will also discuss some of the approaches used to identify the amino acid residues involved in binding the penicillin substrate and to modify its substrate preference for possible industrial application.     

Population structure of the ribbed mussel <Emphasis Type="Italic">Geukensia demissa</Emphasis> in salt marshes in the southern Gulf of St. Lawrence,Canada

The ribbed mussel, Geukensia demissa, is highly dependent on the cordgrass Spartina alterniflora for amelioration from environmental stress and substrate stabilization. Spartina alterniflora is a foundation species in marshes, and G. demissa is typically associated with cordgrass beds. Marshes in the southern Gulf of St. Lawrence are experiencing erosion and degradation, presumably as a result of increases in sea level, which increases salinity exposure and negatively impacts S. alterniflora. The population structure of the ribbed mussel, Geukensia demissa, was studied at nine sites in six estuaries in the southern Gulf of St. Lawrence in Nova Scotia, Canada, where marsh degradation is occurring. Mussel length was used as a proxy for age of G. demissa in three salt marsh zones characterized by density and elevation of Spartina alterniflora: (1) a lower zone in which the S. alterniflora was dead, but where the basal mat was coherent, (2) a zone of living, but low density S. alterniflora at the margin of the living marsh, and (3) a zone of dense S. alterniflora one to three meters back from the edge. Mussel length was significantly different across the three zones in seven of the nine sites. Mean length decreased as elevation increased, and small mussels (i.e., 1–3 cm) were absent at seven sites. The smallest mussels occurred in the dense S. alterniflora zone, higher in the marsh. Mussel length in the two western sites did not differ between zones, and small mussels (i.e., 1–3 cm) were present, but rare. The absence of small mussels in seven of the nine sites, and the size frequency distribution at remaining sites, suggests a lack of recent recruitment and a long-term threat to the survival of G. demissa. Salt marsh degradation and the death of S. alterniflora have negatively impacted G. demissa recruitment, and population decline is evident.     

Substitution of Arg214 at the substrate-binding site of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens.

The gene encoding p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens was cloned in Escherichia coli to provide DNA for mutagenesis studies on the protein product. A plasmid containing a 1.65-kbp insert of P. fluorescens chromosomal DNA was obtained and its nucleotide sequence determined. The DNA-derived amino acid sequence agrees completely with the chemically determined amino acid sequence of the isolated protein. The enzyme is strongly expressed under influence of the vector-encoded lac promotor and is purified to homogeneity in a simple three-step procedure. The relation between substrate binding, the effector role of substrate and hydroxylation efficiency was studied by use of site-directed mutagenesis. Arg214, in ion-pair interaction with the carboxy moiety of p-hydroxybenzoate, was replaced with Lys, Gln and Ala, respectively. The affinity of the free enzymes for NADPH is unchanged, whereas the affinity for the aromatic substrate is strongly decreased. For enzymes Arg214-->Ala and Arg214-->Gln, the effector role of substrate is lost. For enzyme Arg214-->Lys, binding of p-hydroxybenzoate highly stimulates the rate of flavin reduction. In the presence of substrate or substrate analogues, the reduced enzyme Arg214-->Lys fails to stabilize the 4 alpha-hydroperoxyflavin intermediate, essential for efficient hydroxylation. Like the wild-type, enzyme Arg214-->Lys is susceptible to substrate inhibition. From spectral and kinetic results it is suggested that secondary binding of the substrate occurs at the re side of the flavin, where the nicotinamide moiety of NADPH is supposed to bind.     

Establishment and clinical application of a highly sensitive enzyme immunoassay for determination of N‐acetyl‐seryl‐aspartyl‐lysyl‐proline

N‐acetyl‐seryl‐aspartyl‐lysyl‐proline (AcSDKP) is a natural inhibitor of pluripotent hematopoietic stem cell proliferation and is normally found in human plasma. Because AcSDKP is hydrolyzed by the N‐terminal active site of angiotensin converting enzyme and partially eliminated in urine, its plasma level is a result of a complex balance between its production, hydrolysis by ACE, and renal elimination. In this study, we attempted to establish an enzyme immunoassay (EIA) for quantifying AcSDKP‐like immunoreactive substance (IS), which is applicable for monitoring plasma AcSDKP levels in healthy subjects and patients with chronic renal failure. Using β‐ d ‐galactosidase‐labeled Gly‐γAbu‐SDKP as a marker antigen, an anti‐rabbit IgG‐coated immunoplate as a bound/free separator and 4‐methylumbelliferyl‐β‐ d ‐galactopyranoside as a fluorogenic substrate, a highly sensitive and specific EIA was developed for the quantification of AcSDKP‐IS in human plasma. The lower limit of quantification was 0.32 fmol/well, and the sharp inhibition competitive EIA calibration curve obtained was linear between 8.0 and 513 fmol/ml. This EIA was so sensitive that only 10 µl plasma sample was required for a single assay. The coefficients of variation (reproducibility) for human plasma concentrations of 0.2 and 2.1 pmol/ml were 7.2 and 7.7%, respectively, for inter‐assay and 13.3 and 7.8% for intra‐assay comparisons. Plasma AcSDKP‐IS level was significantly higher in patients with chronic renal failure (0.92 ± 0.39 pmol/ml) compared with healthy subjects (0.29 ± 0.07 pmol/ml). These results suggest that our EIA may be useful to evaluate plasma AcSDKP level as a biomarker in various patients. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Membrane‐binding mechanism of a bacterial phospholipid N‐methyltransferase

The membrane lipid phosphatidylcholine (PC) is crucial for stress adaptation and virulence of the plant pathogen Agrobacterium tumefaciens. The phospholipid N‐methyltransferase PmtA catalyzes three successive methylations of phosphatidylethanolamine to yield PC. Here, we asked how PmtA is recruited to its site of action, the inner leaflet of the membrane. We found that the enzyme attaches to the membrane via electrostatic interactions with anionic lipids, which do not serve as substrate for PmtA. Increasing PC concentrations trigger membrane dissociation suggesting that membrane binding of PmtA is negatively regulated by its end product PC. Two predicted alpha‐helical regions (αA and αF) contribute to membrane binding of PmtA. The N‐terminal helix αA binds anionic lipids in vitro with higher affinity than the central helix αF. The latter undergoes a structural transition from disordered to α‐helical conformation in the presence of anionic lipids. The basic amino acids R8 and K12 and the hydrophobic amino acid F19 are critical for membrane binding by αA as well as for activity of full‐length PmtA. We conclude that a combination of electrostatic and hydrophobic forces is responsible for membrane association of the phospholipid‐modifying enzyme.     

First finding of deepwater <Emphasis Type="Italic">profunda</Emphasis> morph of quagga mussel <Emphasis Type="Italic">Dreissena bugensis</Emphasis> in the European part of its range

The deepwater profunda morph of quagga mussel Dreissena bugensis was found for the first time in the European part of its range. The mussels of this morph were found in the Cheboksary Reservoir situated in the midstream of the Volga River (Russian Federation). Traditional and geometric morphometrics confirm the similarity of studied specimens to profunda mussel described from the Great Lakes of North America. In the Cheboksary Reservoir the deepwater mussels live at depth of 26.5 m on sandy-pebbled substrate at conditions of high water velocity (>0.5 m/s), i. e. in the conditions unusual for American profunda. This fact reflects evident ecological plasticity of this morph. Discovery of the deepwater morph of quagga mussel in the European part of its range indicates that possibility of realization of the deepwater phenotype is inherent to this species. It is suggested that quagga mussel may possess two alternative developmental pathways that could be realized in appropriate conditions. Presumably, certain depth and/or water pressure may serve as signaling factors for activating the “deepwater” developmental pathway. The presence of deepwater and shallow-water morphs is very important adaptive feature for sedentary organisms such as dreissenids that are unable to select habitat actively since this feature allows for successful colonization of both deep and shallow water habitats.     

Application of two SH-based methods for metallothionein determination in mussels and intercalibration of the spectrophotometric method: laboratory and field studies in the Mediterranean Sea

Metallothionein (MT) induction is widely used as a biomarker of exposure to metals in mussels. The aims of the present work were first to compare the suitability of spectrophotometry and differential pulse polarography (DPP) for MT detection in mussels exposed to 200 ppb cadmium for 9 days in a laboratory experiment and in mussels sampled in different seasons from expected pollution gradients along the Mediterranean Sea; second, to intercalibrate the widely used spectrophotometric method using mussels from Saronikos Gulf. In the intercalibration of the spectrophotometric method, similar results (p>0.05) were obtained by two different research teams indicating a good reproducibility of the technique. However, polarographic and spectrophotometric methods gave significantly (p<0.05) different results in laboratory and field studies. In the laboratory experiment, MT values detected with DPP were nine times higher than with spectrophotometry. The results obtained by the two methods were significantly correlated. Both methods could discriminate between control and exposed mussels. In field studies, MT values obtained by DPP were 34–38-fold higher than with spectrophotometry, and MT concentrations measured by both methods were not correlated. This discrepancy could be due to several factors, including the low levels of bioavailable metals in the studied areas and the possibility that the different methods can measure MT isoforms differentially. Further work is needed to decipher the functions of MT isoforms in mussels. This information is relevant for the application of MT as a biomarker in biomonitoring programmes.