Isolation and biochemical characterization of heparin-binding proteins from boar seminal plasma: A dual role for spermadhesins in fertilization

Sperm surface-coated heparin-binding proteins originating from secretions of the male sexual accessory glands, are known to play a pivotal role as extrinsic regulatory factors during sperm capacitation in many mammalian species. They interact with glycosaminoglycans present in the female genital tract and enhance the subsequent zona pellucida-induced acrosome reaction. We have isolated heparin-binding proteins from boar seminal plasma by affinity chromatography on heparin–Sepharose and reverse-phase HPLC. N-Terminal sequence analysis of these proteins identified a boar counterpart of the bovine capacitation factors BSP-A1/2 (also called PDC-109) and BSP-A3. Several carbohydrate- and zona pellucida-binding proteins, which belong to the newly described spermadhesin family, were also identified as heparin-binding proteins. Our results imply that, besides other capacitation factors, members of the spermadhesin family may play a dual role in sperm capacitation and fertilization in the pig. © 1993 Wiley-Liss, Inc.     

The pH-dependent unfolding mechanism of P2 myelin protein: an experimental and computational study

The P2 protein is a small, extrinsic protein of the myelin membrane in the peripheral nervous system that structurally belongs to the fatty acid binding proteins (FABPs) family, sharing with them a 10 strands beta-barrel structure. FABPs appear to be involved in cellular fatty acid transport, but very little is known about the role of P2 in the metabolism of peripheral myelin lipids. Study of protein conformation at different pHs is a useful tool for the characterization of the unfolding mechanisms and the intrinsic conformational properties of the protein, and may give insight into factors that guide protein folding pathways. In particular, low pH conditions have been shown to induce partially folded states in several proteins. In this paper, the acidic unfolding of purified P2 protein was studied with both spectroscopic techniques and molecular dynamics simulation. Both experimental and computational results indicate the presence of a partly folded state at low pH, which shows structural changes mainly involving the lid that is formed by the helix-turn-helix domain. The opening of the lid, together with a barrel relaxation, could regulate the ligand exchanges near the cell membrane, supporting the hypothesis that the P2 protein may transport fatty acids between Schwann cells and peripheral myelin.     

The Rop GTPase: an emerging signaling switch in plants

G proteins are ubiquitous molecular switches in eukaryotic signal transduction, but their roles in plant signal transduction had not been clearly established until recent studies of the plant-specific Rop subfamily of RHO GTPases. Rop participates in signaling to an array of physiological processes including cell polarity establishment, cell growth, morphogenesis, actin dynamics, H₂O₂ generation, hormone responses, and probably many other cellular processes in plants. Evidence suggests that plants have developed unique molecular mechanisms to control this universal molecular switch through novel GTPase-activating proteins and potentially through a predominant class of plant receptor-like serine/threonine kinases. Furthermore, the mechanism by which Rop regulates specific processes may also be distinct from that for other GTPases. These advances have raised the exciting possibility that the elucidation of Rop GTPase signaling may lead to the establishment of a new paradigm for G protein-dependent signal transduction in plants.     

Collapse transitions in protein-like lattice polymers: The effect of sequence patterns

The collapse transition of lattice protein-like heteropolymers has been studied by means of the Monte Carlo method. The protein model has been reduced to the α-carbon trace restricted to a high coordination lattice. The sequences of model heteropolymers contain two types of mers: hydrophobic/nonpolar (H) and hydrophilic/polar (P). Interactions of HH and PP pairs were assumed to be negative (weaker attractions of PP pairs) while the contact energy for HP pairs was equal to zero. All sequence-specific short-range interactions have been neglected in the present studies. It has been found that homopolymeric chains undergo a smooth collapse transition to a dense globular state. The globule lacks any signatures of local ordering that could be interpreted as a model of protein secondary structure. Hetero-polymers with the sequences of hydrophilic and hydrophobic residues characteristic for α- and β-type proteins undergo a somewhat sharper (though continuous) collapse transition to a dense globular state with elements of local ordering controlled by the sequence. The helical pattern induces more secondary structure than the β-type pattern. For all examined sequences the level of local ordering was lower than the average secondary structure content of globular proteins. The results are compared with other theoretical work and with known experimental facts. The implications for the reduced modeling of protein systems are briefly discussed. © 1997 John Wiley & Sons, Inc. Biopoly 42: 537–548, 1997     

The role of protein sequence and amino acid composition in amyloid formation: scrambling and backward reading of IAPP amyloid fibrils

The specific functional structure of natural proteins is determined by the way in which amino acids are sequentially connected in the polypeptide. The tight sequence/structure relationship governing protein folding does not seem to apply to amyloid fibril formation because many proteins without any sequence relationship have been shown to assemble into very similar β-sheet-enriched structures. Here, we have characterized the aggregation kinetics, seeding ability, morphology, conformation, stability, and toxicity of amyloid fibrils formed by a 20-residue domain of the islet amyloid polypeptide (IAPP), as well as of a backward and scrambled version of this peptide. The three IAPP peptides readily aggregate into ordered, β-sheet-enriched, amyloid-like fibrils. However, the mechanism of formation and the structural and functional properties of aggregates formed from these three peptides are different in such a way that they do not cross-seed each other despite sharing a common amino acid composition. The results confirm that, as for globular proteins, highly specific polypeptide sequential traits govern the assembly pathway, final fine structure, and cytotoxic properties of amyloid conformations.     

The costs and benefits in an unusual symbiosis: experimental evidence that bitterling fish (Rhodeus sericeus) are parasites of unionid mussels in Europe

Interspecific symbiotic relationships involve a complex network of interactions, and understanding their outcome requires quantification of the costs and benefits to both partners. We experimentally investigated the costs and benefits in the relationship between European bitterling fish (Rhodeus sericeus) and freshwater mussels that are used by R. sericeus for oviposition. This relationship has hitherto been thought mutualistic, on the premise that R. sericeus use mussels as foster parents of their embryos while mussels use R. sericeus as hosts for their larvae. We demonstrate that R. sericeus is a parasite of European mussels, because it (i) avoids the cost of infection by mussel larvae and (ii) imposes a direct cost on mussels. Our experiments also indicate a potential coevolutionary arms race between bitterling fishes and their mussel hosts; the outcome of this relationship may differ between Asia, the centre of distribution of bitterling fishes, and Europe where they have recently invaded.     

The search for stability on narrow supports: an experimental study in cats and dogs

Kinematic and coordination variables were studied in two carnivorans, one with known locomotor capabilities in arboreal substrates (cat), and the other a completely terrestrial species (dog). Two horizontal substrates were used: a flat trackway on the ground (overground locomotion) and an elevated and narrow runway (narrow-support locomotion). Despite their different degree of familiarity with the ‘arboreal’ situation, both species developed a strategy to adapt to narrow supports. The strategy of cats was based on using slower speeds, coupled with modifications to swing phase duration, to keep balance on narrow supports. The strategy of dogs relied on high speeds to gain in dynamic stability, and they increased cycle frequency by reducing swing phase duration. Furthermore, dogs showed a high variability in limb coordination, although a tendency to canter-like coordination was observed, and also avoided whole-body aerial phases. In different ways, both strategies suggested a reduction of peak vertical forces, and hence a reduction of the vertical oscillations of the centre of mass. Finally, lateral oscillation was reduced by the use of a crouched posture.     

The nucleotide sequence of a regulatory gene present on a plasmid in an enterotoxigenic Escherichia coli strain of serotype O167:H5

A DNA fragment that can functionally substitute for cfaD, the positive regulatory gene involved in expression of CFA/I fimbriae, has recently been cloned from an Escherichia coli strain of serotype O167:H5 that produces CS5 fimbriae. Nucleotide sequence determination showed that the fragment contained a gene, csvR (Coli Surface Virulence factor Regulator) homologous to the cfaD gene, which encoded for a protein of 301 amino acid residues. The csvR gene was found to be located between two different insertion sequences. Comparison of the amino acid sequence of the CsvR and CfaD proteins showed that CsvR is 34 amino acid residues longer at the C-terminus and, in the sequence, it also contains an insertion of two amino acid residues. The similarity between CfaD and Rns, the positive regulator of CS1 and CS2 expression, is much higher (97%) than between CsvR and CfaD (87%). This is reflected by the fact that the level of expression of CFA/I fimbriae induced by CsvR is not as high as when expression is induced by CfaD or Rns.     

The potential role of phoresy in the evolution of parasitism: radiolabelling (tritium) evidence from an astigmatid mite

Using tritium as a radiolabel marker of interspecific fluid transfer, we present experimental evidence that the heteromorphic deutonymph of an astigmatid mite (Hemisarcoptes cooremani) acquires materials (at least water) directly from the haemolymph of its beetle host (Chilocorus cacti). This acquisition is above that obtained from atmospheric vapour. The material acquired from the host is necessary for the completion of the ontogeny of H. cooremani and is likely procured through the action of the caudal ventral suckers of the heteromorphic deutonymph (hypopus). On gross morphological criteria, this mite-beetle relationship was previously defined as phoretic (for dispersal). Scanning electron photomicrographs of the physical relationship between the hypopodes and the heetles shed light on the parasitic nature of the hypopus of H. cooremani. Our findings are discussed in terms of the evolution of parasitism from a free-living astigmatid form. This transition into parasitism is facilitated by the heteromorphic hypopus and represents a classic wolf-insheep's-clothing strategy. The heteromorph retains the characteristic phoretic morphology while exploiting the host in transit.     

The disappearance of an hsc70 species in mung bean seed during germination: purification and characterization of the protein

We have purified a 73 kDa protein from the cytosolic fraction of mung bean seeds. It comprises 0.5–1% of the total protein in seeds. This purified protein is a bona fide hsc70 on the basis of several lines of evidence. First, antibodies against bovine brain hsc70 cross-react with the purified 73 kDa protein. Second, the purified protein comigrates on two-dimensional gels with one of the heat-inducible hsc70s in mung bean seedlings. Third, similar to other hsc70 species, the purified 73 kDa protein has a high affinity for ATP. Finally, the hydrolysis of ATP by the purified protein can be stimulated by peptides; ATPase activity increases from 40 nmol/h to 165 nmol/h per mg of protein. The purified mung bean hsc70 autophosphorylates at a substoichiometric level. Moreover, the amount of this hsc70 species diminishes while new species of hsc70s appear after germination, suggesting that the expressionof hsc70 in mung bean is subject to developmental regulation.     

The distribution and localization of an UDP-glucose: Flavonol 3-O-glucosyl transferase activity in pollen

Summary The occurrence of UDP-glucose: flavonol 3-O-glucosyltransferase activity in pollen extracts of various plant species was tested. In case ofAlnus, Quercus, Narcissus andTulipa pollen high enzyme activity could be detected. The high level of enzyme activity inTulipa pollen made short time extraction experiments possible, which showed that the O-glucosyltransferase activity might be located in the pollen wall, possibly in the exine.Abbreviations UDP-glucose uridine diphospho-D-glucose - UDP-rhamnose uridine diphospho-L-rhamnose - DTE dithioerythritol     

The role of trade-off shapes in the evolution of parasites in spatial host populations: an approximate analytical approach

Given the substantial changes in mixing in many populations, there is considerable interest in the role that spatial structure can play in the evolution of disease. Here we examine the role of different trade-off shapes in the evolution of parasites in a spatially structured host population where infection can occur locally or globally. We develop an approximate adaptive dynamic analytical approach, to examine how the evolutionarily stable (ES) virulence depends not only on the fraction of global infection/transmission but also on the shape of the trade-off between transmission and virulence. Our analysis can successfully predict the ES virulence found previously by simulation of the full system. The analysis confirms that when there is a linear trade-off between transmission and virulence spatial structure may lead to an ES virulence that increases as the proportion of global transmission increases. However, we also show that the ESS disappears above a threshold level of global infection, leading to maximization. In addition just below this threshold, there is the possibility of evolutionary bi-stabilities. When we assume the realistic trade-off between transmission and virulence that results in an ESS in the classical mixed model, we find that spatial structure can increase or decrease the ES virulence. A relatively high proportion of local infection reduces virulence but intermediate levels can select for higher virulence. Our work not only emphasizes the importance of spatial structure to the evolution of parasites, but also makes it clear that situations between the local and the global need to be considered. We also emphasize the key role that the shape of trade-offs plays in evolutionary outcomes.     

The use of biomarkers in Daphnia magna toxicity testing. IV. Cellular Energy Allocation: a new methodology to assess the energy budget of toxicant-stressed Daphnia populations

The Cellular Energy Allocation (CEA) methodology wasdeveloped as biomarker technique to assess the effectof toxic stress on the energy budget of testorganisms. This short-term assay is based on thebiochemical assessment of changes in the energyreserves (total carbohydrate, protein and lipidcontent) and the energy consumption (electrontransport activity). The CEA methodology was evaluatedusing Daphnia magna juveniles exposed for 96hto sublethal lindane and mercury chlorideconcentrations. The ecological relevance of the CEAassay was assessed by comparing the sub-organismalresponse with population level parameters (obtainedfrom 21 day life table experiments) such as theintrinsic rate of natural increase (r_m) and themean total offspring per female. Two differentmethodologies were used to assess the effect levels:the no (lowest) observed effect level (NOAECs-LOAECs)approach and the regression-based approach. Bothtoxicants caused a significant decrease in the netenergy budget of D. magna, with a LowestObserved (Adverse) Effect Concentration (LOAEC) of0.18 mg/l and 5.6 µg/l for lindane andHgCl₂,respectively. Changes in the lipid content of theorganisms were detected at toxicant concentrationslower than those affecting the total carbohydrate andprotein content. Toxicant specific effects wereobserved on the electron transport activity.Comparison of the CEA results with those of thepopulation level tests revealed that for mercury theCEA based LOAEC was a three times lower than thatbased on r_m and the total brood size(18 µg/l). For lindane the CEA based LOAEC was twotimes lower than the LOAEC based on r_m(0.32 mg/l) but was higher than that based on thetotal number of offspring produced (0.1 mg/l).Using the regression-based approach, EC₁₀ valueswere calculated using three parameter sigmoid orlogistic models. Comparison between the CEA andr_m based EC₁₀ values demonstrates that forboth chemicals similar effect concentrations areobtained: the CEA-based EC₁₀ (0.20 mg/l) forlindane is 1.5 times higher than the r_m-basedEC₁₀ threshold (0.13 mg/l), while for mercury thebiomarker-based EC₁₀ value (9 µg/l) was 1.4times lower than the population-based EC₁₀ value(12.5 µg/l).From these results, we suggest that the short-term CEAassay may be useful for predicting long-term effectsat the population level. The consequences of theobserved effects on the energy budget of the testorganism are discussed in the context of the effectsemerging at the population and community level.     

Immediate shift towards source dynamics: The pioneer species Conyza canadensis in an initial ecosystem

The aim of this study was to detect a potential temporal shift from sink to source dynamics of pioneer plants during primary succession. The study was done in an initial ecosystem, namely an artificially constructed catchment on Pleistocene sands in eastern Germany. The import and export of seeds (including soil seed bank and seed rain), the development of vegetation cover and the population size of all pioneer species were recorded in 2005-2009. The interplay between the spatial distribution of seed rain and the prevailing wind direction provide evidence that seeds of Conyza canadensis (Asteraceae) first and to a considerable amount left the developing ecosystem. A change from a sink to source dynamics was detected already in the first year of both the existence of the site and occurrence of C. canadensis individuals within the site. Population patterns at the landscape unit scale demonstrated extensive auto-correlations in the coverage of this invasive plant at first. Our results underline the pioneer traits of C. canadensis as a prolific seeder and excellent wind disperser, which enables this species to become rapidly dominant over relatively large areas.     

The chloroplast-located glutamine synthetase of Phaseolus vulgaris L.: nucleotide sequence,expression in different organs and uptake into isolated chloroplasts

Work using a full-length cDNA clone has revealed that the plastid-located glutamine synthetase (GS) of Phaseolus vulgaris is encoded by a single nuclear gene. Nucleotide sequencing has shown that this cDNA is more closely related to a cDNA encoding the plastidic GS of Pisum sativum than to cDNAs encoding three different cytosolic GS subunits of P. vulgaris. The plastid GS subunits are initially synthesized as higher M _r (47000) precursors containing an N-terminal presequence of about 50 amino acids which is structurally similar to the presequences of other nuclear-encoded chloroplast proteins. The precursor has been synthesized in vitro and is imported by isolated pea chloroplasts and processed to two polypeptides of the same size as native P. vulgaris chloroplast GS subunits (M _r 42000). Experiments with fusion proteins show that the N-terminal 68 amino acids of this precursor allow the cytosolic GS subunit also to be imported and processed by isolated chloroplasts. Polyadenylated mRNA specifically related to the plastidic GS gene is most highly abundant in chloroplast-containing organs (leaves and stems) but is also detectable in roots and nodules.     

The glass transition temperatures of amorphous trehalose-water mixtures and the mobility of water: an experimental and in silico study

Isothermal-isobaric molecular dynamics simulations are used to calculate the specific volume of models of trehalose and three amorphous trehalose-water mixtures (2.9%, 4.5% and 5.3% (w/w) water, respectively) as a function of temperature. Plots of specific volume versus temperature exhibit a characteristic change in slope when the amorphous systems change from the glassy to the rubbery state and the intersection of the two regression lines provides an estimate of the glass transition temperature T(g). A comparison of the calculated and experimental T(g) values, as obtained from differential scanning calorimetry, shows that despite the predicted values being systematically higher (about 21-26K), the trend and the incremental differences between the T(g) values have been computed correctly: T(g)(5.3%(w/w))相似文献